Aquaculture 492 (2018) 349–356

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Characteristics of hybrids derived from Channa argus ♀ × Channa maculata ♂ T

⁎
Mi Ou, Jian Zhao, Qing Luo, Xiaoyou Hong, Xinping Zhu, Haiyang Liu, Kunci Chen
Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese
Academy of Fishery Sciences, Guangzhou 510380, China

A R T I C LE I N FO A B S T R A C T

Keywords: Interspecific hybridization has been used as an important tool for genetic manipulation in aquaculture, with the
Interspecific hybridization goal of obtaining new strains with desired traits from both parental species. In this study, a hybrid strain was
Channa argus established by Channa argus (NS) (♀) and Channa maculata (BS) (♂), named NBS hybrid snakehead (NBS). A
Channa maculata comparison of the morphological characteristics (meristic and measurable traits), ploidy (DNA content and
Growth
karyotype), and aquaculture performance (growth and low-temperature tolerance) were examined. NBS re-
Low-temperature tolerance
sembled the male parent (BS) in body stripes and body shape and had moderate values for its meristic traits.
Flow cytometry indicated that NBS is diploid, and the karyotype confirmed that the number of chromosomes in
NBS (2n = 45) was between that in NS (2n = 48) and that in BS (2n = 42). In addition, NBS showed sig-
nificantly higher growth rates compared to its parents. Under the same feeding conditions, the average body
weight of NBS was 421.3 ± 72.9 g at six months of age, which was significantly greater than NS
(187.6 ± 42.8 g) and BS (245.6 ± 52.5 g) by 124.6% and 71.53%, respectively (P < 0.05). The overwintering
culture test proved that there were no significant differences in the low-temperature tolerance between NS and
NBS (P > 0.05), and both lived through the winter when the water temperature dropped to 0–2 °C. In general,
NBS is easily trained to accept commercial feed (a characteristic of BS) and has fast growth rate and strong low-
temperature tolerance (characteristics of NS), and it will be a valuable new strain for the aquaculture industry.

1. Introduction and curative functions in traditional Chinese medicine (Wang et al.,

Interspecific hybridization is a breeding programme for enhancing
various traits or combining valuable traits from different species
(Falconer and Mackay, 1996), and hybrids exhibit heterosis or trans-
gressive inheritance, such as increasing growth rates, improving flesh
quality, strengthening disease resistance and increasing environmental
tolerances (Bartley et al., 2000; Hu et al., 2012). To date, many hybrid
fish have been produced, some are used for aquaculture, and others
have been evaluated for aquaculture performance (Gabriele et al.,
1986; Hulata, 1995; Rosenfield et al., 2000, 2004; Dunham et al., 2008;
You et al., 2009; Nielsen et al., 2010; Granier et al., 2011; Arias et al.,
2012; Dunham et al., 2014; Wang et al., 2015a,b; Huang et al., 2016;
Park et al., 2017).
The northern snakehead (Channa argus, NS) and blotched snakehead
(Channa maculata, BS) are Chinese indigenous species, belonging to the
Channidae family (Zhang et al., 2017). NS is mainly distributed in the
Yangtze River and the river system north of the Yangtze River, while BS
is mainly distributed in the Pearl River and the Hainan River system
(Zhuo et al., 2012). Snakeheads are economically important freshwater
fish due to their high protein content, significant anti-hypoxia capacity
2012; Ou et al., 2017). The total production of snakeheads reached
517,888 tons in 2016 (China's Ministry of Agriculture, 2017), ranking
ninth in terms of the production of all freshwater fish species in China.
NS has fast growth rate, large size, and poor water quality and strong
low-temperature tolerances (Zhou et al., 2012). However, NS currently
feeds on ice fresh fish in aquaculture settings, and it is difficult for them
to be trained to accept commercial feed (Liu et al., 1998; Chen et al.,
2009; Wang et al., 2012). As a result, NS aquaculture costs are rela-
tively high, and it is difficult to maintain good water quality. Although
BS can be trained to accept commercial feed, they have poor low-
temperature tolerances, and mortality rates are relatively high when
water temperature drop below 6 °C. As a result, the areas suitable for BS
aquaculture in China are very limited (Zhang et al., 2015). Over the
past few years, researchers have developed BNS hybrid snakehead (BS
♀ × NS ♂), which has obvious heterosis due to its rapid growth rate,
strong disease resistance, trainability to accept commercial feed, and
high feed conversion ratio (Yang, 2004; Liu et al., 2008; Zhao et al.,
2016). However, field surveys and aquaculture practices have demon-
strated that there is significant sexual dimorphism in the growth rates
and body sizes of males versus females in BNS hybrids, and the quality

⁎
Corresponding author.
E-mail address: chenkunci@prfri.ac.cn (K. Chen).

https://doi.org/10.1016/j.aquaculture.2018.04.038
Received 23 January 2018; Received in revised form 18 April 2018; Accepted 19 April 2018
Available online 22 April 2018
0044-8486/ © 2018 Elsevier B.V. All rights reserved.
M. Ou et al.
and the overall yield of BNS hybrids are negatively impacted by this
sexual dimorphism (Liu et al., 2011a,b; Mei and Gui, 2015; Ou et al.,
2017). Furthermore, BNS cannot live through the winter in northern
China when water temperature drop below 2 °C.
To address the previously mentioned problems, according to the
characteristics of NS and BS, our team conducted an interspecific hy-
bridization of NS ♀ × BS ♂ to produce the NBS hybrids (NBS).
Furthermore, NBS has become a popular breed to culture in the
Channidae family. In this study, we introduced the production process,
morphological characters (meristic and measurable traits), ploidy (DNA
content and chromosome number), and aquaculture performance
(growth and low-temperature tolerance) of NBS, providing guidance for
future Channidae fish breeding.
2. Materials and methods
2.1. Generation of NBS hybrids from NS ♀ × BS ♂
3000 NS and 1000 BS were collected from the wild and fisheries,
then they were bred at the Huinong Aquaculture Fishery (Zhongshan
City, Guanhdong Province, China). Sexually mature and healthy NS
(female) and BS (male) individuals were selected from generation
screening for artificial breeding. The breeding method was employed
according to Kahkesh et al. (2010) with some minor modifications. NS
(♀) were injected with an LH-RH analogue at dosages of 4 μg/kg for the
first injection. Twelve hours later, NS (♀) were injected with 16 μg / kg
LH-RH analogue and 700 IU/kg HCG for the second injection; at the
same time, BS (♂) were injected with 3 μg/kg LH-RH analogue and
60 IU/kg HCG. Then, NS (♀) and BS (♂) were paired together in the
spawning box, and natural mating occurred after approximately 22 h.
Two inbred groups of NS and BS were produced and used as the con-
trols. The eggs were hatched in 29 ± 1 °C freshwater and hatched out
in approximately 48 h. The fry started exogenous feeding with plankton
on day 3 post-hatching, and NS, BS and NBS fry were fed at the Huinong
Aquaculture Fishery under the same conditions.
2.2. Morphological examination of NBS
Thirty one-year-old NS, BS and NBS were randomly selected from
each group for morphological examination, healthy fish were selected,
and the body shapes and stripes were observed and photographed by a
digital camera (Nikon, Japan).
The morphological variations of NS, BS and NBS were studied using
traditional morphometrics, which includes meristic and measurable
traits, and the examination standards were based on Zou et al. (2008).
Ten meristic traits of each fish (number of soft rays of the dorsal fin,
number of soft rays of the pectoral fin, number of soft rays of the pelvic
fin, number of soft rays of the anal fin, number of soft rays of the tail fin,
number of gill rakers, number of lateral line scales, number of scales
above the lateral line, number of scales below the lateral line and
number of vertebra) were recorded. Ten measurable traits (whole
length, body length, body height, body width, head length, caudal
peduncle length, caudal peduncle depth, snout length, eye diameter,
and interorbital width ( ± 0.1 cm)) were recorded.
The average values of whole length to body length (WL / BL), body
length to body height (BL / BH), body length to body width (BL / BW),
body length to head length (BL / HL), body length to caudal peduncle
length (BL / CPL), caudal peduncle length to caudal peduncle height
(CPL / CPH), head length to snout length (HL / SL), head length to eye
diameter (HL / ED), and head length to interorbital width (HL / IW)
were calculated. The hybrid index (HI) of the meristic traits and the
average values of the measurable traits were calculated using the fol-
lowing formula (Matondo et al., 2008): HI = 100 × (Hi-Mi1) / (Mi2-
Mi1), where Hi = mean of hybrid; Mi1 = mean of dam; Mi2 = mean of
sire. When the HI is between 45 and 55, it is an intermediate trait; when
the HI is smaller than 45, it is a maternal trait; when the HI is larger
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Aquaculture 492 (2018) 349–356
than 55, it is a paternal trait; when the HI is larger than 100 or smaller
than 0, it is a transgressive trait. The average values of ten measurable
traits were used for the cluster analysis, and the Euclidean distance of
the population was calculated.
2.3. Ploidy determination of NBS
Individual relative DNA content was determined according to the
technique described by Tao et al. (2012) with some minor modifica-
tions. Peripheral blood was taken from the caudal veins of 15 NS, 15 BS,
and 15 NBS that were older than 6 months. Red blood cells from
chicken (Gallus sp., whose DNA content is 2.50 pg) served as standard
references for DNA quantification. The blood samples were stained
according to the protocol for the Coulter DNA-Prep Reagents Kit
(Beckman Coulter, UK): 100 μL DNA-prep LPR was added to the tube,
the cell was lysed for 1 min, and then, 1 mL DNA-prep stain (propidium
iodide, RNase) was added. While staining, cells were placed in the dark
for 30 min at room temperature. Three replicate analyses were per-
formed per sample. Analyses were performed on Cell Lab Quanta SC
flow cytometer (Beckman Coulter, UK).
Mitotic chromosome preparations were obtained from the kidney
tissues of NS, BS and NBS, and an air-drying technique was adopted
(Foresti et al., 1993; Liu et al., 2011a, 2011b). For each type of fish, 100
good quality metaphase spreads from ten individuals were used for the
analysis of karyotype and chromosomes that were classified by previous
standards (Levan et al., 1964; Liu et al., 2011a,b).
2.4. Aquaculture performance of NBS
2.4.1. Growth performance
On day 7 post-hatching, NS, BS and NBS fry were transferred to
different ponds at a density of 100,000–150,000 fry / 667 m2, and they
were fed to satiation with ice fresh fish twice daily. Then, we gradually
trained NS, BS, and NBS to accept commercial feed and observed their
feed situation. When NS, BS and NBS had been completely trained to
accept commercial feed, after a period of feeding, fish that were 10 cm
in length were selected from each group for growth performance ex-
periments, which were conducted in 500 cm × 500 cm × 150 cm net
cages. Each group of 500 fry / cage was separately fed, and two parallel
groups were set. Body length ( ± 0.1 cm) and body weight ( ± 0.1 g) of
30 randomly selected individuals from each group were measured and
used as the initiative data. All test fish were fed twice daily with
commercial Channidae feed containing 45% crude protein during the
fingerling period and 40% crude protein during the adult period. All
test fish were fed under similar conditions. Body length ( ± 0.1 cm) and
body weight ( ± 0.1 g) of 30 randomly selected individuals of each
group were measured every month from July to November in 2012. The
growth performance experiments were repeated the next year. To better
represent the increase in body weight, the average daily gain (ADG)
was calculated by the following formula (Korkut et al., 2007):
ADG = (Wf − Wi)/(Df − Di), where Wf = mean of final weight;
Wi = mean of initial weight; Df = the final day; Di = the initial day.
2.4.2. Low-temperature tolerance test
To determine the low-temperature tolerance, 60 NS, 60 BS and 60
NBS weighing approximately 95.6 g–106 g and with lengths of
18.2 cm–19.2 cm were first acclimated to the laboratory conditions for
48 h before the tests. The tests were performed in 80 cm × 50 cm
× 40 cm fibre-glass tanks with 50 L of water. Ten individuals were
randomly selected from each of the three groups, those 30 fish were put
in one tank, and three replicates were assigned. To reduce stress caused
by drastic changes in temperature, a temperature regulating device was
used to control the temperature. The cooling process included three
steps. First, the water was cooled at a rate of 1 °C / 3 h when the water
temperature was higher than 12 °C. When the water temperature
reached 12 °C, the cooling process stopped, and all fish acclimated to
M. Ou et al.
the water for 24 h. Second, the water was cooled at a rate of 1 °C / 6 h
until the temperature reached 8 °C. Lastly, the water temperature
dropped by a rate of 2 °C /24 h until 2 °C, then the cooling process
stopped, and the water temperature was maintained at 2 °C for three
days. During the cooling process, fish were checked every 2 h to record
and remove dead ones. Fish that could not freely swim were considered
as cold-shocked, and those that had stopped breathing with their gills
and bodies did not react with glass rod were considered dead. The
cooling experiments were repeated one time, and the mean data were
the results of the cooling experiments.
In 2013–2014, overwintering culture experiments with NBS were
conducted in Shandong Province, which is in northern China, and the
local cultured NS were used as the control. In June 2013, 5000 fin-
gerling NS and NBS that were 5–6 cm in length were cultured in se-
parate ponds (approximately 667 m2), and each group had two ponds.
NBS were fed with commercial feed, while NS were fed with ice fresh
fish. All fish were fed under similar conditions. In December 2013, 30
NS and 30 NBS were randomly selected for measurements before the
ponds froze, and then, the test fish naturally wintered in their original
ponds. In April 2014, when the ponds had thawed, 30 NS and 30 NBS
were randomly selected for measurements before feeding. Then, these
fish continued to feed on commercial fish until October. In 2014–2015,
the low-temperature tolerance tests were conducted again.
2.5. Statistical analyses
All data were presented in the format of X ± SD and analysed with
Student's t-test using the SPSS 19.0 statistical package to determine the
difference between the parameters of the selected strain and the control
group, and significant differences were recognized as valid if P < 0.05.
All graphics were drawn using Sigma Plot 12.5.
3. Results
3.1. Comparison of characters among NS, BS and NBS
The front part of the body of NS, BS and NBS were cylindrical, and
the dorsal margin and ventral margin were flat. However, there were
obvious differences in the stripes among NS, BS and NBS that were
located on the head, the sides of the body and the base of the caudal fin.
NBS resembled the paternal BS much more than the maternal NS in
terms of stripes (Fig. 1). The stripes on the head of NBS were the same
as those on BS and different from those on NS. The spots on the sides of
the body of NS crossed the lateral line, and the stripes obviously in-
terlaced, and the area of the spot was large. However, the spots on the
sides of the body of BS and NBS did not cross the lateral line, and the
stripes appeared in two rows. The area of the spots on BS was much
smaller than that on NS, and the area of spots on NBS was in between
those for NS and BS. There were one or two curve stripes across the base
of the caudal fin of BS; however, no curve stripe crossed the base of the
caudal fin of NS, and the stripe on that of NBS was between that on NS
and BS.
The ten meristic traits of NS, BS and NBS are presented in Table 1.
The number of soft rays of the dorsal fin, soft rays of the pectoral fin,
soft rays of the anal fin, soft rays of the tail fin, lateral line scales, scales
above the lateral line, scales below the lateral line and vertebra in NBS
overlapped with those of NS or BS. For example, the number of soft rays
of the pectoral fin of NBS was 15–18, a value that was between the
15–19 rays of NS and the 16–19 rays of BS. The number of soft rays of
the pelvic fin was 6 for NS, BS and NBS. The number of gill rakers in
NBS (n = 11–14), which resembled BS (n = 9–13) and could be dis-
tinguished from NS (n = 9–10).
With the 10 meristic characters, the HI showed that the number of
lateral line scales and scales above the lateral line inclined to its male
parent species (BS) (HI were 57.79 and 56.42, respectively); the
number of soft rays of the pectoral fin were significantly less than the
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Aquaculture 492 (2018) 349–356
parents (HI was −700.00), which was a deviation from the parents;
other traits (the number of soft rays of the dorsal fin, soft rays of the
anal fin, soft rays of the tail fin and vertebra) were intermediate or
nearly intermediate traits. In general, the average HI of the meristic
traits was calculated to be 54.47, indicating that the meristic traits of
NBS are close to the ideal median.
The comparison analysis for the average values of measurable traits
(Table 2) indicated that BL / BW, BL / HL and HL / IW in NS, BS and
NBS did not have significant differences (P > 0.05); WL / BL and CPL /
CPH were significantly different between NBS and its parents
(P < 0.05). Specifically, BL / BH, BL / CPL and HL / ED in NBS were
not significantly different from BS (P > 0.05), while they were sig-
nificantly different from those in NS (P < 0.05). In terms of HI, BL /
BW (HI = 95.24), BL / HL (HI = 60.87), BL / CPL (HI = 80.91) and
CPL / CPH (HI = 65.00) for NBS were similar to BS. Specifically, WL /
BL (HI = 400.00) was the super-paternal deviation, HL / IW
(HI = 1.03) was similar to NS, and HL / SL (HI = −41.67) was the
super-maternal deviation. The cluster analysis of measurable traits
showed that NBS first clustered with BS, and then, it clustered with NS
(Fig. 2).
3.2. Cellular DNA contents of NS, BS and NBS
The cellular DNA contents of NS, BS and NBS were assayed using a
flow cytometer with the DNA of chicken red blood cells (2.5 pg / 2c) as
a standard. The relative position of the 2n peak between chicken red
blood cells and the red blood cells of NS, BS and NBS are shown in
Fig. 3A–C. The relative cellular DNA contents in NBS and its parents
were 1.520 ± 0.033 pg /2c (NBS), 1.489 ± 0.034 pg / 2c (NS) and
1.488 ± 0.035 pg / 2c (BS). There is no significant difference between
NS and BS (P > 0.05), while there are significant differences between
NBS and its parents (P < 0.05).
3.3. Karyotype analysis of NS, BS and NBS
The chromosome number and karyotype of inbred NS and BS and
hybrid NBS were studied from the kidney cells by an air-flame drying
method. Table 3 shows the distribution of chromosome number for NS,
BS and NBS. The results indicated that the diploid chromosome number
of NS was 2n = 48, the karyotype formula was 4sm + 22st + 22 t, and
the arm number (NF) was 52 (Fig. 4A); BS was 2n = 42, the karyotype
formula was 4 m + 2sm + 16st + 20 t, and NF was 48 (Fig. 4B); NBS
was 2n = 45, the karyotype formula was 2 m + 4sm + 9st + 30 t, and
NF was 51 (Fig. 4C). NBS inherited a set of chromosomes from its
parents, which is the hybrid of NS and BS, and the number of chro-
mosomes could become one identification index of NBS.
3.4. Growth rate of NS, BS and NBS
Table 4 shows the results of the growth performance tests in 2012. After
6 months of feeding, the average body length of NBS was 29.6 ± 2.6 cm,
which was significantly longer than NS (22.5 ± 2.7 cm) and BS
(24.8 ± 2.5 cm) by 31.56% and 19.35%, respectively (P < 0.05). The
average body weight of NBS was 421.3 ± 72.9 g, which was significantly
greater than NS (187.6 ± 42.8 g) and BS (245.6 ± 52.5 g) by 124.6% and
71.53%, respectively (P < .05). Fig. 5A–B presents the growth variation in
NS, BS and the NBS during the test. NBS exhibited a higher growth rate than
its parents over the entire period, especially after 60 days of feeding. The
ADG of NS, BS and NBS were 1.38 g/d, 1.88 g/d and 3.39 g/d, respectively.
The growth performance test was repeated the following year, and
the results, which were consistent with the previous results, are shown
in Table 5 and Fig. 5C. The ADG of NBS was 5.20 g / d, which was
higher than that of NS (2.14 g / d) and that of BS (2.28 g / d) by 143.0%
and 128.1%, respectively. Overall, the growth rate of NBS increased
significantly.
M. Ou et al. Aquaculture 492 (2018) 349–356

Fig. 1. Physical appearance of NS, BS and NBS. (A) The head view of NS. (B) The full view of NS. (C) The head view of BS. (D) The full view of BS. (E) The head view
of NBS. (F) The full view of NBS.

3.5. Low-temperature tolerance of the NBS Table 2

The average values of the measurable traits and hybrid index of NS, BS and
Table 6 shows the behaviour of NS, BS and NBS under different NBS.
water temperatures in the laboratory. The results showed that the low- Items NS BS NBS HI
temperature tolerance ability of BS was the weakest. When the water
1 a a b
temperature was under 10 °C, BS began to move slowly and lay at the WL/BL 1.15 ± 0.01 1.16 ± 0.03 1.19 ± 0.05 400.00
BL/BH1 5.68 ± 0.44a 4.80 ± 0.34b 4.64 ± 0.47b 118.18
bottom; when the water temperature was 5 °C, 41 BS were cold-
BL/BW1 6.44 ± 0.40 6.23 ± 0.67 6.24 ± 0.75 95.24
shocked, accounting for 68.3% of BS, and 12 BS died, accounting for BL/HL1 3.84 ± 0.20 3.61 ± 0.18 3.70 ± 0.09 60.87
20% of BS; there was a substantial number of deaths at 4 °C. In contrast, BL/CPL1 15.40 ± 2.07a 10.11 ± 0.65b 11.12 ± 0.97b 80.91
the low-temperature tolerance of NS and NBS was strong. NS and NBS CPL/CPH1 0.69 ± 0.10a 1.09 ± 0.10a 0.95 ± 0.10b 65.00
were normal until the water temperature was under 5 °C; when the HL/SL1 6.71 ± 0.89ab 5.87 ± 0.59a 7.06 ± 0.61b −41.67
HL/ED1 9.90 ± 0.77a 8.40 ± 0.43b 9.09 ± 0.57b 54.00
water temperature was 4 °C, only 13 NBS were cold-shocked, ac- HL/IW1 4.50 ± 0.33 3.92 ± 0.20 4.32 ± 0.23 31.03
counting for 21.6% of NBS, and 9 NS were cold-shocked, accounting for Mean 95.95
15% of NS; when the water temperature was 3 °C, the remaining BS
a,b
died; however, NS and NBS were only cold-shocked and could respond The means with different superscripts in the same line represents significant
to stimulation with a glass rod; when the water temperature dropped to difference (P < 0.05) (n = 30; ± SD).
1
2 °C, only 4 NBS died, and no NS died. The low-temperature tolerance WL = whole length, BL = body length, BH = body height, BW = body
of NBS was generally equal to that of NS. width, HL = head length, CPL = caudal peduncle length, CPH = caudal ped-
uncle height, SL = snout length, ED = eye diameter, IW = interorbital width.
Table 7 shows the overwintering feeding conditions of NS and NBS
in northern China. After 6 months of feeding, NBS grew faster, with a
mean weight of 371 ± 33.2 g when feeding on commercial feed, than 21,756.35 kg ice fresh fish were used for NS during the test; thus, the
NS, with a mean weight of 258 ± 36.5 g feeding on ice fresh fish. feed conversion rate (FCR) for NS was 3.85; 8102 NBS individuals were
When the water temperature was under 0 °C, the ponds were frozen, obtained, and 11,480 kg compound feed were used for NBS; thus, the
and the test fish naturally wintered in their original ponds. The survival FCR for NBS was 1.58. In general, NBS can survive through the winter
rate of NS and NBS were 100% and 97%, respectively, and there was no under 0 °C, and its FCR was lower than that of NS.
significant difference in the low-temperature tolerances between NS
and NBS (P > .05). Finally, 7865 NS individuals were obtained, and

Table 1
Meristic traits and hybrid index of NS, BS and NBS.
Items NS BS NBS HI

No. of soft rays of the dorsal fin 47–52 (49.25 ± 1.48) 43–46 (44.85 ± 0.99) 46–51 (47.23 ± 1.53) 45.91
No. of soft rays of the pectoral fin 15–19 (17.05 ± 0.83) 16–19 (17.20 ± 0.70) 15–18 (16.0 ± 1.03) −700.00
No. of soft rays of the pelvic fin 6 6 6 /
No. of soft rays of the anal fin 30–34 (32.50 ± 1.10) 28–32 (29.60 ± 1.27) 30–33 (31.08 ± 1.39) 48.96
No. of soft rays of the tail fin 16–20 (17.20 ± 1.01) 15–17 (16.35 ± 0.67) 17–19 (16.79 ± 0.83) 48.23
No. of gill rakers 11–14 (12.55 ± 0.83) 9–13 (10.83 ± 1.00) 9–10 (9.83 ± 0.53) 158.14
No. of lateral line scales 61–68 (62.70 ± 2.10) 53–60 (55.00 ± 1.89) 55–66 (58.25 ± 0.78) 57.79
No. of scales above lateral line 8–10 (8.36 ± 0.57) 5–6 (5.40 ± 0.50) 6–8 (6.69 ± 0.89) 56.42
No. of scales below lateral line 17–20 (18.40 ± 1.00) 13–18 (14.90 ± 1.25) 16–21 (17.36 ± 1.03) 29.71
No. of vertebra 57–60(58.10 ± 0.99) 52–55(53.42 ± 0.79) 52–57 (55.99 ± 0.98) 45.09
Meana 54.47

a
The average HI of meristic traits except for the number of soft rays of the pectoral fin.

352
M. Ou et al. Aquaculture 492 (2018) 349–356

Table 3
Distribution of chromosome numbers of NS, BS and NBS.
Fish type Distribution of chromosome number Total metaphase
spreads
< 42 42 43–44 45 46–47 48 > 48

NS 8 90 2 100
BS 10 87 3 100
NBS 1 16 74 9 100

4.1. Morphological characteristics

NS and BS belong to two species, and they are different in physical

appearance. The stripes of NBS were most similar to BS, the spots on the
sides of the body of NBS did not cross the lateral line, and the stripes
Fig. 2. Dendrogram of the cluster analysis conducted on measurable traits of
appeared in two rows. However, the area of the spot on NBS was be-
NS, BS and NBS. tween that on NS and BS (Fig. 1). Among the ten meristic traits, eight
traits of NBS were within the range of its parents, such as the number of
dorsal fins. The average HI of the meristic traits was 54.47, which can
4. Discussion
be interpreted as the meristic traits approached intermediate values;
thus, it is possible to preliminarily conclude that NBS are the produc-
Hybridization, defined as a cross between two different species or
tion of NS and BS. Of the nine values of the measurable traits, six values
taxa, plays a very important role in the speciation and adaptive radia-
of NBS are significantly different from those of its parents, including
tion of animals (Seehausen, 2004; Mallet, 2007). Hybridization facil-
three that are paternal, one that are maternal and two that deviate from
itates the transfer of genomes between species and gives rise to phe-
the parents. The average HI of the values of the measurable traits was
notypic and genotypic changes in the hybrid offspring (Stelkens et al.,
calculated as 95.95, which indicates that the measurable traits of NBS
2009; Zhang et al., 2014). Commonly, the hybrid offspring performs
are more similar to BS. The cluster analysis showed that NBS has ob-
better than a single or both parental species in terms of growth rate,
vious differences from its parents, appearing to be affected more by
survival, disease resistance, and other traits (Barton, 2001). This study
paternity (Fig. 2). Overall, NBS resembled the male parent in its body
clearly demonstrates that the hybridization between C. argus and C.
stripes and body shape, and it had intermediated values for the meristic
maculata yielded superior progeny than the parental species. Further-
traits.
more, NBS exhibited great heterosis in growth and low-temperature
Liu et al. (2011a,b) conducted studies on the morphological char-
tolerance, which will be very beneficial.
acteristics of the BNS hybrid and found that BNS showed resemblance

Fig. 3. DNA histograms of red blood cells for NS, BS

and NBS. (A) The mean DNA contents of NS (green
line) and chicken (red line). (B) The mean DNA
contents of BS (rose red line) and chicken (red line).
(C) The mean DNA contents of NBS (blue line) and
chicken (red line). (For interpretation of the refer-
ences to colour in this figure legend, the reader is
referred to the web version of this article.)

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M. Ou et al. Aquaculture 492 (2018) 349–356

types of fish had a peak in 100 and a small peak in 200; thus, NBS
should be diploid like its parents. The karyotype confirmed that NBS
inherited a set of chromosomes from maternal NS and paternal BS, and
the number of chromosomes in NBS (2n = 45) was an intermediate
value from that in NS (2n = 48) and that in BS (2n = 42). Liu et al.
(2011a,b) reported that the karyotype formula in BNS was
2n = 45 = 3 m + 6sm + 36 st, t, and NF was 54. The number of
chromosomes in NBS and BNS were the same, while the karyotype
formulas were different. It indicates that reciprocal hybrids may inherit
different chromosomes from their parents under different ways of hy-
bridization, which might explain why there are some differences in
appearances and characteristics between NBS and BNS. Overall, the
hybridization of NS and BS resulted in diploid hybrids, and the DNA
content and the number of chromosomes could become identification
indexes of NBS from its parents.

4.3. Growth

Hybrids often present a good combination of beneficial traits from

Fig. 4. The spread of metaphase chromosomes and the karyotype of NS, BS and
NBS. (A) The karyotype of NS: 2n = 48 = 4sm + 22st + 22 t. (B) The kar-
yotype of BS: 2n = 42 = 4 m + 2sm + 16st + 20 t. (C) The karyotype of NBS:
2n = 45 = 2 m + 4sm + 9st + 30 t.
to its female parent (BS) in body colour, but it resembled the male
parent (NS) in body shape, which is different from the conclusion that
we found. In fact, this phenomenon is universal in the reciprocal cross.
With different ways of hybridization, the same fish may obtain re-
ciprocal hybrids with different morphological characteristics that are
more similar to paternity, maternity or intermediate values, such as
Megalobrama amblycephala and Erythroculter ilishaeformis (Gu et al.,
2008; Zheng et al., 2015), white crucian carp and red crucian carp
(Wang et al., 2015a,b).The results indicate that the morphological
characteristics of hybrids are complex, and the cause of this phenom-
enon needs further study.
4.2. DNA content and karyotype
Fish interspecific hybridization may produce haploid, diploid,
polyploidy, gynogenesis, or androgenesis offspring (Liu, 2010). Flow
cytometry is an optical technique that is valuable for determining
ploidy in fish (Lamatsch et al., 2000). Fig. 3A - 3C showed flow cyto-
metric histograms of red blood cells nuclear relative DNA contents for
NS, BS and NBS. The DNA contents of all the individuals of these three
both parent species, such as heterosis in survival, growth rate, flesh
quality, and disease resistance (Hulata, 2001). An improved growth
rate is probably the most desirable trait for genetic improvement in
aquaculture. NS and BS are carnivorous throughout their life, foraging
on a wide variety of prey (Courtenay and Williams, 2004), and diet
analyses showed that fry and juveniles primarily consume in-
vertebrates, whereas adults are predominantly piscivorous (Wu et al.,
2000). Under long-term artificial feeding, NS can feed on ice fresh fish
(Liu et al., 1998; Chen et al., 2009; Wang et al., 2012), but could not be
trained to eat commercial feed, while BS can be completely trained to
accept commercial feed (Zhang et al., 2015). In our study, an inter-
specific hybridization of NS ♀ × BS ♂ was conducted to produce NBS,
and growth performance showed that NBS was easily domesticated to
commercial feed. Under the same feeding conditions, the same-sized
NS, BS and NBS were cultured. As a result, NBS presented obvious
heterosis in growth rate, whose mean values for body length and body
weight were higher than that of NS and that of BS (Fig. 4A - 4C). Distant
hybridization that results in rapid growth of the hybrid has been widely
used in aquaculture. For example, the hybrids of the silver and big head
carp were reported to grow faster than the parental lines (Marian et al.,
1986); M. amblycephala Yih (BSB) (♀) × Xenocypris davidi Bleeker (YT)
(♂) hybrids grow faster than BSB and YT by 11.2% and 53.1%, re-
spectively (Hu et al., 2012); triploid Epinephelus coioides ♀ × E. lan-
ceolatus ♂ hybrids grow faster than either parent species (Huang et al.,
2016).
During the overwintering culture experiments of NS and NBS in
northern China, NBS, whose mean weight was 896.3 g and FCR was
1.58. However, the mean weight of NS was 718.5 g, and its FCR was
3.85. On the one hand, NBS had a faster growth rate than its parents; on
the other hand, it reduced the aquaculture costs and improved water
quality.
4.4. Low-temperature tolerance
Temperature is one of the most important environmental factors
affecting growth and survival in fish (Kelly and Kohler, 1999); thus, the
low-temperature tolerance of the new strain is critical to determining

Table 4
The growth performance data on body length and body weight from NS, BS and NBS in 2012.
Fish types Initial day 30 days 60 days 90 days 120 days

Length (cm) Weight (g) Length (cm) Weight (g) Length (cm) Weight (g) Length (cm) Weight (g) Length (cm) Weight (g)

NS 10.4 ± 0.2 21.6 ± 3.5 16.7 ± 0.3 75.8 ± 4.7 20.8 ± 0.5 131.3 ± 5.7 21.6 ± 0.34 153.7 ± 6.5 22.5 ± 0.7 187.6 ± 6.8
BS 9.7 ± 0.1 20.1 ± 2.8 16.3 ± 0.3 76.6 ± 2.9 20.5 ± 0.2 136.8 ± 3.2 22.1 ± 0.2 176.9 ± 4.7 24.8 ± 0.5 245.6 ± 6.5
NBS 10.1 ± 0.2 20.7 ± 2.4 17.4 ± 0.3 90.6 ± 3.1 22.6 ± 0.4 177.6 ± 4.8 25.3 ± 0.3 287.2 ± 8.8 29.6 ± 0.4 421.3 ± 5.9

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M. Ou et al. Aquaculture 492 (2018) 349–356

Fig. 5. The results of growth performance among NS, BS and NBS in 2012–2013. (A) The average body length of NS, BS and NBS from July to November in 2012. (B)
The average body weight of NS, BS and NBS from July to November in 2012. (C) The average body weight of NS, BS and NBS from May to September in 2013.

Table 5
The growth performance data for body weight from NS, BS and NBS in 2013.
Fish types Initial weight (g) 30 days weight (g) 60 days weight (g) 90 days weight (g) 120 days weight (g)

NS 9.02 ± 1.24 52.6 ± 9.25 103.2 ± 18.53 172.3 ± 33.29 266 ± 53.24
BS 9.18 ± 1.23 58.1 ± 10.05 123.5 ± 22.57 223.4 ± 43.72 282.3 ± 56.84
NBS 8.66 ± 1.01 122.3 ± 18.83 285.3 ± 47.24 465.2 ± 82.42 632.3 ± 114.5

the waters suitable for its culture. In most of northern China, the surface Table 7
water temperature can drop to 0–2 °C in the winter for extended periods The results of overwintering feeding in 2013–2014.
of up to two weeks. These extreme temperatures significantly affect not Fish Initial Weight before Weight after Final Final
only the growth but also the survival of the hybrids. In this study, a low- types number overwintering (g) overwintering (g) number mean
temperature tolerance test in laboratory determined that NBS had weight
strong low-temperature tolerance like NS, which could survive when (g)

the water temperature was 2 °C. At the same time, we found that BS had NS 10,000 258 ± 36.5 226 ± 26.3 7865 718.5
poor low-temperature tolerance, it expressed discomfort when the NBS 10,000 371 ± 33.2 337 ± 21.5 8102 896.3
water temperature was 7 °C, and experienced substantial mortality
when the water temperature was 4 °C. Thus, BS is only suitable for
culture in warm climate regions. The overwintering culture test also These observations indicate that the low-temperature tolerance trait of
proved that there were no significant differences in the low-tempera- NS partly transferred to NBS, showing maternal effects.
ture tolerances between NS and NBS (P > 0.05), which can survive the In conclusion, NBS was produced from the cross of C. argus (NS) (♀)
winter when the water temperature drops to 0–2 °C in northern China. and C. maculata (BS) (♂). The characteristics of NS, BS and NBS were

Table 6
The behaviour of NS, BS and NBS under different water temperatures.
Temperature (°C) Behaviour BS NBS

NS

> 10 °C normal normal normal

8 °C–10 °C normal began to move slowly and lay on the bottom normal
6 °C–8 °C normal most of the individuals lay on the bottom normal
5 °C began to move slowly and lay on the bottom 41 were in shock, 12 were dead, 7 lay on the bottom began to move slowly and lay in the bottom
4 °C 9 were in shock, other fish lay in the bottom 13 were in shock, 35 were dead 13 were in shock, other fish lay on the bottom
3 °C all shocked, but could respond to stimulation the remaining 13 die all shocked, but could respond to stimulation
2 °C all shocked, but could respond to stimulation / 56 were in shock, 4 were dead

355
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successfully compared by various methods. There were many differ-
ences in body-structure proportions between NBS and its parents. The
aquaculture practices demonstrated that NBS can be trained to accept
commercial feed, and it has fast growth rate, low feed conversion ratio,
and strong low-temperature tolerance, which provides enormous com-
mercial value.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This study was support by China agriculture Research System [grant
number CARS-46] and Pear River S&T Nova Program of Guangzhou
[grant number 201710010174]. We would like to thank all colleagues
in our lab for sample collection and laboratory technical assistance.
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