Biological Characteristics of Jade Perch (Scortumbarcoo)

Jade perch (Scortumbarcoo), also known as Barcoo grunter, is a fish species belonging to thegenus Scortum, the family Terapontidae and the order Perciformes. It originates from the BarcooRiver of the Lake Eyre basin in central Australia and was introduced in China in 2001 [1]. The fleshof jade perch is firm and slightly flaky, sweet and succulent, without intermuscular bones, and isrich in nutrients, especially, highly unsaturated fatty acids. A study conducted by the AustralianCommonwealth Scientific and Industrial Research Organization (CSIRO) in 1998 indicated thatamong 200 seafood species tested, jade perch contained the highest level of omega-3, which wasapproximately 3-fold that in Atlantic salmon and silver bass. Jade perch grow extremely fast andare highly suited for aquaculture in areas with a moderate subtropical to tropical climate. Underartificial aquaculture conditions, they can grow to food size in 6-10 months on a formulated diet.Recently, there is an increasing interest for species diversification to support the development ofsustainable aquaculture. Fast-growing jade perch, which can be stocked at high densities inrecirculating aquaculture systems (RAS) and feeds on grow-out diets with very low levels offishmeal and fish oil, is a promising candidate for aquaculture [2,3]. Currently, this species is beingfarmed in both intensive ponds and recirlulating systems not only in Australian, but also in China, Malaysia, even in Belgium [4-6].Since the introduction of jade perch in China, domestic researchers have conducted severalstudies on the breeding and nutrition of this species. Chen et al. [1,7] studied the artificial


Introduction
Jade perch (Scortumbarcoo), also known as Barcoo grunter, is a fish species belonging to thegenus Scortum, the family Terapontidae and the order Perciformes. It originates from the BarcooRiver of the Lake Eyre basin in central Australia and was introduced in China in 2001 [1]. The fleshof jade perch is firm and slightly flaky, sweet and succulent, without intermuscular bones, and isrich in nutrients, especially, highly unsaturated fatty acids. A study conducted by the AustralianCommonwealth Scientific and Industrial Research Organization (CSIRO) in 1998 indicated thatamong 200 seafood species tested, jade perch contained the highest level of omega-3, which wasapproximately 3-fold that in Atlantic salmon and silver bass. Jade perch grow extremely fast andare highly suited for aquaculture in areas with a moderate subtropical to tropical climate. Underartificial aquaculture conditions, they can grow to food size in 6-10 months on a formulated diet.Recently, there is an increasing interest for species diversification to support the development ofsustainable aquaculture. Fast-growing jade perch, which can be stocked at high densities inrecirculating aquaculture systems (RAS) and feeds on grow-out diets with very low levels offishmeal and fish oil, is a promising candidate for aquaculture [2,3]. Currently, this species is beingfarmed in both intensive ponds and recirlulating systems not only in Australian, but also in China, Malaysia, even in Belgium [4][5][6].Since the introduction of jade perch in China, domestic researchers have conducted severalstudies on the breeding and nutrition of this species. Chen et al. [1,7] studied the artificial propagation of jade perch and rearing of fry and fingerlings. Bao et al. [8] conducted a comparativeanalysis of the composition of typical nutrients (water, ash, crude protein, and crude fat), fatty acids,and amino acids in the muscle and viscera of jade perch, bass, and Chinese perch (Sinipercachuatsi),and Zhao et al. [9] assessed the nutritional value of jade perch. These results indicated thatpolyunsaturated fatty acids, sush as EPA and DHA, is extraordinarily high in jade perch. Feedingand nutrition requirements of jade perch were also investigated in other research. VanHoestenberghe et al. [10] evaluated the effect of weaning age and the use of different sized Artemianauplii as first feed for jade perch. Alkhafaji et al. [6] determined the effect of feeding frequencieson the growth, plasma biochemistry, and liver glycogen of jade perch in a recirculating system. Songet al. [11] set up a study and it was also demonstrated that increasing lipid levels in fish diets waseffective to improve protein utilization and decrease the nitrogen waste outputs and diet costs ofjade perch juveniles. Zhu et al. [12] characterized the Oceanography & Fisheries Open access Journal bacterial community structure associatedwith filter material in the recirculating aquaculture system of jade perch.
There are increasing research done with jade perch, however, studies on basic biological andgenetic characteristics, such as cytogenetic data and DNA content, have not been reported. As jadeperch is native to Australia and was introduced in China for about 17 years now. Currently, athorough knowledge and understanding of germplasm characteristics of this introduced species isof great importance to the protection, improvement, and utilization of germplasm resources inChina. Our previous research used mitochondrial D-loop sequences and simple sequence repeat(SSR) markers to analyze the genetic diversity in 4 reared jade perch populations in Guangdong,China. The results from the 2 methods showed that the 4 Guangdong populations had low geneticdiversity and close phylogenetic relationships [13]. Therefore, there is a need to introduce theoriginal species in order to enrich the genetic diversity of the reared populations in China.
In this study, we aimed to assess relevant germplasm characteristics of jade perch throughobservation of gonadal development, embryonic development and morphological characters,determination of DNA content, and analysis of karyotype, using microscopy, assessment ofmorphological indicators, flow-cytometric analysis, and chromosome preparations from kidneycells of adult fish. From the perspective of basic biology and cytogenetics, a systematic observationand analysis of germplasm characteristics of jade perch not only provides a profound basis forlarge-scale artificial breeding and a reference for germplasm standards, but also offers guidancefor future genetic breeding efforts.

Experimental animals
Jade perch used in this study were obtained from Liheng Aquaculture Farm (Xiqiao Town,Nanhai District, Foshan City, Guangdong Province, China). The parent used in artificial breedinghad been domesticated and cultured for more than five years, weighed approximately 2kg each,possessed a strong physique and high-quality traits, and were sexually mature. All applicableinternational, national, and institutional guidelines for the care and use of animals were followed.

Artificial propagation and embryonic development in jade perch
At a water temperature of 27.5±2 °C, two hormone injections were administered to thethoracic cavity of jade perch at 6h intervals to induce spawning. For female fish, a mixture ofhuman chorionic gonadotropin (HCG, 300-350IU•kg -1 ) and luteinizing hormone-releasinghormone (LRH-A2, 3-3.5µg•kg -1 ) was administered during the first injection, while for the secondinjection was a mixture of domperidone (DOM, 8mg•kg -1 ) and LRH-A2 (8µg•kg -1 ). When the secondinjection was administered to female fish, a quarter-dose was administered to male fish as well.Subsequently, male and female fish were artificially paired for spawning and fertilization, with afemale to male ratio of 1:1. The zygotes were placed in a circular pond for incubation. Throughoutthe incubation period, a portion of zygotes were periodically removed and placed in petri dishesfor observation. Observations, image acquisition, and recording of the process and timeline ofembryonic development were carried out using a ZEISS Stereo Discovery V8 microscope.

Measurements of morphological characters
Morphological characters of jade perch at different developmental stages were measured inaccordance with the People's Republic of China Standard on the Inspection of Germplasm forCultured Fishes -Part 3: Measurement of Characters (GB/T 18654. . Specifically, 30 two-monthold jade perch and 30 six-month-old jade perch were selected for quantification of sevencountable characters (lateral line scales, scales above the lateral line, scales below the lateral line,dorsal fin rays, pectoral fin rays, abdominal fin rays, anal fin rays) as well as determination of ninemeasurable characters (total length, body length, head length, caudal peduncle length, head depth,body depth, caudal peduncle depth, body weight, and weight of fat deposits). Subsequently, thefollowing ratios were calculated: whole length/body length, body length/head length, body length/body depth, head length/head depth, caudal peduncle length/caudal peduncle depth, bodydepth/head depth, and weight of fat deposits/body weight. The data were analyzed by analysis ofvariance using SPSS Statistics 17.0, with P<0.05 regarded as significant.

Analysis of blood DNA content
Ten six-month-old jade perch were randomly selected, and 10µL of blood was collected fromthe caudal veins of each fish. Then, 100µL of PBS was added and the mixture was homogenized byshaking to form a cell suspension. 1mL of 70% ethanol was added for cell fixation at 4°C for 1h.Subsequently, the suspension was centrifuged at 300×gfor 5min at room temperature, and thesupernatant was removed. The remaining liquid (approximately 50µL) was treated with 30µL of1mg/mL propidium iodide (PI) staining solution (CyStain PI Absolute, Partec). Then, the mixturewas diluted to 500µL with PBS and placed in a dark room to facilitate staining in the dark for 1h.
After staining, the sample was filtered through a Partec filter and analyzed using a flow cytometer(Cell Lab Quanta, Beckman Coulter). Another common freshwater bass species, the largemouthbass (Micropterussalmoides), was used as a control. The DNA contents of all samples werecompared using the χ2 test with Yates' continuity correction.

Chromosome preparations of kidney cells
Chromosome preparations of kidney cells were carried out using the protocal reported by Liuet al. [14], with minor modifications. Three six-month-old jade perch were randomly selected andinjected with 8-10µg/g of phytohemagglutinin (PHA) + 2-4µg/g of colchicine (Sigma). 2-3h later,head kidney of the fish was dissected, cut into fragments after removal of blood clots andconnective tissues, and transferred to a centrifuge tube. A small volume of 0.8% saline was added,the mixture was pipetted up and down to generate a flocculated suspension, and saline was addedagain to obtain a final volume of 10mL. The suspension was placed aside for precipitation, thencentrifuged at a speed of 1000-1500rpm. After discarding the supernatant, the remaining liquidwas added to 10mL of hypotonic solution containing 0.0373M KCl and subjected to hypo-osmosisat room temperature for 45-60min. Then, the sample was centrifuged at 1000-1500rpm, thehypotonic solution was removed, and the remaining liquid was fixed using a pre-chilled mixture ofmethanol and acetic acid (3:1) for 30min. The fixation process was repeated 2-3 times. Then, thecell suspension was dripped from a height of approximately 20cm onto a pre-chilled glass slide.
The slide was rapidly baked, and then allowed to air-dry. Lastly, the chromosome preparation wasstained using a Giemsa stain solution and rinsed approximately 30 minutes later. After removal ofthe stain solution using water, the preparations were observed and 50 mitotic metaphase spreadsin each sample were photographed and analyzed under a Leica DM2500 microscope.

Histological observation on the gonads
Each three of females and males at six-month-old and fouryear-old were randomly selectedfor histological observations on the gonad structure by preparing paraffin sections. The gonad ofeach fish was fixed in Bouin's solution, embedded in paraffin, then cut into sections with a LeicaRM2016 and then stained with hematoxylin and eosin. Tissue sections were observed andphotographed with a Leica DM2500. The gonad development of each sample was identifiedaccording to the standards for largemouth bass [15].

Early embryonic development in jade perch
It was observed that zygotes of jade perch are transparent, pelagic, and swell upon absorptionof water to up to 2.1±0.3mm in diameter. Each zygote typically contained one oil globule (~1/4of the diameter of the zygote), with a minority of zygotes containing two to three oil globules. At awater temperature of 27.5±2°C, jade perch fry hatched after 21-26h of normal embryonicdevelopment. Table 1 and Figure 1 show the time and various stages of embryonic development ofjade perch zygotes.   Figure 1 shows various stages of embryonic development of jade perch zygotes. After fertilization,the zygote membrane swells upon absorption of water, and as early as 17-33min after fertilization,the zygote cytoplasm is concentrated at the animal pole and gradually bulges to form an embryonicdisc (Figure 1a). Subsequently, the zygotes go through the cell division stage at 2h40min after fertilization (Figure 1b-1f), then, blastula stage at 5h30min (Figure 1g-1i), gastrula stage at 8h45min ( Figure  1j-1l),neurula stage at 13h26min (Figure 1m-1o), organogenesis stage at 20h45min (Figure 1p-1s) and so on,until hatching at 21h-26h after fertilization (Figure 1t).

Morphological characters of jade perch
Jade perch has a spindle-shaped body, and its back is covered with brilliant green scales, withtypically one or more black spots on both sides of the body (Figure 2). In this study, the mean weightsof two-and six-month-old jade perch were 88.67±31.76g (Figure 2a) and 338.57±72.50g (Figure 2b),respectively. As shown in Table 2, no significant differences were observed in countable charactersof jade perch in different developmental stages (P>0.05). Number of countable characters wereas follows: 13-14 dorsal fin spines, 11-13 dorsal fin rays; 14-16 pectoral fin rays; 1 abdominal finspine, 5 abdominal fin rays; 3 anal fin spines, 8-9 anal fin rays; 65-78 lateral line scales, 13-15scales above the lateral line, and 24-27 scales below the lateral line. Analysis of the ratios ofmeasurable characters indicated that the body length/head length, body depth/head depth, andweight of fat deposits/body weight of two-month-old jade perch were 4.27±0.48, 1.74±0.11, and0.06±0.02, respectively, which were significantly lower than those of six-month-old perch, 5.19±0.53, 2.13±0.17, 0.14±0.01, respectively (P<0.05) ( Table  3). No significant differences (P>0.05)were found for the other ratios, i.e., total length/body length, body length/body depth, headlength/head depth, and caudal peduncle length/caudal peduncle. Compared with two-month-oldfish, the proportion of the head with respect to the body was significantly decreased (P<0.05),while the proportion of fat deposits was significantly increased in six-month-old fish (P<0.05).

DNA Content
Mean DNA content in 5000 blood cells of each sample was determined, using largemouth bassas a control. As shown in Figure 3, for largemouth bass, normal diploid cells accounted for 85.18% ofcells, and the mean DNA content was 175.4 ( Figure   3a), while in jade perch, normal diploid cellsaccounted for 84.4% of cells, and the mean DNA content was 154.7 ( Figure  3b). Statistical analysis ofdata from 10 samples revealed that the DNA content in jade perch (168.27±13.91) wassignificantly lower than that in largemouth bass (194.55±15.85) (P<0.05).

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Normal ovaries in female jade perch at four-year-old were found oblong and soft, which almostfilled the abdominal cavity. Section observation showed that the ovaries of four-year-old jade perchwere in III to IV-stage and the oocytes were full of yolk (Figure 5c). Testes of jade perch were milk-white, bilateral symmetry and well developed. Observing the microstructure of the testes of jadeperch, we found groups of spermatogonia, primary spermatocytes, secondary spermatocytes,spermatids and numerous mature spermatozoa, were filled in seminiferous tubules (Figure 5d).Semen could be squeezed out of these testes of jade perch at four-year-old.

Discussion
Jade perch possesses several advantages, including sweet and succulent flesh, rapid growth,and good suitability for industrial aquaculture. It deserves to be promoted as a profitablecommercial freshwater species. In this study, analysis of gonadal development indicated that jadeperch get to sexually mature at four-year-old, when the ovaries possess partial oocytes in phase IVsuitable for artificial spawing, and mature sperm could be squeezed out of testes in jade perch.
Embryonic development in jade perch was evaluated. It was found that upon absorption of water,the zygotes of jade perch could swell to a average diameter of 2.1mm, which is much larger thanthe zygote diameters of some other species of order Perciformes, e.g., Japanese seabass(Lateolabrax japonicus): 1.35-1.44mm [16], and largemouth bass: 1.32-1.40mm [17], however,significantly smaller than that of Striped Bass, which is 2.45-3.14mm [18]. It was observed that ata water temperature of 27.5 ± 2°C, zygotes of jade perch hatched within 21-26h of fertilization.
For Japanese seabass, the optimum breeding temperature is 12-14°C, and within this temperaturerange, embryonic development takes ~85h. For largemouth bass, when breeding is carried out ina temperature range of 18-20°C, embryonic development takes ~58h. The process of embryonicdevelopment in jade perch is similar to that of seabass [19] and largemouth bass [20], i.e., after thecleavage, blastula, and gastrula stages, the embryoid body is formed, sarcomeres and tail budsemerge, and various organs are formed; subsequently, muscular contraction occurs, and theheartbeat and blood circulation are established before hatching takes place.Body weight, which is correlated with morphological traits at different levels, is an importanttarget in the selective breeding of growth traits in fish species. For instance, in black bream, whichis another member of the family Terapontidae, head depth, body width, caudal peduncle depth, andbody length are four major morphological characters that affect body weight in the adult, with headdepth having the most significant direct impact (P2 = 0.360) [21]. In largemouth bass, body width,body length, and interocular distance are strongly positively correlated with body weight, and arethe major morphological characters that directly or indirectly affect body weight [22]. In addition,differences in developmental stage, habitat or even stress state may cause differences inmorphological characteristics in individuals. For instance, the contribution rates of total length tomorphological characters in four-and six-month-old largemouth bass are 92.29% and 86.40%, respectively; therefore, this indicator can be used as a selection criterion for morphologicalcharacters in this species. The contribution rates of total length/body depth and caudal pedunclelength/caudal peduncle depth to body size in fourmonth-old largemouth bass are 65.94% and34.06%, respectively, while the contribution rates in six-month-old largemouth bass are 69.08%and 30.92%, respectively; therefore, these two indicators can be used as selection criteria for bodysize in largemouth bass [23]. In this study, conventional morphological methods were used tocompare the physical appearances of two-and six-monthold jade perch, and it was found that theproportion of the head with respect to the body was significantly decreased (P<0.05), and that offat deposits was significantly increased in six-monthold fish (P<0.05), whereas differences inother characters were not significant (P<0.05). This suggests that body length/head length andbody depth/head depth are strongly corrlated with body weight in jade perch; therefore, Usingthese parameters as indicators of growth traits for selective breeding may be considered. In six-month-old jade perch, the proportion of abdonimal fat deposits to body weight was as high as 14%,which is close to the average content of fat in whole cutlets of some highly valued fish species. Fatpercentage of a cutlet is 14.6% in Atlantic halibut (Hippoglossushippoglossus L.), which is a highlyvalued fish species [24]. Large fat deposits in the abdomen of adult jade perch may be a species-specific characteristic. Further studies are required to determine if fat content in jade perch can beregulated through changes in nutrient ratios of the feed and breeding methods, and to investigatemethods for the effective exploitation and utilization of unsaturated fatty acids in jade perch.Chromosomes are carriers of genetic material, and chromosome evolution is closely linked tospecies evolution. Changes in chromosome number and structure may lead to mutations ingermplasm characteristics or even the emergence of new species. Knowledge of karyotypecharacteristics allows a better understanding of a species' phylogenetic status. In general, withinspecific taxonomic categories, primitive groups have more telocentric chromosomes, whilespecialized groups have more metacentric or submetacentric chromosomes [25]. More primitivefish species have a higher number of telocentric and subtelocentric chromosomes, fewer or nometacentric and submetacentric chromosomes, and a lower total number of chromosome arms [26]. The karyotype formula of jade perch is 2sm + 2m + 44t. The karyotype formulae of some otherspecies of the order Perciformes are as follows: black bream: 4m + 44t [26], Japanese seabass: 48t,Queensland grouper (Promicropslanceolatus): 4st + 44t, and brown-marbled grouper

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(Epinephelusfuscoguttatus): 2sm + 46t [27]. It can be deduced that among fish species of the order Perciformes,jade perch is a specialized species and has a higher evolutionary status. Largemouth bass has achromosome number of 46, with karyotype formula 2m + 2st + 42t (Germplasm Standard forLargemouth Bass, GB21045-2007), and a DNA content of 194.55±15.85, which is significantlyhigher than the DNA content in jade perch (168.27±13.91) (P<0.05). Compared with largemouthbass, jade perch has more chromosomes but lower DNA content; therefore, we can preliminarilydeduce that jade perch has a smaller genome and low redundancy in genomic information, whichfacilitates future measurement and calculation of jade perch genome size as well as theimplementation of transcriptome and genome sequencing projects.
Chromosome number and karyotype also serve as a biological basis for distant hybridization.Hence, it is worthwhile to study these aspects in further detail. For instance, a study on the distanthybridization between parental species with the same chromosome numbers and betweenparental species with different chromosome numbers revealed that distant hybridization couldresult in offspring with different ploidy levels [28]. On the basis of karyotype analysis, if techniquessuch as G-banding and fluorescent in-situ hybridization can be integrated with studies ofchromosome evolution in fish species, a better understanding of the genetic composition andbiological development can be attained. In addition, such studies will facilitate the identification ofcongeneric species and phylogenetic studies, and are of great significance for breeding and hybridization.

Conclusion
Jade perch, native in Australia, becomes a promising freshwater bass species for aquaculturein China recently. Their populations in southern China have been identified with low degree ofgenetic diversity and differentiation. To protect, improve and utilize the germplasm resources ofthis introduced species, knowledge about the biological characteristics, such as morphological andcytogenetical data, is necessary to be known.
Jade perch zygotes hatched 21-26h after fertilization at a water temperature of 27.5±2°C.The proportion of the head with respect to the body decreased significantly between twoand six-month-old jade perch (P<0.05), while the proportion of abdominal fat deposits increasedsignificantly (P<0.05). Body length/head length and body depth/head depth ratios could serve aspotential indicators of growth traits for selective breeding. Based on chromosome number andDNA content analysis, we deduced that jade perch has a relatively small genome and lowredundancy in genomic information, which facilitates future measurements and calculations ofjade perch genome size as well as the implementation of transcriptome and genome sequencingprojects.

Ethical Approval
All applicable international, national, and institutional guidelines for the care and use ofanimals were followed, and this article does not contain studies with human participants performedby any of the authors.