Comparative analysis of the morphology, karyotypes and biochemical composition of muscle in Siniperca chuatsi, Siniperca scherzeri and the F1 hybrid (S. chuatsi ♀ × S. scherzeri ♂)

Wenzhi Guan, Gaofeng Qiu, Feng Liu

aKey Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture; National Demonstration Center for Experimental Fisheries Science Education;Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, China

bShanghai Fisheries Research Institute, Shanghai Fisheries Technical Extension Station, Shanghai, China

Keywords:

Mandarin fish

Hybrid

Morphology

Karyotype

Nutrition

A B S T R A C T

Combining morphological comparison with karyotyping can make a more accurate and comprehensive study of mandarin fish (Siniperca spp.) and hybrid progenies.

In recent years, the domestication of hybrid mandarin fish has become a hot spot in China, and it is essential to evaluate the nutritional value of the fish after food conversion. In this study, the F1 hybrid (S. chuatsi ♀ ×S.scherzeri♂) were fed with artificial feed for five months. Afterwards, we studied the morphology, karyotypes,and biochemical composition of muscle in the F1 hybrid and its parents. The morphological results showed that total length and body height of the F1 hybrid appear to derive more from S. chuatsi than S. scherzeri. The DNA ploidy and karyotype results indicated that the F1 hybrid have a diploid chromosome number (2n =48) and the same karyotypes (2n =4sm +14th+30t, NF =52) with its parents, suggesting that the chromosome of the mandarin fish has convergence in the process of evolution. The results of biochemical composition of muscle showed that there were no significant difference in other nutritional indicators (P ＞0.05), except the Gly(glycine) content of the F1 hybrid was significantly higher than that of S. chuatsi (P ＜0.05) under the experimental feeding regime, suggesting that the nutritional value of the F1 hybrid with artificial feeding culture is also higher. The study is expected to provide a reference for further research on the domestication, classification,chromosomes, and nutritional requirements of mandarin fish.

1.Introduction

Morphology is a very important indicator in fish classification. In recent years, great progress has been made in the study of interspecific relationships in fish by morphological analysis (Blob et al., 2008; Calabro, Albanese, Lauriano, & Martella, 2005; Guan, Zheng, Wu, & Wang,2017; Zheng et al., 2019). The morphological comparison and identi fication of artificial hybrid offspring contribute to detect the degree of genetic mixing in wild populations and have positive significance for the protection of natural populations (Britz, Conway, & Ruber, 2009; Cabrera Paez, Aguilar Betancourt, & Gonzalez-Sanson, 2008; Camelier,Menezes, Costa-Silva, & Oliveira, 2018; O’Sullivan et al., 2019).Furthermore, three multivariate morphometric analyses have been conducted to assess morphological variations based on statistical data,with the goal of auxiliary seed selection in fish populations (Guan et al.,2017; Guo, Zheng, & Wu, 2018; Matondo, Ovidio, & Poncin, 2008). This method has also been applied to other aquatic animals (Abdel-Baki,2009; Ahmed & Abbas, 2000; Wang, Wu, Yang, & Zhu, 2011). Because of the relatively small habitat ofSinipercaspp, it is easy to mate with each other under natural conditions, which results in the decline and mixing of the germplasm resources of the fish in China.

The analysis of chromosomes and their karyotypes in fish is of great significance and is mainly used in the studies of classification, breeding,evolution and genetics of fishes (Hnatkova et al., 2018; Krysanov &Demidova, 2018; Machado et al., 2018; Mank & Avise, 2006). In China,chromosomes have been studied at least 300 species of fish, mainly from the order Cypriniformes, followed by Perciformes and Siluriformes (Lou,1997). At least more than half of the teleost fish examined had diploid chromosome numbers of 2n =48-50 (Mank & Avise, 2006; Molina ,Martinez, Bertollo, & Bidau, 2014). At present, a wide variety of economic fish species have been identified. These includeMegalobrama amblycephala(Wu, Li, Chen, Jiang, & Zou, 2015),Nibea albi flora(Geng,Xu, Lou, & Chai, 2012),Hemibarbus maculatusBleeker (Li, 2008),Lates niloticus,Siniperca chuatsi(Zhu, Zhang, Min, & Wang, 2009),Pelteobagrus fulvidraco(Mao, Ge, Liu, & Shu, 2012),Epinephelus moara(Zhong, Chen,& Zhuang, 2018) andHucho taimen(Zhang, Sun, Yin, & Yin, 2008).These species are all diploids, and their chromosome number is 2n =48-50. In contrast,Cyprinus carpioandCarassius auratusare also diploid,but they have a higher chromosome number of 2n =100 (Lou, 1997).Surprisingly, few studies have compared the karyotype of chromosomes between hybridised parent species and their offspring. Recently, there have been some studies on mandarin fish. The chromosome number ofS. chuatsiwas 2n =48, but there are differences in the karyotype of this species.S. chuatsiindividuals from TaiHu (Jiangsu Province) have a karyotype of 2n =6sm +12th+30t, NF =54 (Zhu et al., 2009), andS. chuatsiindividuals from Poyanghu (Jiangxi Province) have a karyotype of 2n =24sm +24t, NF =72 (Yang, Li, Xu, & Zhang, 1999).Although the karyotypes of mandarin fish have been reported but they are mainly focus onS. chuatsi.The karyotypes ofS. scherzeriand the F1 hybrid have never been studied.

Mandarin fish, a freshwater predatory fish, is widely favoured as a delicacy in China.S. chuatsiandS. scherzeriare the major species among mandarin fish, owing to their highly palatable meat and economic value(Sun et al., 2017).S. chuatsigrows rapidly, but is susceptible to disease in the breeding process. In contrast,S. scherzerihas the characteristics of slow growth, strong resistance to disease and easily domesticated to eat feed. The superiority of hybrids has prompted many studies on the breeding of fish hybrids in China and abroad (Aboim, Mavarez, Bernatchez, & Coelho, 2010; Chevassus, 1983; Guan, Zheng, Wu, & Wu,2017; Guo et al., 2018; Lou & Li, 2006; Stelkens, Schmid, & Seehausen,2015).S. chuatsiandS. scherzerihave always been used to generate F1 hybrids. The F1 hybrid shows strong disease resistance and easy domestication during culturing, which make it of great commercial value (He et al., 2013; Li et al., 2017; Liu, Guan, & Wang, 2019).Biochemical composition studies of mandarin fish raised by live prey fish have been published (Li et al., 2017; Li, Shi, Li, & Shi, 2015; Mi,Chen, Lian, & Wang, 2009), but there have yet to be studies of mandarin fish raised by artificial feed. In the present study, we analyzed the morphology and karyotypes ofS. chuatsi,S. scherzeri, and their F1 hybrid progeny. Additionally, we studied the biochemical composition of muscle in the three species of fish. We expect that this study will provide guidance for future research on the domestication, classification, chromosomes, and nutritional requirements of mandarin fish.

2.Material and methods

2.1.Fish used in the experiments

Artificial reproduction and insemination of mandarin fish were carried out in May 2018.S. chuatsiandS. scherzeriwere supplied by Sheng Jian Aquaculture Farm of Shanghai, China.F1 hybrid progeny were generated by usedS. chuatsi(♀) andS. scherzeri (♂). F1 individuals were fed with live prey fish until their body length reached 3 cm. Subsequently, artificial feed (Qingdao Qihao Biotechnology Co., Ltd.) containing 40% fish meat was used to raised F1 hybrids for five months. The formula for artificial feeds is as follows: the crude protein and crude lipid content were greater than or equal to 48.0% and 9.0%, and the moisture,crude fiber and ash content were less than or equal to 10.0%, 2.0% and 17.0%, respectively. During the breeding period, we regularly detect the water environment to maintain the indicators within a certain range,including transparency (30-40 cm), temperature (22-26°C), dissolved oxygen (above 8 mg/L), pH (7.0-8.5), ammonia nitrogen (less than 0.2 mg/L) and nitrite (less than 0.1 mg/L).

2.2.Morphological traits

We randomly selected 24 individuals from each group ofS. chuatsi,S. scherzeriand the F1 hybrid for morphological measurement, whose body weight ranged from 81.4 to 325.2 g. An electronic balance was used to measure the body weight (BWE, accurate to 0.01 g), compasses and ruler were used to measure full length, body length (BL), body height (BH) and body width (BWI) (accurate to 0.1 mm). Dorsal fins,including hard fins and soft fins, were counted for each individual.

2.3.DNA ploidy analysis

The DNA ploidy analysis of the three groups (30 fish per group) was measured using fow cytometer (Partec, Germany) according to a previously described method (Liu et al., 2007). Firstly, syringes treated with heparin sodium were used to draw blood from the caudal vein of each fish. Blood samples (3-5 μL) were added to DAPI dye solution (1 mL) and stained for 30-60 s without light. This fuid was then filtered into the upper sample tube using a 500-mesh filter tube. Flow cytometery was used to detect the DNA content of each sample, and it was stopped when between 3000 and 10,000 cells were detected.Megalobrama amblycephalawas selected as the control.

2.4.Preparation of chromosome

Chromosomes were prepared by injecting phytohemagglutinin(PHA) and colchicine in vivo. Three individuals for each group(S. chuatsi,S. scherzeriand F1 hybrids) were selected for the experiment,with each fish weighing about 120g. The fish were placed in a 120 L glass tank and stopped feeding three days before the experiment. PHA was injected into the pectoral fin base of each fish at a concentration of 10 μg/g, then colchicine was injected at 4 mg/g 22 h later. After 4 h, the head-kidney was removed and placed in a saline solution of 0.75%.Using tweezers to tear the kidney tissue until the lymphocytes were released completely and collecting the cell suspension. After centrifugation (1000 r/min, 5 min), it was added to 0.5% KCl solution (6 mL).After treatment with the hypotonic solution for 50 min, the cells were fixed three times with the freshly prepared liquid (methanol: glacial acetic acid =3:1). After the slides were prepared and dried at 37°C for 1h, they were stained with Giemsa. Chromosomes were photographed using a Nikon SMZ1500 fuorescence microscope (Tokyo, Japan). The karyotype analysis method used in this study was adopted from the standard proposed by Levan (Levan A, 1964). By calculating the arm ratio, chromosomes were divided into four types: metacentric (m),submetacentric (sm), subtelocentric (st) and telocentric (t).

2.5.Biochemical composition of muscle

After five months of domestication of F1 hybrid, muscle tissues of the F1 hybrid, undomesticatedS. chuatsiandS. scherzeriwere collected to conduct analysis of biochemical composition. Three fishes (120 g per individuals) were randomly selected from each group. Moisture was determined by oven drying until constant weight (105°C). Crude protein was determined using the Kjeldahl method with an Auto Kjeldahl System (FOSS KJELTEC 2300, Foss Analytics, Hilleroed, Denmark).Crude lipid was determined using the chloroform and methanol extraction method, and ash was created using a muffe furnace at 550°C for 6 h. The hydrolysis of amino acids was determined using an automatic analyser (Sykam S-433D, Skykam, Fürstenfeldbruck, Germany)(Wang et al., 2018).

2.6.Statistical analysis

All the data were analyzed using SPSS 19.0 software and expressed as means ± standard deviation (SD). If significant differences (P ＜0.05)were found in factors, Duncan’s multiple range test was used to rank the means.

3.Results

3.1.Morphological traits

The F1 hybrids and their parents were hatched at different times. In order to eliminate the infuence of different size of fish on morphological analysis, we corrected it by dividing the measured morphological data of each fish by its body length (TL/BL, BH/BL, BWI/BL) according to the previous study (Guan, Zheng, Wu, & Wu, 2017) (Table 1 and Fig. 1). The results showed that there was a significant difference (P ＜0.05) in TL/BL and BH/BL between the F1 hybrid andS. scherzeri, but no significant difference (P ＞0.05) between the F1 hybrid andS. chuatsi.In addition, the F1 hybrid showed a significant difference (P ＜0.05) in BWI/BL when compared with bothS. chuatsiandS. scherzeri.Moreover,S. chuatsiexhibited the largest number of soft fins, which makes it easily distinguishable from other fish species.

Table 1Morphological traits of the three groups.

Fig. 1.The morphology of mandarin fish. A) Siniperca chuatsi, Siniperca scherzeri and F1 hybrid; B). The mapping of morphological measurement. Line 1-4: TL (total length); line 1-3: BL (body length); line 2-5: BH (body height); line 6-7: BWI (body width).

3.2.DNA ploidy analysis

In this study,Megalobrama amblycephalawas used as the control (Wu et al., 2015; Zheng, Zhang, Li, & Chen, 2015). The DNA ploidy analysis was shown in Fig. 2 and Table 2. The results showed that there was no significant difference in DNA content between the three groups (P ＞0.05). The mean DNA content of F1 hybrids have nearly half as much DNA content as their parents (S. chuatsiandS. scherzeri). In addition, the relative DNA content ofS. chuatsi,S. scherzeri, and the F1 hybrid were very similar to the control, suggesting that they are all diploids.

Fig. 2.The relative DNA content of the three groups of mandarin fish analyzed using fow cytometry. A) Siniperca chuatsi; B) Siniperca scherzeri; C) F1 hybrid; D)Megalobrama amblycephala.

Table 2The relative DNA content of the three groups.

3.3.Chromosome karyotype

The head-kidney tissues of fish in the three groups was used to prepare chromosome specimens. After Giemsa staining, 100 cells were analyzed in each individual. Photographs were taken with a Nikon SMZ1500 fuorescence microscope (Tokyo, Japan). The number of cells with chromosomes 2n =48 accounted for 86% of F1 hybrid, 83% ofS. chuatsiand 79% ofS. scherzeri(Table 3). Therefore, the experimental results tend to support the view that the three groups of mandarin fish were all diploid with the chromosome numbers of 2n =48.

Table 3Chromosome number of the three groups.

Photoshop CS5 was used for image matting. Image J was used to analyse and measure the chromosomes, including the long and short arms. Microsoft Excel was used to conduct the statistical analysis.Finally, we obtained the karyotype parameters of the F1 hybrid and its parent species. As shown in Fig. 3 and Table 4, the karyotype results indicated that fish in all three groups have a diploid chromosome number of 2n =48, and they have the same karyotypes (2n =4sm +14th+30t, NF =52). In addition, no metacentric (m), secondary constrictions, satellite or sex chromosomes were found.

Fig. 3.The chromosomes of the three groups of mandarin fish. A) Siniperca chuatsi; B) Siniperca scherzeri; C) F1 hybrid. sm: submetacentric; st: subtelocentric; t: telocentric. Scale bar, 3 μm.

Table 4Indices of karyotypes of the three groups (X±SD, N =10).

3.4.Biochemical composition of muscle

The analysis results of biochemical composition in muscle are shown in Table 5.

Table 5The biochemical composition in muscle tissue of the three groups.

Under the experimental feeding regime, there were no significant difference in moisture, crude protein, crude lipid and ash content in muscle tissue of F1 hybrid compared to their parents (P ＞0.05). However, the crude protein content ofS. scherzeriwas significantly higher than that ofS. chuatsi(P ＜0.05). Additionally, the total amino acid(TAA) and delicious amino acid (DAA) contents of the three fish groups were very similar and showed no significant differences (P ＞0.05). Only the Gly (glycine) content of the muscle tissue of F1 hybrid individuals was significantly higher than that ofS. chuatsiindividuals (P ＜0.05)(Table 6).

Table 6Comparison of Amino acids in the three groups.

4.Discussion

Morphology has often been used in the investigation of fishery resources, including the classification and identification of fish,population differentiation and the assessment of genetic diversity. It is widely used in discriminating fish species all over the world (Arratia,Vila, Lam, & Guerrero, 2017; Guan et al., 2017; Ukenye, Taiwo, &Anyanwu, 2019). Some studies have found that the morphological traits of mandarin fish are related to domestication (Zhang, Liang, & Yi,2013). In the present study, we found that some characteristics of the F1 hybrid (S. chuatsi♀ ×S. scherzeri♂), such as total length and body height, were more partial to the female parent species. The results are similar to those in previous mandarin fish studies (Zhao, Zhu, Chen, &Liu, 2008). These results showed that the contribution ofS. chuatsito the progeny was greater than that ofS. scherzeri. In addition, the somatotype of the F1 hybrid (S. chuatsi♂ ×S. scherzeri♀) were also more similar to theS. chuatsispecies (Xu et al., 2013). Moreover, in our study,S. chuatsiexhibited the greatest number of soft fins among the three species groups(14-15), which makes it easily distinguishable from other fish. This provides a reference for germplasm identification in nature of theSinipercaspp.

Morphological description alone has proved to be insuf ficient in determining genetic relationships within and between species. However, the combination of morphology and karyotype can provide clarity. A study of the clearly evident heterosis and genetic characters of an intergeneric cross and backcross juveniles between blunt snout bream(Megalobrama amblycephala) and topmouth culter (Culter alburnus) has been performed (Zheng et al., 2019). The same analytical method of the karyotype and morphological characteristics was applied to burbot,Lota lota, and it was ultimately speculated that burbot might be an evolutionarily specialized species (Zhou, Jiang, Zhang, & Wang, 2019). Of course, karyotype analysis has wider applications, for example, the biological evolution, genetic variation, sex determination, and hybrid breeding of fish (Canitz, Kirschbaum, & Tiedemann, 2016; Majtanova et al., 2016; Rab et al., 2016; Sanchez, Swarca, & Fenocchio, 2014).Generally, most freshwater fish have diploid chromosome numbers of 2n =48-50 (Lou, 1997). This study also con firmed that the F1 hybrid mandarin fish and its parent have a diploid chromosome number of 2n=48, and they have the same karyotype pattern of 2n =4sm +14th+30t, NF =52. Previous studies have shown that Chinese mandarin fish that come from TaiHu, have a chromosome number of 2n =48 and a karyotype formula of 2n =6sm +12th+30t, NF =54 (Zhu et al., 2009).Additionally, the chromosome number of Poyanghu mandarin fish was also 2n =48, but the karyotype is 2n =24sm +24t, NF =72 (Yang et al.,1999). Compared with the above results, the chromosome number of mandarin fish is 2n =48, but their karyotype and NF are quite different.Similarly,Pelteobagrus fulvidracofrom the Chinese provinces of Heilongjiang, Hubei, and Poyanghu have diploid chromosome numbers of 2n =52, but the karyotypes are also greatly different (Mao et al., 2012).This may be due to different water environments and long-term geographical isolation and heredity, which lead to the occurrence of chromosome polymorphism in fish. Moreover, no metacentric, secondary constriction, satellite or sex chromosome were found in our study.Most studies suggest that the satellite, secondary constriction and sex chromosomes have not been found in many species of freshwater fish(Mao et al., 2012; Wu et al., 2015; Zhou et al., 2019; Zhu et al., 2009).Coilia Nasus, however, is a notable exception, in which the sexual chromosomes and heteromorphic chromosomes were detected, and the sex karyotype formula was ZZ-ZO which is rare not only in fish but also in vertebrates generally (Xu, Li, Fu, & Wu, 2014).

Meanwhile, we used a fow cytometer analyzed the DNA content ofS. chuatsi,S. scherzeriand F1 hybrid. The results demonstrated that there are not much difference in the relative DNA content among the three species groups, and all of which are diploids (P ＞0.05). The DNA content of some fish species is constant and it has species specificity, which can be used as a characteristic parameter of species genetics (Liu et al.,2007; Wu et al., 2015; Yan & Liang, 2014; Zheng et al., 2019). The dual methods of karyotyping and DNA content analysis can provide a direct reference for karyotype operations and fish breeding.

Compared with other fishes, the mandarin fish are a highly nutritious and economically valuable fish (Kim, Lim, Kang, Kim, & Son, 2012;Sagada et al., 2017; Zhao et al., 2018). Through continuous breeding and promotion, hybrids of mandarin fish were produced and have becoming increasingly popular in China recently. An increasing number of studies are being conducted on the reproduction, growth, food transformation and nutrient contents of hybrid mandarin fish (Li et al.,2017; Li et al., 2015; Li, Xu, TH Jeerawat, & Zhao, 2016; Liu, Li, & Guan,2019). In a previous study, the biochemical composition of muscle in the F1 hybrid and its parents (S. chuatsiandS. scherzeri) has been analyzed(Mi et al., 2009). The fishes were all raised on forage fish and come from the same pond environment. Their results showed no significant difference in the muscle moisture, crude lipid, ash and 18 amino acid content amongS. chuatsi,S. scherzeriand their F1 hybrid (P ＞0.05). But,the crude protein content of the F1 hybrid andS. scherzeriwas signi ficantly higher than that ofS. chuatsi(P ＜0.05) (Mi et al., 2009). These results are very similar to our own study. Additionally, there is little difference between F1 hybrids (feeding with forage fish and artificial feed) in the moisture, crude protein, crude lipid and ash content.However, the amino acid content of F1 hybrid in the previous study were lower than that in our study, and this may be caused by different feeding methods that leads to the changes in muscle nutrition in the sample fish.The content of protein and lipid are important indices to evaluate the nutritional value of fish meat. Our results demonstrated that the F1 hybrid cultured with artificial feed still has high nutrition. In addition,no significant difference was found for the TAA and DAA content between the F1 hybrid and its parents. However, the Gly content of F1 was significantly higher than inS. chuatsi,which has a unique sweetness.These results revealed that the nutrition levels of the domesticated hybrid mandarin fish cultured with artificial feed was no big difference withS. chuatsiandS. scherzeri.

5.Conclusion

In summary, F1 hybrid progeny were obtained by crossingS. chuatsi(♀) withS. scherzeri (♂) in this study. It was con firmed that some morphological characteristics of the F1 hybrid fell between their parents, but there was slightly more similarity toS. chuatsi. All three species were diploids. They have the same chromosome number and karyotype,which reveals there was convergence in chromosome karyotype during the evolution of mandarin fish. The biochemical composition in muscle tissue of the domesticated F1 hybrid cultured with artificial feed containing 40% fish meat has no big difference withS. chuatsiandS. scherzeri. The study is expected to provide a reference for further understanding of classification, chromosomes, domestication and nutritional requirements research of the mandarin fish.

CRediT authorship contribution statement

Wenzhi Guan: Formal analysis, Data curation, performed the experiments, analyzed the data, contributed reagents, materials, analysis tools, prepared figures and tables. Gaofeng Qiu: Writing - original draft,conceived and designed the experiments, authored or reviewed drafts of the paper. FengLiu: Formal analysis, Data curation, conceived and designed the experiments, analyzed the data, All authors contributed to finalizing and approving the manuscript.

Declaration of competing interest

The authors declare no competing interests.

Acknowledgements

This research was supported by grants from the Youth Talent Development Plan of Shanghai Municipal Agricultural System, China(Grant No. 20180303), and the Shanghai Agriculture Applied Technology Development Program, China (Grant No. Z20170204).