# A Comparative Study of the Skeletal Development ofEpinephelus fuscoguttatus (♀) and E. tukula (♂) Hybrid Progeny and E. fuscoguttatus

Authors: Xinyi Wang; Yongsheng Tian; Shuai Chen; Linlin Li; Pengfei Duan; Zhontong Li; Linna Wang; Yang Liu; Qingbin Wang; Wensheng Li; Xia Zhao
Journal: Aquaculture Research
 (2023)

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## Abstract

As a new breed of grouper hybrid breeding, EFT has the advantages of delicious flesh, fast growth, and high deformity rate. We explored the metamorphosis and skeletal development ofEpinephelus fuscoguttatus and hybrids (EFT) of E. fuscoguttatus (♀) and E. tukula (♂). EFT grew slightly slower than E. fuscoguttatus during the larval period but grew faster during the juvenile period. The sequences of skeletal development were similar in EFT and E. fuscoguttatus. Newly hatched larvae did not have skeletons until 5 days after hatching (DAH) when head cartilage started to develop and complete at 43 days later. The vertebral column started to develop at 11 DAH and was completed at 38 DAH. Pectoral fin development began at 5 DAH and ossification was completed at 48 DAH in EFT and at 43 DAH in E. fuscoguttatus. Anal fin development started at 11 DAH and ossification was completed earlier in EFT than in E. fuscoguttatus. The caudal fins of the two groupers developed at 8 DAH and ossified earlier in EFT than in E. fuscoguttatus. In contrast, E. fuscoguttatus had one additional epural than EFT. These results provide important basic biological data for the growth and skeletal development of pure and hybrid grouper.


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## Body

## 1. Introduction

Epinephelus fuscoguttatus and E. tukula are groupers belonging to the Perciformes, Serranidae, and Epinephelinae. E. fuscoguttatus has delicious flesh and a high market value; however, its growth rate is slow and its feeding cycle is long. It is often used as the female parent of hybrid groupers owing to its strong disease resistance. E. tukula is a giant grouper, distributed in the southern waters of China; it is not widely cultured, and the breeding resources are mainly derived from wild fishing. We have used frozen sperm of E. tukula to fertilize E. fuscoguttatus eggs, overcoming reproductive isolation, to obtain hybrids (referred to as EFT). EFT has high survival rate, low malformation rate, and fast growth rate. It has an obvious hybridization advantage. Research on EFT to date has focused on its growth and metamorphosis [1], morphological properties [2], genetic diversity [3], hypoxia tolerance [4], and karyotype [5]. The above-given studies also indicated that EFT has rich genetic diversity and low oxygen tolerance. Skeletal development has not been compared between EFT and E. fuscoguttatus.The bones of fish are divided into the central axial skeleton, including the cephalic and vertebral column, and the appendage skeleton, including the even-fin skeleton and odd-fin skeleton [6]. Skeletal development is essential for fry rearing during feeding. The skeleton not only supports the fish body but also protects the internal organs and coordinates movements. In the processes of fish growth and metamorphosis, an initial period of allometric growth of survival-related bones helps to reduce fish mortality and improve survival [7]. In addition, abnormal bone development can result in difficult movement, slow growth, and high rates of mortality.In this study, bone development was observed inE. fuscoguttatus and EFT by the dual staining of hard bone and cartilage, and a comparative analysis was performed to determine the nutritional and environmental factors necessary for growth and metamorphosis and to explore the growth-related advantages of EFT as a hybrid grouper.


## 2. Materials and Methods

### 2.1. Ethic Statement

This study was approved by the Animal Care and Use Committee of the Yellow Sea Fisheries Research Institute [YSFRI-2022027].


### 2.2. Egg Fertilization, Hatching, and Fry Cultivation

EFT was obtained by the artificial fertilization ofE. fuscoguttatus eggs with the thawed frozen sperm of E. tukula. The embryos of the two species of grouper were obtained from Laizhou Mingbo Aquatic Products Co., Ltd., (Shandong Province, China). EFT and E. fuscoguttatus were cultured in circulating water environments with a water temperature of 28°C, salinity of 30‰, and 6 mg/L dissolved oxygen. The ponds were circular with a volume of 40 m3. Before 2 days after hatching (DAH), the yolk sac provides nutrients to the larvae. From 2 to 5 DAH, fish were fed SS rotifers (8–10 individuals mL−1). From 5 to 10 DAH, fish were fed L-rotifers (10–15 individuals mL−1). From 10 to 16 DAH, fish were fed nauplii of Artemia (0.5–2 individuals mL−1). From 16 to 24 DAH, fish were fed adult Artemia (0.5–2 individuals mL−1), followed by a gradual transition to pellets. The feeding amount was adjusted according to the growth of the groupers.


### 2.3. Sample Collection and Treatment

Ten EFT and tenE. fuscoguttatus embryos were obtained for observation every 3 days. After observing the morphology of samples under an inverted biomicroscope and inverted anatomical microscope, the samples were anesthetized with MS-222.


### 2.4. Growth Measurement

Images of samples were obtained after observation under a microscope. Then, the total length of each sample was determined.At the end of metamorphosis, 30E. fuscoguttatus and 30 EFT were randomly selected each month for three consecutive months. Samples were weighed and the total length and body length were determined.


### 2.5. Observation of Skeletal Development

Samples of both fish were stained using previously described bone staining methods [8, 9]. Samples were fixed in 10% formalin for 24 h and then washed with distilled water for 24 h. Next, they were stained with Alcian blue staining solution for cartilage detection until the fish body turned medium blue. Samples were transferred to 95% ethanol for 2-3 h and then washed sequentially in 75%, 40%, and 15% alcohol gradients for 2-3 h per wash. Samples were immersed in distilled water until the fish sank and then placed in a borax-trypsin solution for digestion. When the muscle digestion was completed and the fish body appeared transparent state, the hard bone was stained with alizarin red dye until samples turned red. Samples were washed with 0.5% KOH-glycerol solution and three 0.5% KOH-glycerol solutions (3 : 1, 1 : 1, and 1 : 3) for 24 h each until the melanin was removed (each solution included two drops of 3% H2O2). Finally, the samples were stored in glycerol-containing thymol and skeletal development was observed under an inverted anatomical microscope.The morphological characteristics and skeletal development of EFT andE. fuscoguttatus were observed using the DSY2000X inverted biomicroscope (Chongqing Photoelectric Instrument Co., Ltd.) and SZ810 inverted anatomical microscope (Chongqing Aoter Optical Microscope Co., Ltd.). Image View was used to measure fish lengths. Images were annotated using Adobe Photoshop CS6.


## 2.1. Ethic Statement

This study was approved by the Animal Care and Use Committee of the Yellow Sea Fisheries Research Institute [YSFRI-2022027].


## 2.2. Egg Fertilization, Hatching, and Fry Cultivation

EFT was obtained by the artificial fertilization ofE. fuscoguttatus eggs with the thawed frozen sperm of E. tukula. The embryos of the two species of grouper were obtained from Laizhou Mingbo Aquatic Products Co., Ltd., (Shandong Province, China). EFT and E. fuscoguttatus were cultured in circulating water environments with a water temperature of 28°C, salinity of 30‰, and 6 mg/L dissolved oxygen. The ponds were circular with a volume of 40 m3. Before 2 days after hatching (DAH), the yolk sac provides nutrients to the larvae. From 2 to 5 DAH, fish were fed SS rotifers (8–10 individuals mL−1). From 5 to 10 DAH, fish were fed L-rotifers (10–15 individuals mL−1). From 10 to 16 DAH, fish were fed nauplii of Artemia (0.5–2 individuals mL−1). From 16 to 24 DAH, fish were fed adult Artemia (0.5–2 individuals mL−1), followed by a gradual transition to pellets. The feeding amount was adjusted according to the growth of the groupers.


## 2.3. Sample Collection and Treatment

Ten EFT and tenE. fuscoguttatus embryos were obtained for observation every 3 days. After observing the morphology of samples under an inverted biomicroscope and inverted anatomical microscope, the samples were anesthetized with MS-222.


## 2.4. Growth Measurement

Images of samples were obtained after observation under a microscope. Then, the total length of each sample was determined.At the end of metamorphosis, 30E. fuscoguttatus and 30 EFT were randomly selected each month for three consecutive months. Samples were weighed and the total length and body length were determined.


## 2.5. Observation of Skeletal Development

Samples of both fish were stained using previously described bone staining methods [8, 9]. Samples were fixed in 10% formalin for 24 h and then washed with distilled water for 24 h. Next, they were stained with Alcian blue staining solution for cartilage detection until the fish body turned medium blue. Samples were transferred to 95% ethanol for 2-3 h and then washed sequentially in 75%, 40%, and 15% alcohol gradients for 2-3 h per wash. Samples were immersed in distilled water until the fish sank and then placed in a borax-trypsin solution for digestion. When the muscle digestion was completed and the fish body appeared transparent state, the hard bone was stained with alizarin red dye until samples turned red. Samples were washed with 0.5% KOH-glycerol solution and three 0.5% KOH-glycerol solutions (3 : 1, 1 : 1, and 1 : 3) for 24 h each until the melanin was removed (each solution included two drops of 3% H2O2). Finally, the samples were stored in glycerol-containing thymol and skeletal development was observed under an inverted anatomical microscope.The morphological characteristics and skeletal development of EFT andE. fuscoguttatus were observed using the DSY2000X inverted biomicroscope (Chongqing Photoelectric Instrument Co., Ltd.) and SZ810 inverted anatomical microscope (Chongqing Aoter Optical Microscope Co., Ltd.). Image View was used to measure fish lengths. Images were annotated using Adobe Photoshop CS6.


## 3. Results

### 3.1. Postembryonic Development of EFT andE. fuscoguttatus

The total lengths and weights ofE. fuscoguttatus and EFT are shown in Figures 1 and 2, respectively.Figure 1
Total length of EFT andE. fuscoguttatus. ∗∗P<0.01, ∗P<0.05.Figure 2
Weight of EFT andE. fuscoguttatus.At 2–20 days, the total lengths of EFT andE. fuscoguttatus were similar. Thereafter, E. fuscoguttatus had a longer overall length than that of EFT. After 61 days, the gap between the full length of E. fuscoguttatus and EFT narrowed gradually. At 122 days, the total lengths of EFT (126.47 ± 6.02 mm TL) were significantly longer than that of E. fuscoguttatus (111.92 ± 7.67 mm TL).At 61 DAH, the weight of the EFT was significantly less than that ofE. fuscoguttatus. At 92 DAH, the weight gap between EFT and E. fuscoguttatus narrowed. At 122 DAH, the weight of EFT was not significantly different from that of E. fuscoguttatus, but EFT weighs more than E. fuscoguttatus.


#### 3.1.1. Postembryonic Development ofE. fuscoguttatus

At 2–5 DAH, the egg yolk sac ofE. fuscoguttatus was continually consumed, the oil ball became smaller, and an oral fissure was detected. The digestive tract became thicker, and melanin deposition was observed in the upper part of the digestive tract. The eyeball was completely black, the auditory sac behind the eye was obviously visible, and the bone in the jaw became visible. The fin folds on both sides of the anterior tail were slightly concave, and there was a small amount of melanin deposition in one-third of the body at the front of the tail (Figures 3(a) and 3(b)).Figure 3
Postembryonic development of EFT. (a) 2 DAH (0.28 ± 0.02 mm TL); (b) 5 DAH (0.32 ± 0.02 mm TL); (c) 8 DAH (0.4 ± 0.04 mm TL); (d) 11 DAH (0.44 ± 0.04 mm TL); (e) 14 DAH (0.5 ± 0.08 mm TL); (f) 17 DAH (0.56 ± 0.04 mm TL); (g) 20 DAH (0.64 ± 0.05 mm TL); (h) 23 DAH (0.65 ± 0.06 mm TL); (i) 26 DAH (0.83 ± 0.04 mm TL); (j) 29 DAH (0.84 ± 0.12 mm TL); (k) 32 DAH (1.09 ± 0.15 mm TL); (l) 38 DAH (1.92 ± 0.14 mm TL); (m) 43 DAH (2.72 ± 0.46 mm TL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)At 6–23 DAH, the eyeball became bright and the pupil was clearly visible, the melanin of the internal organ mass decreased gradually, and the area where the melanin was receding appeared silvery-white. The heart area and gills appeared bright red. At 26 DAH, a bright red blood vessel along the spine could be seen and the second dorsal and ventral spines grew. As the fish grew, the second dorsal and ventral spines continued to elongate up to one-half the length of the body, with melanin deposition at the ends of the fin lepidotrichia. The fin folds at the front of the tail continued to recede inward to the narrowest point, the vertebral column appeared segmented, the end of the spine was upturned, and the fin lepidotrichia could be seen in the tail. At 17 DAH, a large number of deadE. fuscoguttatus larvae were observed, with high rates of cannibalism between groups and a survival rate of only 30% (Figures 3(c)–3(g)).At 24–48 DAH, the orbit deepened and a fold formed on the skin around the eyes. The second dorsal and ventral spines began to contract, the melanin at the end gradually receded, the dorsal and ventral fins were clearly visible (except for the contracted fin lepidotrichia), and the pectoral and caudal fins were clearly visible. At 38 DAH, the body ofE. fuscoguttatus was no longer completely transparent, and the body surface had a pattern, with scales covering the body surface. At 43 DAH, the fish body was completely opaque, and the pattern on the body surface was roughly similar to that of adult fish (Figures 3(h)–3(l))At 61 DAH,E. fuscoguttatus was 6.9 ± 0.4 cm in total length and weighed 6.4 ± 1.1 g. At 92 DAH, individuals were 9.2 ± 1.4 cm in total length and weighed 17.1 ± 3.2 g. At 122 DAH, the total length was 11.2 ± 0.8 cm and the weight was 29.7 ± 6.3 g.


#### 3.1.2. Postembryonic Development of EFT

At 2–5 DAH, there were no observable differences in development between EFT andE. fuscoguttatus (Figures 4(a) and 4(b)).Figure 4
Postembryonic development ofE. fuscoguttatus. (a) 2 DAH (2.85 ± 0.1 mm TL); (b) 5 DAH (3.21 ± 0.37 mm TL); (c) 8 DAH (3.61 ± 0.22 mm TL); (d) 11 DAH (4.21 ± 0.40 mm TL); (e) 14 DAH (TL4.76 ± 0.28 mm); (f) 17 DAH (6.00 ± 0.47 mm TL); (g) 20 DAH (6.85 ± 0.59 mm TL); (h) 23 DAH (7.59 ± 0.46 mm TL); (i) 26 DAH (8.07 ± 1.08 mm TL); (j) 29 DAH (9.99 ± 2.46 mm TL); (k) 32 DAH (12.38 ± 1.53 mm TL); (l) 38 DAH (25.16 ± 2.19 mm TL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)At 6–20 DAH, the pupil was clearly visible and the visceral mass melanin receded from the abdomen to the back and was covered in silvery white. The vertebral column was not cylindrical, the neural arch and hemal arch were visible, and the end of the vertebral column were upturned. The second dorsal and ventral spines were elongated, barbs could be seen on the spines of the fins, and melanin was deposited at the ends. The front of the visceral mass, gills, and heart was bright red. The number of EFT deaths was low, and the survival rate was 80% (Figures4(c)–4(h)).At 21–48 DAH, the development of EFT was similar to that ofE. fuscoguttatus, other than an elongated the body of EFT. At 43 DAH, the black pattern appeared on the surface of the body. At 48 DAH, the fish body was completely opaque and the dorsal fin, abdominal fin, anal fin, caudal fin, and pectoral fin were clearly visible. The body surface pattern was not substantially different from that of adult fish (Figure 4(i)).At 61 DAH, EFT was 6.0 ± 0.3 cm in total length and 3.5 ± 0.5 g in weight. At 92 DAH, the total length was 10.2 ± 0.3 cm and the weight was 16.4 ± 1.6 g. At 122 DAH, the total length was 12.6 ± 0.6 cm and the weight was 32.7 ± 5.1 g.


### 3.2. Axial Skeletal Development ofE. fuscoguttatus and EFT

#### 3.2.1. Development of the Cranium

At 2 DAH, juvenile fish lacked head cartilage. At 5 DAH (Figures5(a) and 6(a)), larvae in both groups did not differ substantially, showing the development of the ethmoid plate, trabeculae, Michael’s cartilage, four ceratobranchials, basibranchial cartilage, hypohyal, and hypobranchial. At 8 DAH (Figures 5(a) and 6(b)), both fish had a visible lower pharyngeal bone and supraoccipital, and basibranchial cartilage 2. In addition, in E. fuscoguttatus, nasal and palatoquadrate cartilage were detected. At 11 DAH (Figures 5(c) and 6(c)), two groupers began to develop premaxilla and prosethmoid, and the nasal and palatoquadrate cartilage of EFT began to appear. At 14–20 DAH (Figures 5(d)–5(f), 6(d)–6(f)), the vomer was developed and the maxillary began to ossify. At 23–29 DAH (Figures 5(g)–5(i), 6(g)–6(i)), the branchiostegal was visible, the palatoquadrate began to degenerate, the parietal, maxilla, premaxilla, and preopercular gradually ossified, and dentary appeared. At 32–48 DAH (Figures 5(j)–5(l), 6(j) and 6(k)), bone ossification in each part of the head was completed.Figure 5
Cephalic skeleton development of EFTA1-L1(lateralview) A2-L2(dorsalview) A3-L3(ventralview). (a) 5 DAH (3.2 ± 0.17 mm TL); (b) 8 DAH (3.97 ± 0.4 mm TL); (c) 11 DAH (4.43 ± 0.41 mm TL); (d) 14 DAH (5.04 ± 0.77 mm TL); (e) 17 DAH (5.61 ± 0.41 mm TL); (f) 20 DAH (6.45 ± 0.48 mm TL); (g) 23 DAH (6.53 ± 0.63 mm TL); (h) 26 DAH (8.34 ± 0.44 mm TL); (i) 29 DAH (8.40 ± 1.2 mm TL); (j) 32 DAH (10.87 ± 1.49 mm TL); (k) 38 DAH (19.18 ± 1.40 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: an angular; Bh: basihyal; Br: branchiostegal; Bb1: basibranchial cartilages1; Bb2: basibranchial cartilages2; Ch: ceratohyal; Cb: ceratobranchial; De: dentary; EP: ethmoid plate; EC: ethmoid cartilage; Eh: epihyal; Fr: frontal; Hh: hypohyal; Hb: hypobranchial; Ih: interhyal; LPB: lower pharyngeal bone; LE: lateral ethmoid; MC: Meckel’s cartilage; Ma: maxillary; Na: nasal; MP: mediodorsal; Pe: prosethmoid; Pq: palatoquadrate; Pa: parietal; Po: preopercle; Pm: premaxillary; SOC: supraoccipital Sy: sympletic; Tr: trabeculae; V: vomer.Figure 6
Cephalic skeleton development ofE. fuscoguttatus A1-K1(lateralview) A2-K2(dorsalview) A3-K3(ventralview). (a) 2 DAH (2.85 ± 0.1 mm TL); (b) 5 DAH (3.21 ± 0.37 mm TL); (c) 8 DAH (3.61 ± 0.22 mm TL); (d) 11 DAH (4.21 ± 0.40 mm TL); (e) 14 DAH (TL4.76 ± 0.28 mm); (f) 17 DAH (6.00 ± 0.47 mm TL); (g) 20 DAH (6.85 ± 0.59 mm TL); (h) 23 DAH (7.59 ± 0.46 mm TL); (i) 26 DAH (8.07 ± 1.08 mm TL); (j) 29 DAH (9.99 ± 2.46 mm TL); (k) 32 DAH (12.38 ± 1.53 mm TL).Abbreviations are as follows: An: angular; Bh: basihyal; Br: branchiostegal; Bb1: basibranchial cartilages1; Bb2: basibranchial cartilages2; Ch: ceratohyal; Cb: ceratobranchial; De: dentary; EP: ethmoid plate; EC: ethmoid cartilage; Eh: epihyal; Fr: frontal; Hh: hypohyal; Hb: hypobranchial; Ih: interhyal; LPB: lower pharyngeal bone; LE: lateral ethmoid; MC: Meckel’s cartilage; Ma:maxillary; Na:nasal; MP: mediodorsal Pe: prosethmoid; Pq: palatoquadrate; Pa: parietal; Po:preopercle; Pm: premaxillary; SOC: supraoccipital; Sy: sympletic; Tr: trabeculae; V: vomer.


#### 3.2.2. Development of the Vertebral Column

At 11 DAH (Figures7(a) and 8(a)), the neural arch formed from the front of the back and the hemal arch formed from the front of the abdomen. At 17 DAH (Figures 7(c) and 8(c)), the neural arch and hemal arch were completely formed and the parapophysis was visible. Prior to this point, the two grouper species developed at approximately the same rate. At 20 DAH (Figure 7(d)), the vertebral column of EFT began to ossify, while this was observed as 23 DAH in E. fuscoguttatus (Figure 8(e)). At 29–32 DAH (Figures 7(g), 7(h), 8(g), and 8(h)), two species of grouper had abdominal ribs and dorsal ribs. At 38 DAH (Figures 7(i) and 8(i)), EFT and E. fuscoguttatus vertebrae, neural arches, hemal arches, and parapophysis were completely ossified. When ossification was completed, both EFT and E. fuscoguttatus had 19 vertebrae.Figure 7
Vertebral column development of EFT. (a) 11 DAH (4.43 ± 0.41 mm TL); (b) 14 DAH (5.04 ± 0.77 mm TL); (c) 17 DAH (5.61 ± 0.41 mm TL); (d) 20 DAH (6.45 ± 0.48 mm TL); (e) 23 DAH (6.53 ± 0.63 mm TL); (f) 26 DAH (8.34 ± 0.44 mm TL); (g) 29 DAH (8.40 ± 1.2 mm TL); (h) 32 DAH (10.87 ± 1.49 mm TL); (i) 38 DAH (19.18 ± 1.40 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Dr: dorsal rib; HS: heamal spine; HA: heamal arch; NA: neural arch; NS: neural spine; Pp: parapophysis; Pr: pleural rib; V: vertebral centra.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 8
Vertebral column development ofE. fuscoguttatus. (a) 11 DAH (4.21 ± 0.40 mm TL); (b) 14 DAH (TL4.76 ± 0.28 mm); (c) 17 DAH (6.00 ± 0.47 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 20 DAH (6.85 ± 0.59 mm TL); (f) 23 DAH (7.59 ± 0.46 mm TL); (g) 26 DAH (8.07 ± 1.08 mm TL); (h) 29 DAH (9.99 ± 2.46 mm TL); (i) 32 DAH (12.38 ± 1.53 mm TL); (j) 38 DAH (19.18 ± 1.39 mm TL). Abbreviations are as follows: Dr: dorsal rib; HS: heamal spine; HA: heamal arch; NA: neural arch; NS: neural spine; Pp: parapophysis; Pr: pleural rib; V: vertebral centra.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.3. Appendage Skeletal Development ofE. fuscoguttatus and EFT

#### 3.3.1. Development of the Pectoral Fin

At 5 DAH (Figure9(a)), the EFT coracoid-scapula cartilage and fin plate were visible. At 6–26 DAH (Figures 9(b)–9(h)), the fin part gradually degenerated to form the proximal radius, while the distal radials were visible. At 26 DAH (Figure 9(h)), the pectoral fin was visible. At 48 DAH (Figure 9(m)), the pectoral fin was completely ossified.Figure 9
Pectoral fin development of EFT. (a) 5 DAH (3.2 ± 0.17 mm TL); (b) 8 DAH (3.97 ± 0.4 mm TL); (c) 11 DAH (4.43 ± 0.41 mm TL); (d) 14 DAH (5.04 ± 0.77 mm TL); (e) 17 DAH (5.61 ± 0.41 mm TL); (f) 20 DAH (6.45 ± 0.48 mm TL); (g) 23 DAH (6.53 ± 0.63 mm TL); (h) 26 DAH (8.34 ± 0.44 mm TL); (i) 29 DAH (8.40 ± 1.2 mm TL); (j) 32 DAH (10.87 ± 1.49 mm TL); (k) 38 DAH (19.18 ± 1.40 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL); (m) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: CL, cleithrum; Co-Sca: coracoid-scapula; Co, coracoid; PocLo, metacleithrum lower; Fp, fin plate; ScF, scapular foramen; Sca, scapula; R, lepidotrichium; Rd, distal radial; Prx, proximal pterygiophore; Suc, hypercleithrum.In the development of the pectoral fin ofE. fuscoguttatus, at 5 DAH (Figure 10(a)), coracoid-scapula cartilage was visible. At the 8 DAH (Figure 10(b)), fin plate and cleithrum appeared. At 9–17 DAH (Figures 10(c)–10(e)), part of the fin plate gradually degenerated to form proximal radials. At 20 DAH (Figure 10(f)), the pectoral fin was visible. At 21–43 DAH (Figures 10(g)–10(l)), ossification of the pectoral fin, cleithrum, proximal radials, and other regions was completed.Figure 10
Pectoral fin development ofE. fuscoguttatus. (a) 5 DAH (3.21 ± 0.37 mm TL); (b) 8 DAH (3.61 ± 0.22 mm TL); (c) 11 DAH (4.21 ± 0.40 mm TL); (d) 14 DAH (TL4.76 ± 0.28 mm); (e) 17 DAH (6.00 ± 0.47 mm TL); (f) 20 DAH (6.85 ± 0.59 mm TL); (g) 23 DAH (7.59 ± 0.46 mm TL); (h) 26 DAH (8.07 ± 1.08 mm TL); (i) 29 DAH (9.99 ± 2.46 mm TL); (j) 32 DAH (12.38 ± 1.53 mm TL); (k) 38 DAH (25.16 ± 2.19 mm TL); (l) 43 DAH (30.49 ± 3.16 mm TL). Abbreviations are as follows: CL, cleithrum; Co-Sca: coracoid-scapula; Co, coracoid; PocLo, metacleithrum lower; Fp, fin plate; ScF, scapular foramen; Sca, scapula; R, lepidotrichium; Rd, distal radial; Prx, proximal pterygiophore; Suc, hypercleithrum.


#### 3.3.2. Development of the Dorsal Fin

In EFT, at 8 DAH (Figure11(a)), the second dorsal spine and its fin bones appeared. At 11 DAH (Figure 11(b)), the first dorsal spine and fin bone appeared, the second dorsal spine began to elongate continuously, and barbs appeared. At 17 DAH (Figure 11(d)), the third dorsal spine and fin bone were visible and the first dorsal spine and second dorsal spine began to ossify. At 23 DAH (Figure 11(f)), the fourth dorsal spine developed. At 26 DAH (Figure 11(g)), the rest of the dorsal spines, dorsal fin strips, and dorsal fin distal radials were observed, while the second dorsal fin spine underwent a degenerative process, gradually shortening. At 32 DAH (Figure 11(i)), stay cartilage appeared. At 38 DAH (Figure 11(j)), the second dorsal spine degenerated and was reduced to the length of the other dorsal spines. The dorsal fin also began to ossify. At 48 DAH (Figure 11(m)), the dorsal fin was completely ossified.Figure 11
Dorsal fin development of EFT Dorsal fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL); (m) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: Ba, barb; Prd, predosal; R, lepidotrichium; Rd, distal radial; S, hard spine; Sp, spinule; stay.InE. fuscoguttatus, at 8–17 DAH (Figures 12(b)–12(e)), the degree of development was comparable to that of EFT. At 20 DAH (Figure 12(e)), the rest of the dorsal spines, dorsal fin strips, and distal radius of the dorsal fin were visible, and the second dorsal spine degenerated. At 23 DAH (Figure 12(f)), the stay cartilage was visible. At 43 DAH (Figure 12(k)), the second dorsal spine was shortened to the length of other dorsal spines, slightly longer than the first dorsal spine. The dorsal fin spine also began to ossify.Figure 12
Dorsal fin development ofE. fuscoguttatus. (a) 8 DAH (3.61 ± 0.22 mm TL); (b) 11 DAH (4.21 ± 0.40 mm TL); (c) 14 DAH (4.76 ± 0.28 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 20 DAH (6.85 ± 0.59 mm TL); (f) 23 DAH (7.59 ± 0.46 mm TL); (g) 26 DAH (8.07 ± 1.08 mm TL); (h) 29 DAH (9.99 ± 2.46 mm TL); (i) 32 DAH (12.38 ± 1.53 mm TL); (j) 38 DAH (25.16 ± 2.19 mm TL); (k) 43 DAH (30.49 ± 3.16 mm TL). (l) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Ba, barb; Prd, predosal; R, lepidotrichium; Rd, distal radial; S, hard spine; Sp, spinule; stay.


#### 3.3.3. Development of the Pelvic Fin

In EFT, at 8 DAH (Figure13(a)), the abdominal spines and fin-based cartilage emerged. At 14 DAH (Figure 13(c)), abdominal spines were elongated, accompanied by barb growth. At 17 DAH, abdominal spines began to ossify. At 23 DAH (Figure 13(f)), ventral fin development was observed. At 26 DAH (Figure 13(g)), fin-based cartilage ossification was observed. Abdominal spines began to shorten until they were comparable in length to other fin spines.Figure 13
Pelvic fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Bp, basipterygium; Bb, barb; R, lepidotrichium S, hard spine; Sp, spinule; In, innominatum.InE. fuscoguttatus, at 8–14 DAH, development was similar to that in EFT. At 17 DAH (Figure 14(c)), the abdominal spines began to ossify, and ventral fin development was observed. At 20 DAH (Figure 14(d)), the basipterygiua began to ossify, and the abdominal spine underwent a degenerative shortening process until it was comparable in length to those of other fin spines.Figure 14
Pelvic fin development ofE. fuscoguttatus. (a) 11 DAH (4.21 ± 0.40 mm TL); (b) 14 DAH (4.76 ± 0.28 mm TL); (c) 17 DAH (6.00 ± 0.47 mm TL); (d) 20 DAH (6.85 ± 0.59 mm TL); (e) 23 DAH (7.59 ± 0.46 mm TL); (f) 26 DAH (8.07 ± 1.08 mm TL); (g) 29 DAH (9.99 ± 2.46 mm TL); (h) 32 DAH (12.38 ± 1.53 mm TL); (i) 38 DAH (25.16 ± 2.19 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL); (k) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Bp, basipterygium; Bb, barb; R, lepidotrichium S, hard spine; Sp, spinule; In, innominatum.


#### 3.3.4. Development of the Anal Fin

In EFT, at 11 DAH (Figure15(a)), proximal radials appeared, developing from the front of the body to the tail. At 20 DAH (Figure 15(d)), 10 pieces of proximal radials developed, with visible lepidotrichia. At 23 DAH (Figure 15(e)), the distal radials developed. At 26 DAH (Figure 15(f)), the first and second fin spines were visible and began to ossify, and stay cartilage appeared. At 32 DAH (Figure 15(h)), ossification of other spines and basipterygia began. At 43 DAH (Figure 15(j)), ossification was completed.Figure 15
Anal fin development of EFT. (a) 11 DAH (4.43 ± 0.41 mm TL); (b) 14 DAH (5.04 ± 0.77 mm TL); (c) 17 DAH (5.61 ± 0.41 mm TL); (d) 20 DAH (6.45 ± 0.48 mm TL); (e) 23 DAH (6.53 ± 0.63 mm TL); (f) 26 DAH (8.34 ± 0.44 mm TL); (g) 29 DAH (8.40 ± 1.2 mm TL); (h) 32 DAH (10.87 ± 1.49 mm TL); (i) 38 DAH (19.18 ± 1.40 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL); (k) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: Dr, distal radial; R, lepidotrichium; S, hard spine; Prx, proximal radial; stay.InE. fuscoguttatus, at 17 DAH (Figure 16(b)), proximal radials developed. At 20 DAH (Figure 17(c)), the first and second fin spines, distal radials, anal fin spines, and stay cartilage were visible. At 29 DAH (Figure 16(g)), the first and second fin spines began to ossify. At 32 DAH (Figure 16(h)), other spines and pterygiophore began to ossify.Figure 16
Anal fin development ofE. fuscoguttatus. (a) 14 DAH (TL4.76 ± 0.28 mm); (b) 17 DAH (6.00 ± 0.47 mm TL); (c) 20 DAH (6.85 ± 0.59 mm TL); (d) 20 DAH (6.85 ± 0.59 mm TL); (e) 23 DAH (7.59 ± 0.46 mm TL); (f) 26 DAH (8.07 ± 1.08 mm TL); (g) 29 DAH (9.99 ± 2.46 mm TL); (h) 32 DAH (12.38 ± 1.53 mm TL); (i) 38 DAH (25.16 ± 2.19 mm TL); (j) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Dr, distal radial; R, lepidotrichium; S, hard spine; Prx, proximal radial; stay.Figure 17
Caudal fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Ac, accessory; Ep, epural; Hy, hypural; PCR, caudal lepidotrichia; PrH, parhyural; SNA, specialzed neural; Ur, urostyle.


#### 3.3.5. Development of the Caudal Fin

In EFT, at 8 DAH (Figure17(a)), hypural 1 formed. At 11 DAH (Figure 17(b)), hypural 2-3 and parhypural formed. At 17 DAH (Figure 17(d)), parhypural 1–3 formed and caudal fin rays were somewhat visible. At 26 DAH (Figure 17(g)), hypural 4 developed. At 29 DAH (Figure 17(h)), epural 3 and epural 4 fused into a single piece and the caudal fin ray and urostyle started to ossify. At 38 DAH (Figure 17(j)), urostyle osteosis was completed. At 48 DAH (Figure 17(k)), caudal fin ray ossification was nearly complete.InE. fuscoguttatus, at 8 DAH (Figure 18(a)), hypural 1-2 formation was observed. At 11–14 DAH (Figures 18(b) and 18(c)), hypural 3 and parhypural formed. At 17 DAH (Figure 18(d)), epural 1–3 developed. At 20 DAH (Figure 18(f)), epural 4-5 and hypural 4-5 formed; at the same time, caudal fin rays could be seen. At 29 DAH (Figure 18(i)), epural 4-5 fused into a single piece, and the urostyle and caudal fin rays began to ossify. At 38 DAH (Figure 18(k)), urostyle ossification was completed. By 48 DAH (Figure 18(l)), ossification of the caudal fin rays was still incomplete.Figure 18
Caudal fin development ofE. fuscoguttatus. (a) 8 DAH (3.61 ± 0.22 mm TL); (b) 11 DAH (4.21 ± 0.40 mm TL); (c) 14 DAH (4.76 ± 0.28 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 17 DAH (6.00 ± 0.47 mm TL); (f) 20 DAH (6.85 ± 0.59 mm TL); (g) 23 DAH (7.59 ± 0.46 mm TL); (h) 26 DAH (8.07 ± 1.08 mm TL); (i) 29 DAH (9.99 ± 2.46 mm TL); (j) 32 DAH (12.38 ± 1.53 mm TL); (k) 38 DAH (25.16 ± 2.19 mm TL); (l) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Ac, accessory; Ep, epural; Hy, hypural; PCR, caudal lepidotrichia; PrH, parhyural; SNA, specialzed neural; Ur, urostyle.


## 3.1. Postembryonic Development of EFT andE. fuscoguttatus

The total lengths and weights ofE. fuscoguttatus and EFT are shown in Figures 1 and 2, respectively.Figure 1
Total length of EFT andE. fuscoguttatus. ∗∗P<0.01, ∗P<0.05.Figure 2
Weight of EFT andE. fuscoguttatus.At 2–20 days, the total lengths of EFT andE. fuscoguttatus were similar. Thereafter, E. fuscoguttatus had a longer overall length than that of EFT. After 61 days, the gap between the full length of E. fuscoguttatus and EFT narrowed gradually. At 122 days, the total lengths of EFT (126.47 ± 6.02 mm TL) were significantly longer than that of E. fuscoguttatus (111.92 ± 7.67 mm TL).At 61 DAH, the weight of the EFT was significantly less than that ofE. fuscoguttatus. At 92 DAH, the weight gap between EFT and E. fuscoguttatus narrowed. At 122 DAH, the weight of EFT was not significantly different from that of E. fuscoguttatus, but EFT weighs more than E. fuscoguttatus.


### 3.1.1. Postembryonic Development ofE. fuscoguttatus

At 2–5 DAH, the egg yolk sac ofE. fuscoguttatus was continually consumed, the oil ball became smaller, and an oral fissure was detected. The digestive tract became thicker, and melanin deposition was observed in the upper part of the digestive tract. The eyeball was completely black, the auditory sac behind the eye was obviously visible, and the bone in the jaw became visible. The fin folds on both sides of the anterior tail were slightly concave, and there was a small amount of melanin deposition in one-third of the body at the front of the tail (Figures 3(a) and 3(b)).Figure 3
Postembryonic development of EFT. (a) 2 DAH (0.28 ± 0.02 mm TL); (b) 5 DAH (0.32 ± 0.02 mm TL); (c) 8 DAH (0.4 ± 0.04 mm TL); (d) 11 DAH (0.44 ± 0.04 mm TL); (e) 14 DAH (0.5 ± 0.08 mm TL); (f) 17 DAH (0.56 ± 0.04 mm TL); (g) 20 DAH (0.64 ± 0.05 mm TL); (h) 23 DAH (0.65 ± 0.06 mm TL); (i) 26 DAH (0.83 ± 0.04 mm TL); (j) 29 DAH (0.84 ± 0.12 mm TL); (k) 32 DAH (1.09 ± 0.15 mm TL); (l) 38 DAH (1.92 ± 0.14 mm TL); (m) 43 DAH (2.72 ± 0.46 mm TL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)At 6–23 DAH, the eyeball became bright and the pupil was clearly visible, the melanin of the internal organ mass decreased gradually, and the area where the melanin was receding appeared silvery-white. The heart area and gills appeared bright red. At 26 DAH, a bright red blood vessel along the spine could be seen and the second dorsal and ventral spines grew. As the fish grew, the second dorsal and ventral spines continued to elongate up to one-half the length of the body, with melanin deposition at the ends of the fin lepidotrichia. The fin folds at the front of the tail continued to recede inward to the narrowest point, the vertebral column appeared segmented, the end of the spine was upturned, and the fin lepidotrichia could be seen in the tail. At 17 DAH, a large number of deadE. fuscoguttatus larvae were observed, with high rates of cannibalism between groups and a survival rate of only 30% (Figures 3(c)–3(g)).At 24–48 DAH, the orbit deepened and a fold formed on the skin around the eyes. The second dorsal and ventral spines began to contract, the melanin at the end gradually receded, the dorsal and ventral fins were clearly visible (except for the contracted fin lepidotrichia), and the pectoral and caudal fins were clearly visible. At 38 DAH, the body ofE. fuscoguttatus was no longer completely transparent, and the body surface had a pattern, with scales covering the body surface. At 43 DAH, the fish body was completely opaque, and the pattern on the body surface was roughly similar to that of adult fish (Figures 3(h)–3(l))At 61 DAH,E. fuscoguttatus was 6.9 ± 0.4 cm in total length and weighed 6.4 ± 1.1 g. At 92 DAH, individuals were 9.2 ± 1.4 cm in total length and weighed 17.1 ± 3.2 g. At 122 DAH, the total length was 11.2 ± 0.8 cm and the weight was 29.7 ± 6.3 g.


### 3.1.2. Postembryonic Development of EFT

At 2–5 DAH, there were no observable differences in development between EFT andE. fuscoguttatus (Figures 4(a) and 4(b)).Figure 4
Postembryonic development ofE. fuscoguttatus. (a) 2 DAH (2.85 ± 0.1 mm TL); (b) 5 DAH (3.21 ± 0.37 mm TL); (c) 8 DAH (3.61 ± 0.22 mm TL); (d) 11 DAH (4.21 ± 0.40 mm TL); (e) 14 DAH (TL4.76 ± 0.28 mm); (f) 17 DAH (6.00 ± 0.47 mm TL); (g) 20 DAH (6.85 ± 0.59 mm TL); (h) 23 DAH (7.59 ± 0.46 mm TL); (i) 26 DAH (8.07 ± 1.08 mm TL); (j) 29 DAH (9.99 ± 2.46 mm TL); (k) 32 DAH (12.38 ± 1.53 mm TL); (l) 38 DAH (25.16 ± 2.19 mm TL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)At 6–20 DAH, the pupil was clearly visible and the visceral mass melanin receded from the abdomen to the back and was covered in silvery white. The vertebral column was not cylindrical, the neural arch and hemal arch were visible, and the end of the vertebral column were upturned. The second dorsal and ventral spines were elongated, barbs could be seen on the spines of the fins, and melanin was deposited at the ends. The front of the visceral mass, gills, and heart was bright red. The number of EFT deaths was low, and the survival rate was 80% (Figures4(c)–4(h)).At 21–48 DAH, the development of EFT was similar to that ofE. fuscoguttatus, other than an elongated the body of EFT. At 43 DAH, the black pattern appeared on the surface of the body. At 48 DAH, the fish body was completely opaque and the dorsal fin, abdominal fin, anal fin, caudal fin, and pectoral fin were clearly visible. The body surface pattern was not substantially different from that of adult fish (Figure 4(i)).At 61 DAH, EFT was 6.0 ± 0.3 cm in total length and 3.5 ± 0.5 g in weight. At 92 DAH, the total length was 10.2 ± 0.3 cm and the weight was 16.4 ± 1.6 g. At 122 DAH, the total length was 12.6 ± 0.6 cm and the weight was 32.7 ± 5.1 g.


## 3.1.1. Postembryonic Development ofE. fuscoguttatus

At 2–5 DAH, the egg yolk sac ofE. fuscoguttatus was continually consumed, the oil ball became smaller, and an oral fissure was detected. The digestive tract became thicker, and melanin deposition was observed in the upper part of the digestive tract. The eyeball was completely black, the auditory sac behind the eye was obviously visible, and the bone in the jaw became visible. The fin folds on both sides of the anterior tail were slightly concave, and there was a small amount of melanin deposition in one-third of the body at the front of the tail (Figures 3(a) and 3(b)).Figure 3
Postembryonic development of EFT. (a) 2 DAH (0.28 ± 0.02 mm TL); (b) 5 DAH (0.32 ± 0.02 mm TL); (c) 8 DAH (0.4 ± 0.04 mm TL); (d) 11 DAH (0.44 ± 0.04 mm TL); (e) 14 DAH (0.5 ± 0.08 mm TL); (f) 17 DAH (0.56 ± 0.04 mm TL); (g) 20 DAH (0.64 ± 0.05 mm TL); (h) 23 DAH (0.65 ± 0.06 mm TL); (i) 26 DAH (0.83 ± 0.04 mm TL); (j) 29 DAH (0.84 ± 0.12 mm TL); (k) 32 DAH (1.09 ± 0.15 mm TL); (l) 38 DAH (1.92 ± 0.14 mm TL); (m) 43 DAH (2.72 ± 0.46 mm TL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)At 6–23 DAH, the eyeball became bright and the pupil was clearly visible, the melanin of the internal organ mass decreased gradually, and the area where the melanin was receding appeared silvery-white. The heart area and gills appeared bright red. At 26 DAH, a bright red blood vessel along the spine could be seen and the second dorsal and ventral spines grew. As the fish grew, the second dorsal and ventral spines continued to elongate up to one-half the length of the body, with melanin deposition at the ends of the fin lepidotrichia. The fin folds at the front of the tail continued to recede inward to the narrowest point, the vertebral column appeared segmented, the end of the spine was upturned, and the fin lepidotrichia could be seen in the tail. At 17 DAH, a large number of deadE. fuscoguttatus larvae were observed, with high rates of cannibalism between groups and a survival rate of only 30% (Figures 3(c)–3(g)).At 24–48 DAH, the orbit deepened and a fold formed on the skin around the eyes. The second dorsal and ventral spines began to contract, the melanin at the end gradually receded, the dorsal and ventral fins were clearly visible (except for the contracted fin lepidotrichia), and the pectoral and caudal fins were clearly visible. At 38 DAH, the body ofE. fuscoguttatus was no longer completely transparent, and the body surface had a pattern, with scales covering the body surface. At 43 DAH, the fish body was completely opaque, and the pattern on the body surface was roughly similar to that of adult fish (Figures 3(h)–3(l))At 61 DAH,E. fuscoguttatus was 6.9 ± 0.4 cm in total length and weighed 6.4 ± 1.1 g. At 92 DAH, individuals were 9.2 ± 1.4 cm in total length and weighed 17.1 ± 3.2 g. At 122 DAH, the total length was 11.2 ± 0.8 cm and the weight was 29.7 ± 6.3 g.


## 3.1.2. Postembryonic Development of EFT

At 2–5 DAH, there were no observable differences in development between EFT andE. fuscoguttatus (Figures 4(a) and 4(b)).Figure 4
Postembryonic development ofE. fuscoguttatus. (a) 2 DAH (2.85 ± 0.1 mm TL); (b) 5 DAH (3.21 ± 0.37 mm TL); (c) 8 DAH (3.61 ± 0.22 mm TL); (d) 11 DAH (4.21 ± 0.40 mm TL); (e) 14 DAH (TL4.76 ± 0.28 mm); (f) 17 DAH (6.00 ± 0.47 mm TL); (g) 20 DAH (6.85 ± 0.59 mm TL); (h) 23 DAH (7.59 ± 0.46 mm TL); (i) 26 DAH (8.07 ± 1.08 mm TL); (j) 29 DAH (9.99 ± 2.46 mm TL); (k) 32 DAH (12.38 ± 1.53 mm TL); (l) 38 DAH (25.16 ± 2.19 mm TL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)At 6–20 DAH, the pupil was clearly visible and the visceral mass melanin receded from the abdomen to the back and was covered in silvery white. The vertebral column was not cylindrical, the neural arch and hemal arch were visible, and the end of the vertebral column were upturned. The second dorsal and ventral spines were elongated, barbs could be seen on the spines of the fins, and melanin was deposited at the ends. The front of the visceral mass, gills, and heart was bright red. The number of EFT deaths was low, and the survival rate was 80% (Figures4(c)–4(h)).At 21–48 DAH, the development of EFT was similar to that ofE. fuscoguttatus, other than an elongated the body of EFT. At 43 DAH, the black pattern appeared on the surface of the body. At 48 DAH, the fish body was completely opaque and the dorsal fin, abdominal fin, anal fin, caudal fin, and pectoral fin were clearly visible. The body surface pattern was not substantially different from that of adult fish (Figure 4(i)).At 61 DAH, EFT was 6.0 ± 0.3 cm in total length and 3.5 ± 0.5 g in weight. At 92 DAH, the total length was 10.2 ± 0.3 cm and the weight was 16.4 ± 1.6 g. At 122 DAH, the total length was 12.6 ± 0.6 cm and the weight was 32.7 ± 5.1 g.


## 3.2. Axial Skeletal Development ofE. fuscoguttatus and EFT

### 3.2.1. Development of the Cranium

At 2 DAH, juvenile fish lacked head cartilage. At 5 DAH (Figures5(a) and 6(a)), larvae in both groups did not differ substantially, showing the development of the ethmoid plate, trabeculae, Michael’s cartilage, four ceratobranchials, basibranchial cartilage, hypohyal, and hypobranchial. At 8 DAH (Figures 5(a) and 6(b)), both fish had a visible lower pharyngeal bone and supraoccipital, and basibranchial cartilage 2. In addition, in E. fuscoguttatus, nasal and palatoquadrate cartilage were detected. At 11 DAH (Figures 5(c) and 6(c)), two groupers began to develop premaxilla and prosethmoid, and the nasal and palatoquadrate cartilage of EFT began to appear. At 14–20 DAH (Figures 5(d)–5(f), 6(d)–6(f)), the vomer was developed and the maxillary began to ossify. At 23–29 DAH (Figures 5(g)–5(i), 6(g)–6(i)), the branchiostegal was visible, the palatoquadrate began to degenerate, the parietal, maxilla, premaxilla, and preopercular gradually ossified, and dentary appeared. At 32–48 DAH (Figures 5(j)–5(l), 6(j) and 6(k)), bone ossification in each part of the head was completed.Figure 5
Cephalic skeleton development of EFTA1-L1(lateralview) A2-L2(dorsalview) A3-L3(ventralview). (a) 5 DAH (3.2 ± 0.17 mm TL); (b) 8 DAH (3.97 ± 0.4 mm TL); (c) 11 DAH (4.43 ± 0.41 mm TL); (d) 14 DAH (5.04 ± 0.77 mm TL); (e) 17 DAH (5.61 ± 0.41 mm TL); (f) 20 DAH (6.45 ± 0.48 mm TL); (g) 23 DAH (6.53 ± 0.63 mm TL); (h) 26 DAH (8.34 ± 0.44 mm TL); (i) 29 DAH (8.40 ± 1.2 mm TL); (j) 32 DAH (10.87 ± 1.49 mm TL); (k) 38 DAH (19.18 ± 1.40 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: an angular; Bh: basihyal; Br: branchiostegal; Bb1: basibranchial cartilages1; Bb2: basibranchial cartilages2; Ch: ceratohyal; Cb: ceratobranchial; De: dentary; EP: ethmoid plate; EC: ethmoid cartilage; Eh: epihyal; Fr: frontal; Hh: hypohyal; Hb: hypobranchial; Ih: interhyal; LPB: lower pharyngeal bone; LE: lateral ethmoid; MC: Meckel’s cartilage; Ma: maxillary; Na: nasal; MP: mediodorsal; Pe: prosethmoid; Pq: palatoquadrate; Pa: parietal; Po: preopercle; Pm: premaxillary; SOC: supraoccipital Sy: sympletic; Tr: trabeculae; V: vomer.Figure 6
Cephalic skeleton development ofE. fuscoguttatus A1-K1(lateralview) A2-K2(dorsalview) A3-K3(ventralview). (a) 2 DAH (2.85 ± 0.1 mm TL); (b) 5 DAH (3.21 ± 0.37 mm TL); (c) 8 DAH (3.61 ± 0.22 mm TL); (d) 11 DAH (4.21 ± 0.40 mm TL); (e) 14 DAH (TL4.76 ± 0.28 mm); (f) 17 DAH (6.00 ± 0.47 mm TL); (g) 20 DAH (6.85 ± 0.59 mm TL); (h) 23 DAH (7.59 ± 0.46 mm TL); (i) 26 DAH (8.07 ± 1.08 mm TL); (j) 29 DAH (9.99 ± 2.46 mm TL); (k) 32 DAH (12.38 ± 1.53 mm TL).Abbreviations are as follows: An: angular; Bh: basihyal; Br: branchiostegal; Bb1: basibranchial cartilages1; Bb2: basibranchial cartilages2; Ch: ceratohyal; Cb: ceratobranchial; De: dentary; EP: ethmoid plate; EC: ethmoid cartilage; Eh: epihyal; Fr: frontal; Hh: hypohyal; Hb: hypobranchial; Ih: interhyal; LPB: lower pharyngeal bone; LE: lateral ethmoid; MC: Meckel’s cartilage; Ma:maxillary; Na:nasal; MP: mediodorsal Pe: prosethmoid; Pq: palatoquadrate; Pa: parietal; Po:preopercle; Pm: premaxillary; SOC: supraoccipital; Sy: sympletic; Tr: trabeculae; V: vomer.


### 3.2.2. Development of the Vertebral Column

At 11 DAH (Figures7(a) and 8(a)), the neural arch formed from the front of the back and the hemal arch formed from the front of the abdomen. At 17 DAH (Figures 7(c) and 8(c)), the neural arch and hemal arch were completely formed and the parapophysis was visible. Prior to this point, the two grouper species developed at approximately the same rate. At 20 DAH (Figure 7(d)), the vertebral column of EFT began to ossify, while this was observed as 23 DAH in E. fuscoguttatus (Figure 8(e)). At 29–32 DAH (Figures 7(g), 7(h), 8(g), and 8(h)), two species of grouper had abdominal ribs and dorsal ribs. At 38 DAH (Figures 7(i) and 8(i)), EFT and E. fuscoguttatus vertebrae, neural arches, hemal arches, and parapophysis were completely ossified. When ossification was completed, both EFT and E. fuscoguttatus had 19 vertebrae.Figure 7
Vertebral column development of EFT. (a) 11 DAH (4.43 ± 0.41 mm TL); (b) 14 DAH (5.04 ± 0.77 mm TL); (c) 17 DAH (5.61 ± 0.41 mm TL); (d) 20 DAH (6.45 ± 0.48 mm TL); (e) 23 DAH (6.53 ± 0.63 mm TL); (f) 26 DAH (8.34 ± 0.44 mm TL); (g) 29 DAH (8.40 ± 1.2 mm TL); (h) 32 DAH (10.87 ± 1.49 mm TL); (i) 38 DAH (19.18 ± 1.40 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Dr: dorsal rib; HS: heamal spine; HA: heamal arch; NA: neural arch; NS: neural spine; Pp: parapophysis; Pr: pleural rib; V: vertebral centra.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 8
Vertebral column development ofE. fuscoguttatus. (a) 11 DAH (4.21 ± 0.40 mm TL); (b) 14 DAH (TL4.76 ± 0.28 mm); (c) 17 DAH (6.00 ± 0.47 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 20 DAH (6.85 ± 0.59 mm TL); (f) 23 DAH (7.59 ± 0.46 mm TL); (g) 26 DAH (8.07 ± 1.08 mm TL); (h) 29 DAH (9.99 ± 2.46 mm TL); (i) 32 DAH (12.38 ± 1.53 mm TL); (j) 38 DAH (19.18 ± 1.39 mm TL). Abbreviations are as follows: Dr: dorsal rib; HS: heamal spine; HA: heamal arch; NA: neural arch; NS: neural spine; Pp: parapophysis; Pr: pleural rib; V: vertebral centra.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.2.1. Development of the Cranium

At 2 DAH, juvenile fish lacked head cartilage. At 5 DAH (Figures5(a) and 6(a)), larvae in both groups did not differ substantially, showing the development of the ethmoid plate, trabeculae, Michael’s cartilage, four ceratobranchials, basibranchial cartilage, hypohyal, and hypobranchial. At 8 DAH (Figures 5(a) and 6(b)), both fish had a visible lower pharyngeal bone and supraoccipital, and basibranchial cartilage 2. In addition, in E. fuscoguttatus, nasal and palatoquadrate cartilage were detected. At 11 DAH (Figures 5(c) and 6(c)), two groupers began to develop premaxilla and prosethmoid, and the nasal and palatoquadrate cartilage of EFT began to appear. At 14–20 DAH (Figures 5(d)–5(f), 6(d)–6(f)), the vomer was developed and the maxillary began to ossify. At 23–29 DAH (Figures 5(g)–5(i), 6(g)–6(i)), the branchiostegal was visible, the palatoquadrate began to degenerate, the parietal, maxilla, premaxilla, and preopercular gradually ossified, and dentary appeared. At 32–48 DAH (Figures 5(j)–5(l), 6(j) and 6(k)), bone ossification in each part of the head was completed.Figure 5
Cephalic skeleton development of EFTA1-L1(lateralview) A2-L2(dorsalview) A3-L3(ventralview). (a) 5 DAH (3.2 ± 0.17 mm TL); (b) 8 DAH (3.97 ± 0.4 mm TL); (c) 11 DAH (4.43 ± 0.41 mm TL); (d) 14 DAH (5.04 ± 0.77 mm TL); (e) 17 DAH (5.61 ± 0.41 mm TL); (f) 20 DAH (6.45 ± 0.48 mm TL); (g) 23 DAH (6.53 ± 0.63 mm TL); (h) 26 DAH (8.34 ± 0.44 mm TL); (i) 29 DAH (8.40 ± 1.2 mm TL); (j) 32 DAH (10.87 ± 1.49 mm TL); (k) 38 DAH (19.18 ± 1.40 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: an angular; Bh: basihyal; Br: branchiostegal; Bb1: basibranchial cartilages1; Bb2: basibranchial cartilages2; Ch: ceratohyal; Cb: ceratobranchial; De: dentary; EP: ethmoid plate; EC: ethmoid cartilage; Eh: epihyal; Fr: frontal; Hh: hypohyal; Hb: hypobranchial; Ih: interhyal; LPB: lower pharyngeal bone; LE: lateral ethmoid; MC: Meckel’s cartilage; Ma: maxillary; Na: nasal; MP: mediodorsal; Pe: prosethmoid; Pq: palatoquadrate; Pa: parietal; Po: preopercle; Pm: premaxillary; SOC: supraoccipital Sy: sympletic; Tr: trabeculae; V: vomer.Figure 6
Cephalic skeleton development ofE. fuscoguttatus A1-K1(lateralview) A2-K2(dorsalview) A3-K3(ventralview). (a) 2 DAH (2.85 ± 0.1 mm TL); (b) 5 DAH (3.21 ± 0.37 mm TL); (c) 8 DAH (3.61 ± 0.22 mm TL); (d) 11 DAH (4.21 ± 0.40 mm TL); (e) 14 DAH (TL4.76 ± 0.28 mm); (f) 17 DAH (6.00 ± 0.47 mm TL); (g) 20 DAH (6.85 ± 0.59 mm TL); (h) 23 DAH (7.59 ± 0.46 mm TL); (i) 26 DAH (8.07 ± 1.08 mm TL); (j) 29 DAH (9.99 ± 2.46 mm TL); (k) 32 DAH (12.38 ± 1.53 mm TL).Abbreviations are as follows: An: angular; Bh: basihyal; Br: branchiostegal; Bb1: basibranchial cartilages1; Bb2: basibranchial cartilages2; Ch: ceratohyal; Cb: ceratobranchial; De: dentary; EP: ethmoid plate; EC: ethmoid cartilage; Eh: epihyal; Fr: frontal; Hh: hypohyal; Hb: hypobranchial; Ih: interhyal; LPB: lower pharyngeal bone; LE: lateral ethmoid; MC: Meckel’s cartilage; Ma:maxillary; Na:nasal; MP: mediodorsal Pe: prosethmoid; Pq: palatoquadrate; Pa: parietal; Po:preopercle; Pm: premaxillary; SOC: supraoccipital; Sy: sympletic; Tr: trabeculae; V: vomer.


## 3.2.2. Development of the Vertebral Column

At 11 DAH (Figures7(a) and 8(a)), the neural arch formed from the front of the back and the hemal arch formed from the front of the abdomen. At 17 DAH (Figures 7(c) and 8(c)), the neural arch and hemal arch were completely formed and the parapophysis was visible. Prior to this point, the two grouper species developed at approximately the same rate. At 20 DAH (Figure 7(d)), the vertebral column of EFT began to ossify, while this was observed as 23 DAH in E. fuscoguttatus (Figure 8(e)). At 29–32 DAH (Figures 7(g), 7(h), 8(g), and 8(h)), two species of grouper had abdominal ribs and dorsal ribs. At 38 DAH (Figures 7(i) and 8(i)), EFT and E. fuscoguttatus vertebrae, neural arches, hemal arches, and parapophysis were completely ossified. When ossification was completed, both EFT and E. fuscoguttatus had 19 vertebrae.Figure 7
Vertebral column development of EFT. (a) 11 DAH (4.43 ± 0.41 mm TL); (b) 14 DAH (5.04 ± 0.77 mm TL); (c) 17 DAH (5.61 ± 0.41 mm TL); (d) 20 DAH (6.45 ± 0.48 mm TL); (e) 23 DAH (6.53 ± 0.63 mm TL); (f) 26 DAH (8.34 ± 0.44 mm TL); (g) 29 DAH (8.40 ± 1.2 mm TL); (h) 32 DAH (10.87 ± 1.49 mm TL); (i) 38 DAH (19.18 ± 1.40 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Dr: dorsal rib; HS: heamal spine; HA: heamal arch; NA: neural arch; NS: neural spine; Pp: parapophysis; Pr: pleural rib; V: vertebral centra.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 8
Vertebral column development ofE. fuscoguttatus. (a) 11 DAH (4.21 ± 0.40 mm TL); (b) 14 DAH (TL4.76 ± 0.28 mm); (c) 17 DAH (6.00 ± 0.47 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 20 DAH (6.85 ± 0.59 mm TL); (f) 23 DAH (7.59 ± 0.46 mm TL); (g) 26 DAH (8.07 ± 1.08 mm TL); (h) 29 DAH (9.99 ± 2.46 mm TL); (i) 32 DAH (12.38 ± 1.53 mm TL); (j) 38 DAH (19.18 ± 1.39 mm TL). Abbreviations are as follows: Dr: dorsal rib; HS: heamal spine; HA: heamal arch; NA: neural arch; NS: neural spine; Pp: parapophysis; Pr: pleural rib; V: vertebral centra.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.3. Appendage Skeletal Development ofE. fuscoguttatus and EFT

### 3.3.1. Development of the Pectoral Fin

At 5 DAH (Figure9(a)), the EFT coracoid-scapula cartilage and fin plate were visible. At 6–26 DAH (Figures 9(b)–9(h)), the fin part gradually degenerated to form the proximal radius, while the distal radials were visible. At 26 DAH (Figure 9(h)), the pectoral fin was visible. At 48 DAH (Figure 9(m)), the pectoral fin was completely ossified.Figure 9
Pectoral fin development of EFT. (a) 5 DAH (3.2 ± 0.17 mm TL); (b) 8 DAH (3.97 ± 0.4 mm TL); (c) 11 DAH (4.43 ± 0.41 mm TL); (d) 14 DAH (5.04 ± 0.77 mm TL); (e) 17 DAH (5.61 ± 0.41 mm TL); (f) 20 DAH (6.45 ± 0.48 mm TL); (g) 23 DAH (6.53 ± 0.63 mm TL); (h) 26 DAH (8.34 ± 0.44 mm TL); (i) 29 DAH (8.40 ± 1.2 mm TL); (j) 32 DAH (10.87 ± 1.49 mm TL); (k) 38 DAH (19.18 ± 1.40 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL); (m) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: CL, cleithrum; Co-Sca: coracoid-scapula; Co, coracoid; PocLo, metacleithrum lower; Fp, fin plate; ScF, scapular foramen; Sca, scapula; R, lepidotrichium; Rd, distal radial; Prx, proximal pterygiophore; Suc, hypercleithrum.In the development of the pectoral fin ofE. fuscoguttatus, at 5 DAH (Figure 10(a)), coracoid-scapula cartilage was visible. At the 8 DAH (Figure 10(b)), fin plate and cleithrum appeared. At 9–17 DAH (Figures 10(c)–10(e)), part of the fin plate gradually degenerated to form proximal radials. At 20 DAH (Figure 10(f)), the pectoral fin was visible. At 21–43 DAH (Figures 10(g)–10(l)), ossification of the pectoral fin, cleithrum, proximal radials, and other regions was completed.Figure 10
Pectoral fin development ofE. fuscoguttatus. (a) 5 DAH (3.21 ± 0.37 mm TL); (b) 8 DAH (3.61 ± 0.22 mm TL); (c) 11 DAH (4.21 ± 0.40 mm TL); (d) 14 DAH (TL4.76 ± 0.28 mm); (e) 17 DAH (6.00 ± 0.47 mm TL); (f) 20 DAH (6.85 ± 0.59 mm TL); (g) 23 DAH (7.59 ± 0.46 mm TL); (h) 26 DAH (8.07 ± 1.08 mm TL); (i) 29 DAH (9.99 ± 2.46 mm TL); (j) 32 DAH (12.38 ± 1.53 mm TL); (k) 38 DAH (25.16 ± 2.19 mm TL); (l) 43 DAH (30.49 ± 3.16 mm TL). Abbreviations are as follows: CL, cleithrum; Co-Sca: coracoid-scapula; Co, coracoid; PocLo, metacleithrum lower; Fp, fin plate; ScF, scapular foramen; Sca, scapula; R, lepidotrichium; Rd, distal radial; Prx, proximal pterygiophore; Suc, hypercleithrum.


### 3.3.2. Development of the Dorsal Fin

In EFT, at 8 DAH (Figure11(a)), the second dorsal spine and its fin bones appeared. At 11 DAH (Figure 11(b)), the first dorsal spine and fin bone appeared, the second dorsal spine began to elongate continuously, and barbs appeared. At 17 DAH (Figure 11(d)), the third dorsal spine and fin bone were visible and the first dorsal spine and second dorsal spine began to ossify. At 23 DAH (Figure 11(f)), the fourth dorsal spine developed. At 26 DAH (Figure 11(g)), the rest of the dorsal spines, dorsal fin strips, and dorsal fin distal radials were observed, while the second dorsal fin spine underwent a degenerative process, gradually shortening. At 32 DAH (Figure 11(i)), stay cartilage appeared. At 38 DAH (Figure 11(j)), the second dorsal spine degenerated and was reduced to the length of the other dorsal spines. The dorsal fin also began to ossify. At 48 DAH (Figure 11(m)), the dorsal fin was completely ossified.Figure 11
Dorsal fin development of EFT Dorsal fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL); (m) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: Ba, barb; Prd, predosal; R, lepidotrichium; Rd, distal radial; S, hard spine; Sp, spinule; stay.InE. fuscoguttatus, at 8–17 DAH (Figures 12(b)–12(e)), the degree of development was comparable to that of EFT. At 20 DAH (Figure 12(e)), the rest of the dorsal spines, dorsal fin strips, and distal radius of the dorsal fin were visible, and the second dorsal spine degenerated. At 23 DAH (Figure 12(f)), the stay cartilage was visible. At 43 DAH (Figure 12(k)), the second dorsal spine was shortened to the length of other dorsal spines, slightly longer than the first dorsal spine. The dorsal fin spine also began to ossify.Figure 12
Dorsal fin development ofE. fuscoguttatus. (a) 8 DAH (3.61 ± 0.22 mm TL); (b) 11 DAH (4.21 ± 0.40 mm TL); (c) 14 DAH (4.76 ± 0.28 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 20 DAH (6.85 ± 0.59 mm TL); (f) 23 DAH (7.59 ± 0.46 mm TL); (g) 26 DAH (8.07 ± 1.08 mm TL); (h) 29 DAH (9.99 ± 2.46 mm TL); (i) 32 DAH (12.38 ± 1.53 mm TL); (j) 38 DAH (25.16 ± 2.19 mm TL); (k) 43 DAH (30.49 ± 3.16 mm TL). (l) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Ba, barb; Prd, predosal; R, lepidotrichium; Rd, distal radial; S, hard spine; Sp, spinule; stay.


### 3.3.3. Development of the Pelvic Fin

In EFT, at 8 DAH (Figure13(a)), the abdominal spines and fin-based cartilage emerged. At 14 DAH (Figure 13(c)), abdominal spines were elongated, accompanied by barb growth. At 17 DAH, abdominal spines began to ossify. At 23 DAH (Figure 13(f)), ventral fin development was observed. At 26 DAH (Figure 13(g)), fin-based cartilage ossification was observed. Abdominal spines began to shorten until they were comparable in length to other fin spines.Figure 13
Pelvic fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Bp, basipterygium; Bb, barb; R, lepidotrichium S, hard spine; Sp, spinule; In, innominatum.InE. fuscoguttatus, at 8–14 DAH, development was similar to that in EFT. At 17 DAH (Figure 14(c)), the abdominal spines began to ossify, and ventral fin development was observed. At 20 DAH (Figure 14(d)), the basipterygiua began to ossify, and the abdominal spine underwent a degenerative shortening process until it was comparable in length to those of other fin spines.Figure 14
Pelvic fin development ofE. fuscoguttatus. (a) 11 DAH (4.21 ± 0.40 mm TL); (b) 14 DAH (4.76 ± 0.28 mm TL); (c) 17 DAH (6.00 ± 0.47 mm TL); (d) 20 DAH (6.85 ± 0.59 mm TL); (e) 23 DAH (7.59 ± 0.46 mm TL); (f) 26 DAH (8.07 ± 1.08 mm TL); (g) 29 DAH (9.99 ± 2.46 mm TL); (h) 32 DAH (12.38 ± 1.53 mm TL); (i) 38 DAH (25.16 ± 2.19 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL); (k) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Bp, basipterygium; Bb, barb; R, lepidotrichium S, hard spine; Sp, spinule; In, innominatum.


### 3.3.4. Development of the Anal Fin

In EFT, at 11 DAH (Figure15(a)), proximal radials appeared, developing from the front of the body to the tail. At 20 DAH (Figure 15(d)), 10 pieces of proximal radials developed, with visible lepidotrichia. At 23 DAH (Figure 15(e)), the distal radials developed. At 26 DAH (Figure 15(f)), the first and second fin spines were visible and began to ossify, and stay cartilage appeared. At 32 DAH (Figure 15(h)), ossification of other spines and basipterygia began. At 43 DAH (Figure 15(j)), ossification was completed.Figure 15
Anal fin development of EFT. (a) 11 DAH (4.43 ± 0.41 mm TL); (b) 14 DAH (5.04 ± 0.77 mm TL); (c) 17 DAH (5.61 ± 0.41 mm TL); (d) 20 DAH (6.45 ± 0.48 mm TL); (e) 23 DAH (6.53 ± 0.63 mm TL); (f) 26 DAH (8.34 ± 0.44 mm TL); (g) 29 DAH (8.40 ± 1.2 mm TL); (h) 32 DAH (10.87 ± 1.49 mm TL); (i) 38 DAH (19.18 ± 1.40 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL); (k) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: Dr, distal radial; R, lepidotrichium; S, hard spine; Prx, proximal radial; stay.InE. fuscoguttatus, at 17 DAH (Figure 16(b)), proximal radials developed. At 20 DAH (Figure 17(c)), the first and second fin spines, distal radials, anal fin spines, and stay cartilage were visible. At 29 DAH (Figure 16(g)), the first and second fin spines began to ossify. At 32 DAH (Figure 16(h)), other spines and pterygiophore began to ossify.Figure 16
Anal fin development ofE. fuscoguttatus. (a) 14 DAH (TL4.76 ± 0.28 mm); (b) 17 DAH (6.00 ± 0.47 mm TL); (c) 20 DAH (6.85 ± 0.59 mm TL); (d) 20 DAH (6.85 ± 0.59 mm TL); (e) 23 DAH (7.59 ± 0.46 mm TL); (f) 26 DAH (8.07 ± 1.08 mm TL); (g) 29 DAH (9.99 ± 2.46 mm TL); (h) 32 DAH (12.38 ± 1.53 mm TL); (i) 38 DAH (25.16 ± 2.19 mm TL); (j) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Dr, distal radial; R, lepidotrichium; S, hard spine; Prx, proximal radial; stay.Figure 17
Caudal fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Ac, accessory; Ep, epural; Hy, hypural; PCR, caudal lepidotrichia; PrH, parhyural; SNA, specialzed neural; Ur, urostyle.


### 3.3.5. Development of the Caudal Fin

In EFT, at 8 DAH (Figure17(a)), hypural 1 formed. At 11 DAH (Figure 17(b)), hypural 2-3 and parhypural formed. At 17 DAH (Figure 17(d)), parhypural 1–3 formed and caudal fin rays were somewhat visible. At 26 DAH (Figure 17(g)), hypural 4 developed. At 29 DAH (Figure 17(h)), epural 3 and epural 4 fused into a single piece and the caudal fin ray and urostyle started to ossify. At 38 DAH (Figure 17(j)), urostyle osteosis was completed. At 48 DAH (Figure 17(k)), caudal fin ray ossification was nearly complete.InE. fuscoguttatus, at 8 DAH (Figure 18(a)), hypural 1-2 formation was observed. At 11–14 DAH (Figures 18(b) and 18(c)), hypural 3 and parhypural formed. At 17 DAH (Figure 18(d)), epural 1–3 developed. At 20 DAH (Figure 18(f)), epural 4-5 and hypural 4-5 formed; at the same time, caudal fin rays could be seen. At 29 DAH (Figure 18(i)), epural 4-5 fused into a single piece, and the urostyle and caudal fin rays began to ossify. At 38 DAH (Figure 18(k)), urostyle ossification was completed. By 48 DAH (Figure 18(l)), ossification of the caudal fin rays was still incomplete.Figure 18
Caudal fin development ofE. fuscoguttatus. (a) 8 DAH (3.61 ± 0.22 mm TL); (b) 11 DAH (4.21 ± 0.40 mm TL); (c) 14 DAH (4.76 ± 0.28 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 17 DAH (6.00 ± 0.47 mm TL); (f) 20 DAH (6.85 ± 0.59 mm TL); (g) 23 DAH (7.59 ± 0.46 mm TL); (h) 26 DAH (8.07 ± 1.08 mm TL); (i) 29 DAH (9.99 ± 2.46 mm TL); (j) 32 DAH (12.38 ± 1.53 mm TL); (k) 38 DAH (25.16 ± 2.19 mm TL); (l) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Ac, accessory; Ep, epural; Hy, hypural; PCR, caudal lepidotrichia; PrH, parhyural; SNA, specialzed neural; Ur, urostyle.


## 3.3.1. Development of the Pectoral Fin

At 5 DAH (Figure9(a)), the EFT coracoid-scapula cartilage and fin plate were visible. At 6–26 DAH (Figures 9(b)–9(h)), the fin part gradually degenerated to form the proximal radius, while the distal radials were visible. At 26 DAH (Figure 9(h)), the pectoral fin was visible. At 48 DAH (Figure 9(m)), the pectoral fin was completely ossified.Figure 9
Pectoral fin development of EFT. (a) 5 DAH (3.2 ± 0.17 mm TL); (b) 8 DAH (3.97 ± 0.4 mm TL); (c) 11 DAH (4.43 ± 0.41 mm TL); (d) 14 DAH (5.04 ± 0.77 mm TL); (e) 17 DAH (5.61 ± 0.41 mm TL); (f) 20 DAH (6.45 ± 0.48 mm TL); (g) 23 DAH (6.53 ± 0.63 mm TL); (h) 26 DAH (8.34 ± 0.44 mm TL); (i) 29 DAH (8.40 ± 1.2 mm TL); (j) 32 DAH (10.87 ± 1.49 mm TL); (k) 38 DAH (19.18 ± 1.40 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL); (m) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: CL, cleithrum; Co-Sca: coracoid-scapula; Co, coracoid; PocLo, metacleithrum lower; Fp, fin plate; ScF, scapular foramen; Sca, scapula; R, lepidotrichium; Rd, distal radial; Prx, proximal pterygiophore; Suc, hypercleithrum.In the development of the pectoral fin ofE. fuscoguttatus, at 5 DAH (Figure 10(a)), coracoid-scapula cartilage was visible. At the 8 DAH (Figure 10(b)), fin plate and cleithrum appeared. At 9–17 DAH (Figures 10(c)–10(e)), part of the fin plate gradually degenerated to form proximal radials. At 20 DAH (Figure 10(f)), the pectoral fin was visible. At 21–43 DAH (Figures 10(g)–10(l)), ossification of the pectoral fin, cleithrum, proximal radials, and other regions was completed.Figure 10
Pectoral fin development ofE. fuscoguttatus. (a) 5 DAH (3.21 ± 0.37 mm TL); (b) 8 DAH (3.61 ± 0.22 mm TL); (c) 11 DAH (4.21 ± 0.40 mm TL); (d) 14 DAH (TL4.76 ± 0.28 mm); (e) 17 DAH (6.00 ± 0.47 mm TL); (f) 20 DAH (6.85 ± 0.59 mm TL); (g) 23 DAH (7.59 ± 0.46 mm TL); (h) 26 DAH (8.07 ± 1.08 mm TL); (i) 29 DAH (9.99 ± 2.46 mm TL); (j) 32 DAH (12.38 ± 1.53 mm TL); (k) 38 DAH (25.16 ± 2.19 mm TL); (l) 43 DAH (30.49 ± 3.16 mm TL). Abbreviations are as follows: CL, cleithrum; Co-Sca: coracoid-scapula; Co, coracoid; PocLo, metacleithrum lower; Fp, fin plate; ScF, scapular foramen; Sca, scapula; R, lepidotrichium; Rd, distal radial; Prx, proximal pterygiophore; Suc, hypercleithrum.


## 3.3.2. Development of the Dorsal Fin

In EFT, at 8 DAH (Figure11(a)), the second dorsal spine and its fin bones appeared. At 11 DAH (Figure 11(b)), the first dorsal spine and fin bone appeared, the second dorsal spine began to elongate continuously, and barbs appeared. At 17 DAH (Figure 11(d)), the third dorsal spine and fin bone were visible and the first dorsal spine and second dorsal spine began to ossify. At 23 DAH (Figure 11(f)), the fourth dorsal spine developed. At 26 DAH (Figure 11(g)), the rest of the dorsal spines, dorsal fin strips, and dorsal fin distal radials were observed, while the second dorsal fin spine underwent a degenerative process, gradually shortening. At 32 DAH (Figure 11(i)), stay cartilage appeared. At 38 DAH (Figure 11(j)), the second dorsal spine degenerated and was reduced to the length of the other dorsal spines. The dorsal fin also began to ossify. At 48 DAH (Figure 11(m)), the dorsal fin was completely ossified.Figure 11
Dorsal fin development of EFT Dorsal fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL); (l) 43 DAH (27.21 ± 4.6 mm TL); (m) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: Ba, barb; Prd, predosal; R, lepidotrichium; Rd, distal radial; S, hard spine; Sp, spinule; stay.InE. fuscoguttatus, at 8–17 DAH (Figures 12(b)–12(e)), the degree of development was comparable to that of EFT. At 20 DAH (Figure 12(e)), the rest of the dorsal spines, dorsal fin strips, and distal radius of the dorsal fin were visible, and the second dorsal spine degenerated. At 23 DAH (Figure 12(f)), the stay cartilage was visible. At 43 DAH (Figure 12(k)), the second dorsal spine was shortened to the length of other dorsal spines, slightly longer than the first dorsal spine. The dorsal fin spine also began to ossify.Figure 12
Dorsal fin development ofE. fuscoguttatus. (a) 8 DAH (3.61 ± 0.22 mm TL); (b) 11 DAH (4.21 ± 0.40 mm TL); (c) 14 DAH (4.76 ± 0.28 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 20 DAH (6.85 ± 0.59 mm TL); (f) 23 DAH (7.59 ± 0.46 mm TL); (g) 26 DAH (8.07 ± 1.08 mm TL); (h) 29 DAH (9.99 ± 2.46 mm TL); (i) 32 DAH (12.38 ± 1.53 mm TL); (j) 38 DAH (25.16 ± 2.19 mm TL); (k) 43 DAH (30.49 ± 3.16 mm TL). (l) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Ba, barb; Prd, predosal; R, lepidotrichium; Rd, distal radial; S, hard spine; Sp, spinule; stay.


## 3.3.3. Development of the Pelvic Fin

In EFT, at 8 DAH (Figure13(a)), the abdominal spines and fin-based cartilage emerged. At 14 DAH (Figure 13(c)), abdominal spines were elongated, accompanied by barb growth. At 17 DAH, abdominal spines began to ossify. At 23 DAH (Figure 13(f)), ventral fin development was observed. At 26 DAH (Figure 13(g)), fin-based cartilage ossification was observed. Abdominal spines began to shorten until they were comparable in length to other fin spines.Figure 13
Pelvic fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Bp, basipterygium; Bb, barb; R, lepidotrichium S, hard spine; Sp, spinule; In, innominatum.InE. fuscoguttatus, at 8–14 DAH, development was similar to that in EFT. At 17 DAH (Figure 14(c)), the abdominal spines began to ossify, and ventral fin development was observed. At 20 DAH (Figure 14(d)), the basipterygiua began to ossify, and the abdominal spine underwent a degenerative shortening process until it was comparable in length to those of other fin spines.Figure 14
Pelvic fin development ofE. fuscoguttatus. (a) 11 DAH (4.21 ± 0.40 mm TL); (b) 14 DAH (4.76 ± 0.28 mm TL); (c) 17 DAH (6.00 ± 0.47 mm TL); (d) 20 DAH (6.85 ± 0.59 mm TL); (e) 23 DAH (7.59 ± 0.46 mm TL); (f) 26 DAH (8.07 ± 1.08 mm TL); (g) 29 DAH (9.99 ± 2.46 mm TL); (h) 32 DAH (12.38 ± 1.53 mm TL); (i) 38 DAH (25.16 ± 2.19 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL); (k) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Bp, basipterygium; Bb, barb; R, lepidotrichium S, hard spine; Sp, spinule; In, innominatum.


## 3.3.4. Development of the Anal Fin

In EFT, at 11 DAH (Figure15(a)), proximal radials appeared, developing from the front of the body to the tail. At 20 DAH (Figure 15(d)), 10 pieces of proximal radials developed, with visible lepidotrichia. At 23 DAH (Figure 15(e)), the distal radials developed. At 26 DAH (Figure 15(f)), the first and second fin spines were visible and began to ossify, and stay cartilage appeared. At 32 DAH (Figure 15(h)), ossification of other spines and basipterygia began. At 43 DAH (Figure 15(j)), ossification was completed.Figure 15
Anal fin development of EFT. (a) 11 DAH (4.43 ± 0.41 mm TL); (b) 14 DAH (5.04 ± 0.77 mm TL); (c) 17 DAH (5.61 ± 0.41 mm TL); (d) 20 DAH (6.45 ± 0.48 mm TL); (e) 23 DAH (6.53 ± 0.63 mm TL); (f) 26 DAH (8.34 ± 0.44 mm TL); (g) 29 DAH (8.40 ± 1.2 mm TL); (h) 32 DAH (10.87 ± 1.49 mm TL); (i) 38 DAH (19.18 ± 1.40 mm TL); (j) 43 DAH (27.21 ± 4.6 mm TL); (k) 48 DAH (31.09 ± 2.15 mm TL). Abbreviations are as follows: Dr, distal radial; R, lepidotrichium; S, hard spine; Prx, proximal radial; stay.InE. fuscoguttatus, at 17 DAH (Figure 16(b)), proximal radials developed. At 20 DAH (Figure 17(c)), the first and second fin spines, distal radials, anal fin spines, and stay cartilage were visible. At 29 DAH (Figure 16(g)), the first and second fin spines began to ossify. At 32 DAH (Figure 16(h)), other spines and pterygiophore began to ossify.Figure 16
Anal fin development ofE. fuscoguttatus. (a) 14 DAH (TL4.76 ± 0.28 mm); (b) 17 DAH (6.00 ± 0.47 mm TL); (c) 20 DAH (6.85 ± 0.59 mm TL); (d) 20 DAH (6.85 ± 0.59 mm TL); (e) 23 DAH (7.59 ± 0.46 mm TL); (f) 26 DAH (8.07 ± 1.08 mm TL); (g) 29 DAH (9.99 ± 2.46 mm TL); (h) 32 DAH (12.38 ± 1.53 mm TL); (i) 38 DAH (25.16 ± 2.19 mm TL); (j) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Dr, distal radial; R, lepidotrichium; S, hard spine; Prx, proximal radial; stay.Figure 17
Caudal fin development of EFT. (a) 8 DAH (3.97 ± 0.4 mm TL); (b) 11 DAH (4.43 ± 0.41 mm TL); (c) 14 DAH (5.04 ± 0.77 mm TL); (d) 17 DAH (5.61 ± 0.41 mm TL); (e) 20 DAH (6.45 ± 0.48 mm TL); (f) 23 DAH (6.53 ± 0.63 mm TL); (g) 26 DAH (8.34 ± 0.44 mm TL); (h) 29 DAH (8.40 ± 1.2 mm TL); (i) 32 DAH (10.87 ± 1.49 mm TL); (j) 38 DAH (19.18 ± 1.40 mm TL); (k) 43 DAH (27.21 ± 4.6 mm TL). Abbreviations are as follows: Ac, accessory; Ep, epural; Hy, hypural; PCR, caudal lepidotrichia; PrH, parhyural; SNA, specialzed neural; Ur, urostyle.


## 3.3.5. Development of the Caudal Fin

In EFT, at 8 DAH (Figure17(a)), hypural 1 formed. At 11 DAH (Figure 17(b)), hypural 2-3 and parhypural formed. At 17 DAH (Figure 17(d)), parhypural 1–3 formed and caudal fin rays were somewhat visible. At 26 DAH (Figure 17(g)), hypural 4 developed. At 29 DAH (Figure 17(h)), epural 3 and epural 4 fused into a single piece and the caudal fin ray and urostyle started to ossify. At 38 DAH (Figure 17(j)), urostyle osteosis was completed. At 48 DAH (Figure 17(k)), caudal fin ray ossification was nearly complete.InE. fuscoguttatus, at 8 DAH (Figure 18(a)), hypural 1-2 formation was observed. At 11–14 DAH (Figures 18(b) and 18(c)), hypural 3 and parhypural formed. At 17 DAH (Figure 18(d)), epural 1–3 developed. At 20 DAH (Figure 18(f)), epural 4-5 and hypural 4-5 formed; at the same time, caudal fin rays could be seen. At 29 DAH (Figure 18(i)), epural 4-5 fused into a single piece, and the urostyle and caudal fin rays began to ossify. At 38 DAH (Figure 18(k)), urostyle ossification was completed. By 48 DAH (Figure 18(l)), ossification of the caudal fin rays was still incomplete.Figure 18
Caudal fin development ofE. fuscoguttatus. (a) 8 DAH (3.61 ± 0.22 mm TL); (b) 11 DAH (4.21 ± 0.40 mm TL); (c) 14 DAH (4.76 ± 0.28 mm TL); (d) 17 DAH (6.00 ± 0.47 mm TL); (e) 17 DAH (6.00 ± 0.47 mm TL); (f) 20 DAH (6.85 ± 0.59 mm TL); (g) 23 DAH (7.59 ± 0.46 mm TL); (h) 26 DAH (8.07 ± 1.08 mm TL); (i) 29 DAH (9.99 ± 2.46 mm TL); (j) 32 DAH (12.38 ± 1.53 mm TL); (k) 38 DAH (25.16 ± 2.19 mm TL); (l) 48 DAH (37.69 ± 4.58 mm TL). Abbreviations are as follows: Ac, accessory; Ep, epural; Hy, hypural; PCR, caudal lepidotrichia; PrH, parhyural; SNA, specialzed neural; Ur, urostyle.


## 4. Discussion

### 4.1. Postembryonic Development of EFT andE. fuscoguttatus

Recently, to improve grouper germplasm and obtain groupers with economic traits, hybrid breeding has been widely used. Many hybrid groupers have been reported, such asE. lanceolatus (♂) × E. brumenus (♀) [10], E. lanceolatus (♂) × E. moara (♀) [11], E. moara (♀) × E. akaara (♂) [12], E. fuscoguttatus (♀) × E. lanceolatus (♂) [13], E. moara (♀) × E. septemfasciatus (♂) [14], and E. fuscoguttatus (♀) × E. polyphekadion (♂) [15]. The main hybrids used for production are E. lanceolatus (♂) × E. moara (♀) and E. fuscoguttatus (♀) × E. lanceolatus (♂). At 2–20 DAH, there was no difference in the total length of EFT and E. fuscoguttatus. At 23 DAH, E. fuscoguttatus larval survival was low. This low survival was not observed in EFT under the same breeding conditions, suggesting that the E. fuscoguttatus was affected by the disease, and surviving fry had strong disease resistance. Subsequent cannibalism resulted in the rapid growth of E. fuscoguttatus, resulting in a longer total length than that of EFT. However, at 3 months after hatching, the total length of EFT exceeded that of E. fuscoguttatus, and at 4 months after hatching, the total length of EFT was significantly longer than that of E. fuscoguttatus. The weight of EFT at this point was 32.7 ± 5.07 g, while that of E. fuscoguttatus was 29.7 ± 6.26 g. These results show that EFT grew faster than their female parents, and the mortality rate during growth was lower than that of E. fuscoguttatus.


### 4.2. Skeletal Development of EFT andE. fuscoguttatus

#### 4.2.1. Axial Skeletal Development

The skeletal development of fish is closely related to the functional needs of juveniles at different stages, and the prioritization of bone development is related to the survival needs of different periods [16, 17]. During the larval period, the larvae had oral fissures; at this stage, breathing and feeding abilities were crucial and the skeletal features associated with food ingestion and respiration showed preferential development. The cranium developed first in both E. fuscoguttatus and EFT, with similar rates of development, enabling larval respiration and feeding. The ossification of the maxilla, premaxilla, and preopercle occurred before that of other bones of the head. A similar pattern has been discovered in E. lanceolatus [18] and E. akaara [19].The vertebral column of a fish is the main support structure in the fish body, usually consisting of the vertebral centrum, neural arch, neural spine, hemal arch, hemal spine, parapophysis, and ribs. Vertebral ossification differs among fish. The development of the vertebral column proceeds from the head to tail inSebastiscus marmoratus [20], Larimichthys crocea [6], and Rachycentron canadum [21] and from the head to tail to middle portion of Alosa sapidissima [22]. Spine ossification in E. fuscoguttatus and EFT also occurred from the head to tail.


#### 4.2.2. Appendage Skeletal Development

The pectoral fins are the only fins that exist before larvae open their mouths to feed in various species, such asScyliorhinus canicula and Pagellus erythrinus [23]. In this study, the pectoral fin was indeed the first appendage to develop in E. fuscoguttatus and EFT, as observed in E. lanceolatus [24] and E. awoara (♀) × E. tukula (♂) [25]. Early development of the pectoral fin may enable simple swimming in the early larval period.During the development of groupers, the dorsal fin spines and ventral fin spines lengthen and contract substantially; this is a special feature of grouper development [26]. This feature is also often used to divide groupers into larval and juvenile stages [27, 28].InHexagrammos otakii [29] and Chaeturichthys stigmatia [30], the dorsal fin and pelvic fins develop after the pectoral and caudal fins. Dorsal and pelvic fins developed earlier than caudal fins in EFT and E. fuscoguttatus, similar to the developmental sequence in E. fuscoguttatus (♀) × E. lanceolatus (♂) [13] and E. lanceolatus [31]. During the larval stage, the elongation of the second dorsal fin spine and pelvic fin spine, usually in a natural open state, helps larvae to maintain body stability in the current. The second dorsal fin spine of EFT began to degenerate at 26 DAH, which is later than the corresponding time point in E. fuscoguttatus (20 DAH), enabling EFT to swim stably during the growth process, thereby improving survival. The dorsal fin spines of EFT shrink less than those of E. fuscoguttatus, further indicating that EFT can undergo a rapid metamorphosis.The anal fin functions in the maintenance of balance, preventing tilting and rocking, and coordinating swimming. The caudal fin is an important driving organ of the fish, providing power for the fish body to swim forward, and the caudal fin contributes to the swimming direction. The anal fin and caudal fin of EFT had shorter ossification times than those inE. fuscoguttatus, suggesting that EFT had an advantage with respect to swimming and enemy avoidance. There is a fusion of the hypural and epural during the development of the caudal fin. The hypural of Hexagrammos otakii [29] and Favonigobius gymnauchen [32] fuse. This fusion may promote a stronger tail thrust, thereby improving the ability to catch prey and avoid predators. In this study, both E. fuscoguttatus and EFT had epural fusion. After the fusion of the caudal bones in EFT, there were three epural bones, as observed in most groupers, while E. fuscoguttatus had four epural bones when the ossification of the caudal fin was completed. In addition, Salanx ariakensis [33] and Solea senegalensis [34] have one epural bone, Thunnus tonggol [35] has four epural bones, Siniperca chuatsi [36] has three epural bones, E. awoara [37] has three epural bones, and Takifugu flavidus [38] has no epural bone. At present, there are no reports on the function of the epural, and further research is needed to evaluate the number of epural bones and the functional significance of fusion.


## 4.1. Postembryonic Development of EFT andE. fuscoguttatus

Recently, to improve grouper germplasm and obtain groupers with economic traits, hybrid breeding has been widely used. Many hybrid groupers have been reported, such asE. lanceolatus (♂) × E. brumenus (♀) [10], E. lanceolatus (♂) × E. moara (♀) [11], E. moara (♀) × E. akaara (♂) [12], E. fuscoguttatus (♀) × E. lanceolatus (♂) [13], E. moara (♀) × E. septemfasciatus (♂) [14], and E. fuscoguttatus (♀) × E. polyphekadion (♂) [15]. The main hybrids used for production are E. lanceolatus (♂) × E. moara (♀) and E. fuscoguttatus (♀) × E. lanceolatus (♂). At 2–20 DAH, there was no difference in the total length of EFT and E. fuscoguttatus. At 23 DAH, E. fuscoguttatus larval survival was low. This low survival was not observed in EFT under the same breeding conditions, suggesting that the E. fuscoguttatus was affected by the disease, and surviving fry had strong disease resistance. Subsequent cannibalism resulted in the rapid growth of E. fuscoguttatus, resulting in a longer total length than that of EFT. However, at 3 months after hatching, the total length of EFT exceeded that of E. fuscoguttatus, and at 4 months after hatching, the total length of EFT was significantly longer than that of E. fuscoguttatus. The weight of EFT at this point was 32.7 ± 5.07 g, while that of E. fuscoguttatus was 29.7 ± 6.26 g. These results show that EFT grew faster than their female parents, and the mortality rate during growth was lower than that of E. fuscoguttatus.


## 4.2. Skeletal Development of EFT andE. fuscoguttatus

### 4.2.1. Axial Skeletal Development

The skeletal development of fish is closely related to the functional needs of juveniles at different stages, and the prioritization of bone development is related to the survival needs of different periods [16, 17]. During the larval period, the larvae had oral fissures; at this stage, breathing and feeding abilities were crucial and the skeletal features associated with food ingestion and respiration showed preferential development. The cranium developed first in both E. fuscoguttatus and EFT, with similar rates of development, enabling larval respiration and feeding. The ossification of the maxilla, premaxilla, and preopercle occurred before that of other bones of the head. A similar pattern has been discovered in E. lanceolatus [18] and E. akaara [19].The vertebral column of a fish is the main support structure in the fish body, usually consisting of the vertebral centrum, neural arch, neural spine, hemal arch, hemal spine, parapophysis, and ribs. Vertebral ossification differs among fish. The development of the vertebral column proceeds from the head to tail inSebastiscus marmoratus [20], Larimichthys crocea [6], and Rachycentron canadum [21] and from the head to tail to middle portion of Alosa sapidissima [22]. Spine ossification in E. fuscoguttatus and EFT also occurred from the head to tail.


### 4.2.2. Appendage Skeletal Development

The pectoral fins are the only fins that exist before larvae open their mouths to feed in various species, such asScyliorhinus canicula and Pagellus erythrinus [23]. In this study, the pectoral fin was indeed the first appendage to develop in E. fuscoguttatus and EFT, as observed in E. lanceolatus [24] and E. awoara (♀) × E. tukula (♂) [25]. Early development of the pectoral fin may enable simple swimming in the early larval period.During the development of groupers, the dorsal fin spines and ventral fin spines lengthen and contract substantially; this is a special feature of grouper development [26]. This feature is also often used to divide groupers into larval and juvenile stages [27, 28].InHexagrammos otakii [29] and Chaeturichthys stigmatia [30], the dorsal fin and pelvic fins develop after the pectoral and caudal fins. Dorsal and pelvic fins developed earlier than caudal fins in EFT and E. fuscoguttatus, similar to the developmental sequence in E. fuscoguttatus (♀) × E. lanceolatus (♂) [13] and E. lanceolatus [31]. During the larval stage, the elongation of the second dorsal fin spine and pelvic fin spine, usually in a natural open state, helps larvae to maintain body stability in the current. The second dorsal fin spine of EFT began to degenerate at 26 DAH, which is later than the corresponding time point in E. fuscoguttatus (20 DAH), enabling EFT to swim stably during the growth process, thereby improving survival. The dorsal fin spines of EFT shrink less than those of E. fuscoguttatus, further indicating that EFT can undergo a rapid metamorphosis.The anal fin functions in the maintenance of balance, preventing tilting and rocking, and coordinating swimming. The caudal fin is an important driving organ of the fish, providing power for the fish body to swim forward, and the caudal fin contributes to the swimming direction. The anal fin and caudal fin of EFT had shorter ossification times than those inE. fuscoguttatus, suggesting that EFT had an advantage with respect to swimming and enemy avoidance. There is a fusion of the hypural and epural during the development of the caudal fin. The hypural of Hexagrammos otakii [29] and Favonigobius gymnauchen [32] fuse. This fusion may promote a stronger tail thrust, thereby improving the ability to catch prey and avoid predators. In this study, both E. fuscoguttatus and EFT had epural fusion. After the fusion of the caudal bones in EFT, there were three epural bones, as observed in most groupers, while E. fuscoguttatus had four epural bones when the ossification of the caudal fin was completed. In addition, Salanx ariakensis [33] and Solea senegalensis [34] have one epural bone, Thunnus tonggol [35] has four epural bones, Siniperca chuatsi [36] has three epural bones, E. awoara [37] has three epural bones, and Takifugu flavidus [38] has no epural bone. At present, there are no reports on the function of the epural, and further research is needed to evaluate the number of epural bones and the functional significance of fusion.


## 4.2.1. Axial Skeletal Development

The skeletal development of fish is closely related to the functional needs of juveniles at different stages, and the prioritization of bone development is related to the survival needs of different periods [16, 17]. During the larval period, the larvae had oral fissures; at this stage, breathing and feeding abilities were crucial and the skeletal features associated with food ingestion and respiration showed preferential development. The cranium developed first in both E. fuscoguttatus and EFT, with similar rates of development, enabling larval respiration and feeding. The ossification of the maxilla, premaxilla, and preopercle occurred before that of other bones of the head. A similar pattern has been discovered in E. lanceolatus [18] and E. akaara [19].The vertebral column of a fish is the main support structure in the fish body, usually consisting of the vertebral centrum, neural arch, neural spine, hemal arch, hemal spine, parapophysis, and ribs. Vertebral ossification differs among fish. The development of the vertebral column proceeds from the head to tail inSebastiscus marmoratus [20], Larimichthys crocea [6], and Rachycentron canadum [21] and from the head to tail to middle portion of Alosa sapidissima [22]. Spine ossification in E. fuscoguttatus and EFT also occurred from the head to tail.


## 4.2.2. Appendage Skeletal Development

The pectoral fins are the only fins that exist before larvae open their mouths to feed in various species, such asScyliorhinus canicula and Pagellus erythrinus [23]. In this study, the pectoral fin was indeed the first appendage to develop in E. fuscoguttatus and EFT, as observed in E. lanceolatus [24] and E. awoara (♀) × E. tukula (♂) [25]. Early development of the pectoral fin may enable simple swimming in the early larval period.During the development of groupers, the dorsal fin spines and ventral fin spines lengthen and contract substantially; this is a special feature of grouper development [26]. This feature is also often used to divide groupers into larval and juvenile stages [27, 28].InHexagrammos otakii [29] and Chaeturichthys stigmatia [30], the dorsal fin and pelvic fins develop after the pectoral and caudal fins. Dorsal and pelvic fins developed earlier than caudal fins in EFT and E. fuscoguttatus, similar to the developmental sequence in E. fuscoguttatus (♀) × E. lanceolatus (♂) [13] and E. lanceolatus [31]. During the larval stage, the elongation of the second dorsal fin spine and pelvic fin spine, usually in a natural open state, helps larvae to maintain body stability in the current. The second dorsal fin spine of EFT began to degenerate at 26 DAH, which is later than the corresponding time point in E. fuscoguttatus (20 DAH), enabling EFT to swim stably during the growth process, thereby improving survival. The dorsal fin spines of EFT shrink less than those of E. fuscoguttatus, further indicating that EFT can undergo a rapid metamorphosis.The anal fin functions in the maintenance of balance, preventing tilting and rocking, and coordinating swimming. The caudal fin is an important driving organ of the fish, providing power for the fish body to swim forward, and the caudal fin contributes to the swimming direction. The anal fin and caudal fin of EFT had shorter ossification times than those inE. fuscoguttatus, suggesting that EFT had an advantage with respect to swimming and enemy avoidance. There is a fusion of the hypural and epural during the development of the caudal fin. The hypural of Hexagrammos otakii [29] and Favonigobius gymnauchen [32] fuse. This fusion may promote a stronger tail thrust, thereby improving the ability to catch prey and avoid predators. In this study, both E. fuscoguttatus and EFT had epural fusion. After the fusion of the caudal bones in EFT, there were three epural bones, as observed in most groupers, while E. fuscoguttatus had four epural bones when the ossification of the caudal fin was completed. In addition, Salanx ariakensis [33] and Solea senegalensis [34] have one epural bone, Thunnus tonggol [35] has four epural bones, Siniperca chuatsi [36] has three epural bones, E. awoara [37] has three epural bones, and Takifugu flavidus [38] has no epural bone. At present, there are no reports on the function of the epural, and further research is needed to evaluate the number of epural bones and the functional significance of fusion.


## 5. Conclusion

In this study, the postembryonic development and skeletal development ofE. fuscoguttatus and the hybrid EFT were observed, revealing that the development of EFT lagged behind that of E. fuscoguttatus slightly in the larval and juvenile stages, while the growth rate of EFT was faster than that of E. fuscoguttatus in the juvenile stage, indicating that EFT had a growth advantage. The order of skeletal development was similar in EFT and E. fuscoguttatus, indicating that skeletal development in EFT was normal. In addition, compared with that morphology of other groupers, E. fuscoguttatus had an extra epural bone; the molecular mechanism and significance of this extra bone remain to be explored.



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*Source: 5740050-2023-02-21.xml*