# Early Development of the Spinal Vertebrae and Appendicular Skeleton inMiichthys miiuy Larvae

Authors: Fengfang Zhou; Weiqing Huang; Xiujuan Shan; Lei Shi; Zheng Liu
Journal: Aquaculture Research
 (2023)

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## Abstract

We investigate the early developmental characteristics of the skeletal system inMiichthys miiuy larvae using a double staining method on the whole larval skeletons (aged 1–30 days). We systematically observed and described the morphological development of both the spinal vertebrae and appendicular skeleton. The results showed that there were 25 spinal vertebrae in M. miiuy, which consist of 12 abdominal vertebrae and 13 coccyges; The development of spinal vertebrae began at 12 days after hatching (dah), with the emergence of ossified neural and haemal arches, where ossification appeared at 17 dah; and the appendicular skeletons were developed from the pectoral fins to ventral fins. The coracoid-scapula cartilage was clearly observable at three dah, and fin rays were clearly differentiated at 21 dah. The cartilage of appendicular of the ventral fins was formed until 19 dah, and the ventral fins were completely ossified at 21 dah. Subsequently, the caudal, dorsal, and anal fins appeared at 10, 13, and 14 dah, respectively. Additionally, the appendicular skeleton associated with the movement of M.miiuy was completely developed by 28 dah. Finally we observed that there are four types of spinal vertebrae abnormalities during the early development of M.miiuy, which included spinal bulging, splitting of vertebrae, and supernumerary and bifurcated haemal and neural spines. Skeletal length and age (in days) were highly correlated (r2 = 0.942). There was a strong relationship between total length and skeletal length (r2 = 0.979) and between body length and skeletal length (r2 = 0.9779).


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## Body

## 1. Introduction

In fish, the skeleton plays a pivotal role in interior protection, coordinating movement, respiration, feeding, systematic evolution and classification [1]. Skeletal development is affected by genetic, environmental, and nutritional factors [2]. Further, skeletal deformities seriously affect fish morphology, slowing the growth and lowering the market value of the stock [3]. Many studies have been conducted on the developmental characteristics of fish skeletons. For example, the growth and development of the spinal joints in Larimichthys crocea was analysed using the double staining method [4]. In another study, cartilage bone clearing and staining technique were used to describe the skeletal developmental characteristics and skeletal malformations of cobia larvae in detail [5]. At present, fish osteology mainly includes the identification and occurrence of bone deformities, and the effects of genetic, environmental, and nutritional factors on fish deformity and mortality.Miichthys miiuy (Perciformes, Sciaenidae, Miichthys), is a commercially important offshore warm-water demersal species [6]. The species is mainly distributed in the Zhejiang, Taiwan, Fujian, Shandong, and Jiangsu provinces in China, with the Zhoushan Islands in the Zhejiang province being the largest fish-producing area [7]. With high nutritional and economic value and rapid growth, M. miiuy has become an important stock for capture fisheries and aquaculture. Experimental studies on artificial reproduction and breeding of M. miiuy have been conducted in coastal areas such as Fujian and Zhejiang provinces since the 1990s [8]. At present, research on M.miiuy is mainly focused on improving aquaculture techniques [9], muscle nutritional composition [10], gene function and regulation [11] and processing technology [12, 13].The skeletal development ofM.miiuy has been studied in terms of basic morphology and morphometrics. Here, we study the growth and development patterns in further detail, as well as ecological significance of these patterns, by observing and quantifying the early ontogenesis of both larval and juvenile M. miiuy (aged 1–53 days) [14]. Morphological characteristics of the early skeleton development (including hyoid arch, branchial arch, and opercular apparatus) in relation to their feeding habit were observed in M. miiuy larvae using standard clearing and staining methods [15]. However, the early development of the spinal vertebrae and appendicular skeleton in M. miiuy larvae has not yet been explored.With the continuous development and expansion of theM. miiuy aquaculture industry, the exploration of skeletal developmental are essential for the evidence-based guidance in large-scale aquaculture, particularly for improving breeding and increasing production. The early developmental characteristics of spinal vertebrae and appendicular skeleton in the larval and juvenile M. miiuy, as well as the detailed observations and descriptions of some abnormal phenomena during the development process of the vertebrae, were quantified in this study. We aim to provide further understanding of the developmental biology of the species, which will have useful applications for the commercial breeding of high quality M. miiuy stock.


## 2. Materials and Methods

### 2.1. Ethics Statement

All procedures complied with ethic committee of Ningde Normal University regarding animal experimentation.


### 2.2. Sample Collection

Larvae ofM. miiuy in this study were provided by Ning De Ding Cheng Aquatic Product Company Ltd. (Ningde, China). The M. miiuy sampling and experiments were conducted in accordance with the ethical requirements and followed the guidelines for the Animal Ethics Committee of Ningde Normal University. Larvae were reared in 40 m3 tanks with water up to a depth of 1 m. Rearing density was c. 125 larvae l−1. During the experimental period, oxygen, salinity, and pH were 7.0–8.5 mg l−1, 22–25, and 6.5–7.0, respectively, and were monitored daily. Larvae were fed rotifers (Brachionus plicatilis). Next, 10 larvae aged 1–30 days were randomly sampled at each day posthatching. They were placed in 10% neutral buffered formalin for at least 24 h to ensure complete fixation of samples.


### 2.3. Larval and Juvenile Skeleton Specimens

The cartilage bone clearing and staining technique was used as described by Huang et al. [4], after some modifications. The specific steps used are as follows:


#### 2.3.1. Fixation and Cleaning

The samples were continually soaked in 10% formalin for three days and then in distilled water for one day.


#### 2.3.2. Graded Dehydration

Samples were dehydrated with graded ethanol (20% ⟶ 40% ⟶ 60% ⟶ 75%) for 12–24 h of each gradient. The dehydration time depended on the sample size. Subsequently, the samples were stored at 4°C and stained in 95% alcohol for another 24 h before soaking.


#### 2.3.3. Cartilage Staining

The cartilage staining solution was prepared by adding 20 ml of glacial acetic acid, 80 ml of 95% alcohol, and 10 mg of Alisin Blue Stain. The samples were stained in this solution for approximately 5 h until the body surface and fin base of the fish was clearly blue.


#### 2.3.4. Neutralization

The samples were soaked in saturated sodium borate solution for 1 day.


#### 2.3.5. Decolouration

The samples were soaked in the mixture of 20 ml of 3% H2O2 solution and 80 ml of 1% KOH solution for one day to decolorize.


#### 2.3.6. Bone Staining

The bone staining solution was prepared by adding 5 mg of alizarin red and 100 ml of 1% KOH. The samples were stained in this solution for 5–9 h, depending on the sample volume.


#### 2.3.7. Sample Transparency

The sample was placed in 1% KOH solution for transparency until the surface was unpigmented and the bones could be clearly observed.


#### 2.3.8. Storage

The samples were transferred sequentially into a mixture of glycerol and 1% KOH in a ratio of 1 : 3, 1 : 1, and 3 : 1 to elute excess stain for one day each. The samples were stored in pure glycerine when the fish became transparent.


### 2.4. Larval and Juvenile Observation of Transparent Specimens

The developmental process and morphological characteristics of the spinal vertebrae and appendicular skeleton were observed using a Leica S9i Digital Stereo Microscope (Leica S9i, Germany), photographed, and recorded. We used the naming conventions for each skeleton as in Deng et al. [16]; and Mao [5]. We named the different vertebrae deformation types according to Gai et al. [13].


### 2.5. Data Processing

The total length, body length, and skeletal length of larvae and juveniles were measured (the total length: the length from snout to the end of caudal fin; the body length: the length from snout to the front of caudal fin; the skeletal length: the length from the the first abdominal vertebra to the coccyx). Correlations were calculated using the Microsoft Excel software (2010), and recorded photographs were processed using Adobe Photoshop CC (2017). Based on the observation and analysis of those photos, the developmental characteristics of spinal vertebrae and appendicular skeleton, as well as spinal vertebrae abnormalities, were summarized.


## 2.1. Ethics Statement

All procedures complied with ethic committee of Ningde Normal University regarding animal experimentation.


## 2.2. Sample Collection

Larvae ofM. miiuy in this study were provided by Ning De Ding Cheng Aquatic Product Company Ltd. (Ningde, China). The M. miiuy sampling and experiments were conducted in accordance with the ethical requirements and followed the guidelines for the Animal Ethics Committee of Ningde Normal University. Larvae were reared in 40 m3 tanks with water up to a depth of 1 m. Rearing density was c. 125 larvae l−1. During the experimental period, oxygen, salinity, and pH were 7.0–8.5 mg l−1, 22–25, and 6.5–7.0, respectively, and were monitored daily. Larvae were fed rotifers (Brachionus plicatilis). Next, 10 larvae aged 1–30 days were randomly sampled at each day posthatching. They were placed in 10% neutral buffered formalin for at least 24 h to ensure complete fixation of samples.


## 2.3. Larval and Juvenile Skeleton Specimens

The cartilage bone clearing and staining technique was used as described by Huang et al. [4], after some modifications. The specific steps used are as follows:


### 2.3.1. Fixation and Cleaning

The samples were continually soaked in 10% formalin for three days and then in distilled water for one day.


### 2.3.2. Graded Dehydration

Samples were dehydrated with graded ethanol (20% ⟶ 40% ⟶ 60% ⟶ 75%) for 12–24 h of each gradient. The dehydration time depended on the sample size. Subsequently, the samples were stored at 4°C and stained in 95% alcohol for another 24 h before soaking.


### 2.3.3. Cartilage Staining

The cartilage staining solution was prepared by adding 20 ml of glacial acetic acid, 80 ml of 95% alcohol, and 10 mg of Alisin Blue Stain. The samples were stained in this solution for approximately 5 h until the body surface and fin base of the fish was clearly blue.


### 2.3.4. Neutralization

The samples were soaked in saturated sodium borate solution for 1 day.


### 2.3.5. Decolouration

The samples were soaked in the mixture of 20 ml of 3% H2O2 solution and 80 ml of 1% KOH solution for one day to decolorize.


### 2.3.6. Bone Staining

The bone staining solution was prepared by adding 5 mg of alizarin red and 100 ml of 1% KOH. The samples were stained in this solution for 5–9 h, depending on the sample volume.


### 2.3.7. Sample Transparency

The sample was placed in 1% KOH solution for transparency until the surface was unpigmented and the bones could be clearly observed.


### 2.3.8. Storage

The samples were transferred sequentially into a mixture of glycerol and 1% KOH in a ratio of 1 : 3, 1 : 1, and 3 : 1 to elute excess stain for one day each. The samples were stored in pure glycerine when the fish became transparent.


## 2.3.1. Fixation and Cleaning

The samples were continually soaked in 10% formalin for three days and then in distilled water for one day.


## 2.3.2. Graded Dehydration

Samples were dehydrated with graded ethanol (20% ⟶ 40% ⟶ 60% ⟶ 75%) for 12–24 h of each gradient. The dehydration time depended on the sample size. Subsequently, the samples were stored at 4°C and stained in 95% alcohol for another 24 h before soaking.


## 2.3.3. Cartilage Staining

The cartilage staining solution was prepared by adding 20 ml of glacial acetic acid, 80 ml of 95% alcohol, and 10 mg of Alisin Blue Stain. The samples were stained in this solution for approximately 5 h until the body surface and fin base of the fish was clearly blue.


## 2.3.4. Neutralization

The samples were soaked in saturated sodium borate solution for 1 day.


## 2.3.5. Decolouration

The samples were soaked in the mixture of 20 ml of 3% H2O2 solution and 80 ml of 1% KOH solution for one day to decolorize.


## 2.3.6. Bone Staining

The bone staining solution was prepared by adding 5 mg of alizarin red and 100 ml of 1% KOH. The samples were stained in this solution for 5–9 h, depending on the sample volume.


## 2.3.7. Sample Transparency

The sample was placed in 1% KOH solution for transparency until the surface was unpigmented and the bones could be clearly observed.


## 2.3.8. Storage

The samples were transferred sequentially into a mixture of glycerol and 1% KOH in a ratio of 1 : 3, 1 : 1, and 3 : 1 to elute excess stain for one day each. The samples were stored in pure glycerine when the fish became transparent.


## 2.4. Larval and Juvenile Observation of Transparent Specimens

The developmental process and morphological characteristics of the spinal vertebrae and appendicular skeleton were observed using a Leica S9i Digital Stereo Microscope (Leica S9i, Germany), photographed, and recorded. We used the naming conventions for each skeleton as in Deng et al. [16]; and Mao [5]. We named the different vertebrae deformation types according to Gai et al. [13].


## 2.5. Data Processing

The total length, body length, and skeletal length of larvae and juveniles were measured (the total length: the length from snout to the end of caudal fin; the body length: the length from snout to the front of caudal fin; the skeletal length: the length from the the first abdominal vertebra to the coccyx). Correlations were calculated using the Microsoft Excel software (2010), and recorded photographs were processed using Adobe Photoshop CC (2017). Based on the observation and analysis of those photos, the developmental characteristics of spinal vertebrae and appendicular skeleton, as well as spinal vertebrae abnormalities, were summarized.


## 3. Results

### 3.1. Developmental Characteristics of Spinal Vertebrae

The spinal vertebrae of larvalM. miiuy were clearly visible and the ossification appeared at 17 days, but the development of the spinal vertebrae was completed at 28 days after hatching (dah). The developmental characteristics and spinal joints of spinal vertebrae were observed and counted from 17 to 30 dah. There were 25 spinal vertebrae in M. miiuy, which consist of 12 abdominal vertebrae and 13 coccyges.Ossification did not appear in the skeleton of larvalM. miiuy at one dah, with a straight notochord and no appendicular skeleton. An uneven notochord was observed at five dah, and the neural arch appeared ahead of the notochord (Figure 1(a)). At 12 dah, the neural arch cartilage on the dorsal spine and haemal arch cartilage on the ventral spine were clearly visible, and a pair of those cartilage arches formed a neural arch, which was located on both sides of the dorsal spine. The caudal part of neural arch was fused, and the vertebral cartilage also became abjoint at this time. With the development of the skeleton, the neural arch and haemal arch were extended to neural and haemal spines, and the vertebral column completely ossified from anterior to posterior (Figure 1(b)). At 17 dah, all joints in spinal vertebrae of larval M. miiuy were stained with alizarin red, which indicates that ossification was complete. Additionally, the biconcave joint combined to form the vertebrae and protect the notochord. We clearly observed 25 nodes, neural and haemal spines (Figure 1(c)). By 28 dah, the development of vertebrae in juvenile M. miiuy was completed, and no significant difference was observed in the biological morphology of vertebrae of juvenile M. miiuy of 28 dah compared with that of vertebrae of juvenile M. miiuy of 29 and 30 dah (Figure 1(d)).Figure 1
Vertebral column development timing and progression of larval and juvenileMiichthys miiuy. (a) 5 dah; (b) 12 dah; (c) 17 dah; (d) 28 dah. Nc: notochord; Na: neural arch; Ha: haemal arch; Ns: neural spine; Hs: haemal spine; V; vertebra. Bars = 1 mm.
(a)(b)(c)(d)


### 3.2. Developmental Characteristics of Appendicular Skeleton

#### 3.2.1. Developmental Characteristics of Paired Fins

The pectoral fins ofM. miiuy were formed before the ventral fins. At three dah, the coracoid-scapula cartilages were clearly visible in larvae (Figure 2(a)). At 12 dah, the pectoral fin plate of larvae was clear with a fissure in the middle, and the cleithrum and supracleithrum were visible (Figure 2(b)). As the larvae grew and developed, the fin fold gradually widened. By the 21 dah, the fissures on the fin plate were increased to three, which were fan-shaped, and dozens of fin rays were clearly differentiated radially (Figure 2(c)). At 28 dah, the pectoral fin bone was completely developed, 21 fin rays in juveniles, and no significant morphological changes were observed between the juveniles at 28 and 27 dah (Figure 2(d)).Figure 2
Pectoral fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 3 dah; (b) 12 dah; (c) 21 dah; (d) 28 dah. Cl: cleithrum; CS: coracoid-scapula cartilage; Fp: fin plate; Fr: fin ray; Scl: supracleithrum; SF. scapula foramen. Bars = 1 mm.
(a)(b)(c)(d)The development of ventral fins began with the appearance of cartilages of fin plate at 19 dah (Figure3(a)). Five ventral fin rays were clearly visible in larvae at 21 dah when ossification also occurred in fin spines and rays (Figure 3(b)). Next, the fin spines and rays gradually ossified from the base to the distal end. Twenty-eight dah, the ossification was complete, with clear pelvic bones and fin rays (Figure 3(c)).Figure 3
Ventral fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 19 dah; (b) 21 dah; (c) 28 dah. Bars = 1 mm.
(a)(b)(c)


#### 3.2.2. Developmental Characteristics of the Median Fin

The median fins ofM. miiuy is formed in the order of caudal, dorsal and anal fins. Three hypural cartilaginous spines were formed at the ventral posterior notochord (Figure 4(a)). The hypural caudal fins differentiated further and increased to five at 14 dah, four of which were wide and flat. The last one was the smallest and rod-like with a tapered bottom. The gap between the second and third hypural fins was slighter larger than that between others. Moreover, there were 18 radial primitive fin rays, which indicated that the caudal fins tended to mature. The last caudal vertebra curved upward to form the urostyle, and three rod-like epural cartilaginous spines were clearly visible between the urostyle and medullary spine at third from bottom (Figure 4(b)). At 17 dah, the caudal fin rays were abjoint obviously and ossified in the direction from base to the distal end (Figure 4(c)). At 28 dah, the caudal fins were fully developed (Figure 4(d)).Figure 4
Caudal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 10 dah; (b) 14 dah; (c) 17 dah; (d) 28 dah. Hy: hypural; Ep: epural; Ur: urostyle; Urn: Uroneural. Bars = 1 mm.
(a)(b)(c)(d)The fin plate was formed from the middle part of the dorsal fin spine at 13 dah, and the numbers of dorsal fin plates were >20 by 14 dah (Figure5(a)). At 17 dah, the radial primitive fin rays appeared (Figure 5(b)). The basipterygium was nearly fully developed, and the fin rays were ossified from the base to the distal end (Figure 5(c)). By 28 dah, the ossification was completed, indicating that the dorsal fin was fully developed (Figure 5(d)).Figure 5
Dorsal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 14 dah; (b) 17 dah; (c) 21 dah; (d) 28 dah. Bars = 1 mm.
(a)(b)(c)(d)The fin plate and fin ray appeared at 14 dah (Figure6(a)) and 17 dah (Figure 6(b)), respectively. Ossification began from base to the distal end with the development of fins. By 28 dah, ossification was complete and identical to that of adult individuals (Figure 6(c)).Figure 6
Anal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 14 dah; (b) 17 dah; (c) 28 dah. Bars = 1 mm.
(a)(b)(c)


### 3.3. Spinal Vertebrae Abnormalities

Four types of deformities were observed: spinal bulging, splitting of vertebrae, and supernumerary and bifurcated neural spines in vertebrae. Abnormalities primarily occurred at 13–28 dah.The redundancy of spines was observed in the middle of spinal vertebrae both at 13 and 21 dah, with two spines appearing at adjacent or similar nodes (Figure7(a)). Lordosis was observed at 18, 19, and 20 dah (Figure 7(b)), while kyphosis was observed at 21 dah (Figure 7(c)). Lordosis was featured with upward curvature, while kyphosis was featured with downward curvature when observed laterally. Vertebral deformity was found in the 16th node at 21 dah and in the 7th and 9th nodes at 26 dah. Notably, some bones of the vertebrae were split but not completely separated (Figure 7(c)). The bifurcated neural spines were observed in 4th node at 21, 24, and 25 dah at the middle of the spine. The neural spine was divided into two parts from middle and upper part, with a dendritic bifurcation at the end (Figure 7(d)).Figure 7
Vertebral column deformities of larval and juvenileMiichthys miiuy. (a) 13 dah; (b) 19 dah; (c) 21 dah; (d) 24 dah. SNS: supernumerary neural spines; BNS: bifurcated neural spines; LO: lordosis; KY: kyphosis; VD: vertebra deformity. Bars = 1 mm.
(a)(b)(c)(d)


### 3.4. Skeletal Growth of Larvae and Juveniles

We found that the relationship between corresponding day-age (x1) and skeletal length (y) was y = 0.0032x12 + 0.0971x1 + 1.1353 (r2 = 0.942). The skeletal length of larvae at 1 dah was 1.63 ± 0.02 mm and reached 7.18 ± 0.52 mm after 29 days, with an average growth rate of 0.191 mm/d (Figure 8). The relationship between total length (x2) and skeletal length (y) was y = −0.0049x22 + 0.5361x2 + 0.2444 (r2 = 0.979; Figure 9). Additionally, the relationship between body length (x3) and skeletal length (y) was y = −0.002x32 − 0.6506x3 + 0.1465 (r2 = 0.978; Figure 10).Figure 8
Relationship between skeleton length and day-age of the juvenileMiichthys miiuy.Figure 9
Relationship between skeleton length and full length of the juvenileMiichthys miiuy.Figure 10
Relationship between skeleton length and body length of juvenileMiichthys miiuy.


## 3.1. Developmental Characteristics of Spinal Vertebrae

The spinal vertebrae of larvalM. miiuy were clearly visible and the ossification appeared at 17 days, but the development of the spinal vertebrae was completed at 28 days after hatching (dah). The developmental characteristics and spinal joints of spinal vertebrae were observed and counted from 17 to 30 dah. There were 25 spinal vertebrae in M. miiuy, which consist of 12 abdominal vertebrae and 13 coccyges.Ossification did not appear in the skeleton of larvalM. miiuy at one dah, with a straight notochord and no appendicular skeleton. An uneven notochord was observed at five dah, and the neural arch appeared ahead of the notochord (Figure 1(a)). At 12 dah, the neural arch cartilage on the dorsal spine and haemal arch cartilage on the ventral spine were clearly visible, and a pair of those cartilage arches formed a neural arch, which was located on both sides of the dorsal spine. The caudal part of neural arch was fused, and the vertebral cartilage also became abjoint at this time. With the development of the skeleton, the neural arch and haemal arch were extended to neural and haemal spines, and the vertebral column completely ossified from anterior to posterior (Figure 1(b)). At 17 dah, all joints in spinal vertebrae of larval M. miiuy were stained with alizarin red, which indicates that ossification was complete. Additionally, the biconcave joint combined to form the vertebrae and protect the notochord. We clearly observed 25 nodes, neural and haemal spines (Figure 1(c)). By 28 dah, the development of vertebrae in juvenile M. miiuy was completed, and no significant difference was observed in the biological morphology of vertebrae of juvenile M. miiuy of 28 dah compared with that of vertebrae of juvenile M. miiuy of 29 and 30 dah (Figure 1(d)).Figure 1
Vertebral column development timing and progression of larval and juvenileMiichthys miiuy. (a) 5 dah; (b) 12 dah; (c) 17 dah; (d) 28 dah. Nc: notochord; Na: neural arch; Ha: haemal arch; Ns: neural spine; Hs: haemal spine; V; vertebra. Bars = 1 mm.
(a)(b)(c)(d)


## 3.2. Developmental Characteristics of Appendicular Skeleton

### 3.2.1. Developmental Characteristics of Paired Fins

The pectoral fins ofM. miiuy were formed before the ventral fins. At three dah, the coracoid-scapula cartilages were clearly visible in larvae (Figure 2(a)). At 12 dah, the pectoral fin plate of larvae was clear with a fissure in the middle, and the cleithrum and supracleithrum were visible (Figure 2(b)). As the larvae grew and developed, the fin fold gradually widened. By the 21 dah, the fissures on the fin plate were increased to three, which were fan-shaped, and dozens of fin rays were clearly differentiated radially (Figure 2(c)). At 28 dah, the pectoral fin bone was completely developed, 21 fin rays in juveniles, and no significant morphological changes were observed between the juveniles at 28 and 27 dah (Figure 2(d)).Figure 2
Pectoral fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 3 dah; (b) 12 dah; (c) 21 dah; (d) 28 dah. Cl: cleithrum; CS: coracoid-scapula cartilage; Fp: fin plate; Fr: fin ray; Scl: supracleithrum; SF. scapula foramen. Bars = 1 mm.
(a)(b)(c)(d)The development of ventral fins began with the appearance of cartilages of fin plate at 19 dah (Figure3(a)). Five ventral fin rays were clearly visible in larvae at 21 dah when ossification also occurred in fin spines and rays (Figure 3(b)). Next, the fin spines and rays gradually ossified from the base to the distal end. Twenty-eight dah, the ossification was complete, with clear pelvic bones and fin rays (Figure 3(c)).Figure 3
Ventral fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 19 dah; (b) 21 dah; (c) 28 dah. Bars = 1 mm.
(a)(b)(c)


### 3.2.2. Developmental Characteristics of the Median Fin

The median fins ofM. miiuy is formed in the order of caudal, dorsal and anal fins. Three hypural cartilaginous spines were formed at the ventral posterior notochord (Figure 4(a)). The hypural caudal fins differentiated further and increased to five at 14 dah, four of which were wide and flat. The last one was the smallest and rod-like with a tapered bottom. The gap between the second and third hypural fins was slighter larger than that between others. Moreover, there were 18 radial primitive fin rays, which indicated that the caudal fins tended to mature. The last caudal vertebra curved upward to form the urostyle, and three rod-like epural cartilaginous spines were clearly visible between the urostyle and medullary spine at third from bottom (Figure 4(b)). At 17 dah, the caudal fin rays were abjoint obviously and ossified in the direction from base to the distal end (Figure 4(c)). At 28 dah, the caudal fins were fully developed (Figure 4(d)).Figure 4
Caudal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 10 dah; (b) 14 dah; (c) 17 dah; (d) 28 dah. Hy: hypural; Ep: epural; Ur: urostyle; Urn: Uroneural. Bars = 1 mm.
(a)(b)(c)(d)The fin plate was formed from the middle part of the dorsal fin spine at 13 dah, and the numbers of dorsal fin plates were >20 by 14 dah (Figure5(a)). At 17 dah, the radial primitive fin rays appeared (Figure 5(b)). The basipterygium was nearly fully developed, and the fin rays were ossified from the base to the distal end (Figure 5(c)). By 28 dah, the ossification was completed, indicating that the dorsal fin was fully developed (Figure 5(d)).Figure 5
Dorsal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 14 dah; (b) 17 dah; (c) 21 dah; (d) 28 dah. Bars = 1 mm.
(a)(b)(c)(d)The fin plate and fin ray appeared at 14 dah (Figure6(a)) and 17 dah (Figure 6(b)), respectively. Ossification began from base to the distal end with the development of fins. By 28 dah, ossification was complete and identical to that of adult individuals (Figure 6(c)).Figure 6
Anal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 14 dah; (b) 17 dah; (c) 28 dah. Bars = 1 mm.
(a)(b)(c)


## 3.2.1. Developmental Characteristics of Paired Fins

The pectoral fins ofM. miiuy were formed before the ventral fins. At three dah, the coracoid-scapula cartilages were clearly visible in larvae (Figure 2(a)). At 12 dah, the pectoral fin plate of larvae was clear with a fissure in the middle, and the cleithrum and supracleithrum were visible (Figure 2(b)). As the larvae grew and developed, the fin fold gradually widened. By the 21 dah, the fissures on the fin plate were increased to three, which were fan-shaped, and dozens of fin rays were clearly differentiated radially (Figure 2(c)). At 28 dah, the pectoral fin bone was completely developed, 21 fin rays in juveniles, and no significant morphological changes were observed between the juveniles at 28 and 27 dah (Figure 2(d)).Figure 2
Pectoral fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 3 dah; (b) 12 dah; (c) 21 dah; (d) 28 dah. Cl: cleithrum; CS: coracoid-scapula cartilage; Fp: fin plate; Fr: fin ray; Scl: supracleithrum; SF. scapula foramen. Bars = 1 mm.
(a)(b)(c)(d)The development of ventral fins began with the appearance of cartilages of fin plate at 19 dah (Figure3(a)). Five ventral fin rays were clearly visible in larvae at 21 dah when ossification also occurred in fin spines and rays (Figure 3(b)). Next, the fin spines and rays gradually ossified from the base to the distal end. Twenty-eight dah, the ossification was complete, with clear pelvic bones and fin rays (Figure 3(c)).Figure 3
Ventral fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 19 dah; (b) 21 dah; (c) 28 dah. Bars = 1 mm.
(a)(b)(c)


## 3.2.2. Developmental Characteristics of the Median Fin

The median fins ofM. miiuy is formed in the order of caudal, dorsal and anal fins. Three hypural cartilaginous spines were formed at the ventral posterior notochord (Figure 4(a)). The hypural caudal fins differentiated further and increased to five at 14 dah, four of which were wide and flat. The last one was the smallest and rod-like with a tapered bottom. The gap between the second and third hypural fins was slighter larger than that between others. Moreover, there were 18 radial primitive fin rays, which indicated that the caudal fins tended to mature. The last caudal vertebra curved upward to form the urostyle, and three rod-like epural cartilaginous spines were clearly visible between the urostyle and medullary spine at third from bottom (Figure 4(b)). At 17 dah, the caudal fin rays were abjoint obviously and ossified in the direction from base to the distal end (Figure 4(c)). At 28 dah, the caudal fins were fully developed (Figure 4(d)).Figure 4
Caudal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 10 dah; (b) 14 dah; (c) 17 dah; (d) 28 dah. Hy: hypural; Ep: epural; Ur: urostyle; Urn: Uroneural. Bars = 1 mm.
(a)(b)(c)(d)The fin plate was formed from the middle part of the dorsal fin spine at 13 dah, and the numbers of dorsal fin plates were >20 by 14 dah (Figure5(a)). At 17 dah, the radial primitive fin rays appeared (Figure 5(b)). The basipterygium was nearly fully developed, and the fin rays were ossified from the base to the distal end (Figure 5(c)). By 28 dah, the ossification was completed, indicating that the dorsal fin was fully developed (Figure 5(d)).Figure 5
Dorsal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 14 dah; (b) 17 dah; (c) 21 dah; (d) 28 dah. Bars = 1 mm.
(a)(b)(c)(d)The fin plate and fin ray appeared at 14 dah (Figure6(a)) and 17 dah (Figure 6(b)), respectively. Ossification began from base to the distal end with the development of fins. By 28 dah, ossification was complete and identical to that of adult individuals (Figure 6(c)).Figure 6
Anal fin development timing and progression of larval and juvenileMiichthys miiuy. (a) 14 dah; (b) 17 dah; (c) 28 dah. Bars = 1 mm.
(a)(b)(c)


## 3.3. Spinal Vertebrae Abnormalities

Four types of deformities were observed: spinal bulging, splitting of vertebrae, and supernumerary and bifurcated neural spines in vertebrae. Abnormalities primarily occurred at 13–28 dah.The redundancy of spines was observed in the middle of spinal vertebrae both at 13 and 21 dah, with two spines appearing at adjacent or similar nodes (Figure7(a)). Lordosis was observed at 18, 19, and 20 dah (Figure 7(b)), while kyphosis was observed at 21 dah (Figure 7(c)). Lordosis was featured with upward curvature, while kyphosis was featured with downward curvature when observed laterally. Vertebral deformity was found in the 16th node at 21 dah and in the 7th and 9th nodes at 26 dah. Notably, some bones of the vertebrae were split but not completely separated (Figure 7(c)). The bifurcated neural spines were observed in 4th node at 21, 24, and 25 dah at the middle of the spine. The neural spine was divided into two parts from middle and upper part, with a dendritic bifurcation at the end (Figure 7(d)).Figure 7
Vertebral column deformities of larval and juvenileMiichthys miiuy. (a) 13 dah; (b) 19 dah; (c) 21 dah; (d) 24 dah. SNS: supernumerary neural spines; BNS: bifurcated neural spines; LO: lordosis; KY: kyphosis; VD: vertebra deformity. Bars = 1 mm.
(a)(b)(c)(d)


## 3.4. Skeletal Growth of Larvae and Juveniles

We found that the relationship between corresponding day-age (x1) and skeletal length (y) was y = 0.0032x12 + 0.0971x1 + 1.1353 (r2 = 0.942). The skeletal length of larvae at 1 dah was 1.63 ± 0.02 mm and reached 7.18 ± 0.52 mm after 29 days, with an average growth rate of 0.191 mm/d (Figure 8). The relationship between total length (x2) and skeletal length (y) was y = −0.0049x22 + 0.5361x2 + 0.2444 (r2 = 0.979; Figure 9). Additionally, the relationship between body length (x3) and skeletal length (y) was y = −0.002x32 − 0.6506x3 + 0.1465 (r2 = 0.978; Figure 10).Figure 8
Relationship between skeleton length and day-age of the juvenileMiichthys miiuy.Figure 9
Relationship between skeleton length and full length of the juvenileMiichthys miiuy.Figure 10
Relationship between skeleton length and body length of juvenileMiichthys miiuy.


## 4. Discussion

### 4.1. Analysis of Developmental Characteristics on Spinal Vertebrae and Appendicular Skeleton

To date, many studies on skeletal developmental characteristics of larval and juvenile fish have been reported. For instance, the developmental pattern of spinal vertebrae, pectoral fin, and caudal fin of the large yellow croaker (Larimichthys crocea) have been studied using the double staining method [17]. The results showed that the larval notochord appeared segment at 6 dah, and the neural and haemal archs developed sequentially. The vertebral cartilage was formed at 14 dah and ossified completely at 28 dah from the base to the distal end. The pectoral fin was the earliest to develop among the appendicular skeleton. The supracleithrum, cleithrum, and postcleithrum appeared at 3 dah, and proximal pterygrophores and fin rays developed completely at 27 dah. Further, the ossification was complete at 28 dah. The results of our study are consistent with those of Wang et al. [17]; likely because L. crocea and M. miiuy both belong to Sciaenidae and have a similar developmental sequence.In a previous study, the early morphological development ofM. miiuy was observed and analysed [18]. The results showed that the median fins were formed in the order of caudal, anal, and dorsal fins, and the appendicular skeleton was fully developed by 17 dah. All fins were completely differentiated at 28 dah, which also indicated the transformation of M. miiuy from larval to juvenile stage. The main reasons for these differences may be attributed to the differences in water temperature, light intensity, fish bait abundance, and feeding method during the breeding period, which may lead to a different developmental sequence in the different parts of larval and juvenile M. miiuy. Moreover, the difference in the developmental stages may be due to the use of different classifications.The development of spinal vertebrae varies among different fish species. In our study, there were 25 spinal vertebrae inM. miiuy, which consist of 12 abdominal vertebrae and 13 coccyges. While Lates calcarifer have 24 spinal vertebrae during the developmental process of larvae and juveniles aged 0–23 days [19], euthynnus affinis have 20 trunk vertebrae and 18 tail vertebrae [20].The development of the vertebral column and appendicular skeleton of the larvae and juveniles of Chaeturichthys stigmatias was also described [21]. The results revealed 40–43 spinal vertebrae, which consisted of 15 trunk vertebrae and 25–28 tail vertebrae. The number of spinal vertebrae had an important influence on biological characteristics like size, length, and height [22]. This data can provide a theoretical basis for the study of fish traits and biological classification through the analysis of developmental characteristics of fish vertebrae [23].


### 4.2. Reasons of Vertebral Column Deformities

The skeletal deformities can not only affect the movement, and external characteristics of fish but also their economic value [24]. The research development on skeletal deformities of larval and juvenile fish in recent years have been summarized by a recent review [25]. The results showed that skeletal deformities may cause poor food intake, poor growth, weak physical ability, and low survival, which may ultimately lead to large economic losses. Generally, common skeletal deformities in fish include lordosis, kyphosis, vertebrae ectopic, and neural and haemal spines deformities, among which the rate of vertebral deformity is the highest. The influencing factors of skeletal deformities include heredity, environment, and nutrition. Many studies have confirmed that proteins, lipids, and vitamins can affect the development of larval and juvenile fish to a certain extent. Notably, vitamins have considerable effects on skeletal development. For example, vitamin D can promote cartilage formation. Further, vitamin C deficiency has been shown to increase the probability of developmental deformities such as lordosis and kyphosis in larvae and juvenile fish [26]. Several skeletal abnormal phenomena such as lordosis, kyphosis, neural and haemal spines deformities, and vertebrae split were observed in our study, among which the vertebrae split was relatively uncommon. These skeletal deformities may be related to the feed nutrients provided during the artificial cultivation of larval and juvenile M. miiuy. However, other factors such as environment and disease can not be excluded. The relationship between skeletal deformities and heredity has not been investigated in this study due to the lack of observation and analysis on parent skeleton of M. miiuy.


## 4.1. Analysis of Developmental Characteristics on Spinal Vertebrae and Appendicular Skeleton

To date, many studies on skeletal developmental characteristics of larval and juvenile fish have been reported. For instance, the developmental pattern of spinal vertebrae, pectoral fin, and caudal fin of the large yellow croaker (Larimichthys crocea) have been studied using the double staining method [17]. The results showed that the larval notochord appeared segment at 6 dah, and the neural and haemal archs developed sequentially. The vertebral cartilage was formed at 14 dah and ossified completely at 28 dah from the base to the distal end. The pectoral fin was the earliest to develop among the appendicular skeleton. The supracleithrum, cleithrum, and postcleithrum appeared at 3 dah, and proximal pterygrophores and fin rays developed completely at 27 dah. Further, the ossification was complete at 28 dah. The results of our study are consistent with those of Wang et al. [17]; likely because L. crocea and M. miiuy both belong to Sciaenidae and have a similar developmental sequence.In a previous study, the early morphological development ofM. miiuy was observed and analysed [18]. The results showed that the median fins were formed in the order of caudal, anal, and dorsal fins, and the appendicular skeleton was fully developed by 17 dah. All fins were completely differentiated at 28 dah, which also indicated the transformation of M. miiuy from larval to juvenile stage. The main reasons for these differences may be attributed to the differences in water temperature, light intensity, fish bait abundance, and feeding method during the breeding period, which may lead to a different developmental sequence in the different parts of larval and juvenile M. miiuy. Moreover, the difference in the developmental stages may be due to the use of different classifications.The development of spinal vertebrae varies among different fish species. In our study, there were 25 spinal vertebrae inM. miiuy, which consist of 12 abdominal vertebrae and 13 coccyges. While Lates calcarifer have 24 spinal vertebrae during the developmental process of larvae and juveniles aged 0–23 days [19], euthynnus affinis have 20 trunk vertebrae and 18 tail vertebrae [20].The development of the vertebral column and appendicular skeleton of the larvae and juveniles of Chaeturichthys stigmatias was also described [21]. The results revealed 40–43 spinal vertebrae, which consisted of 15 trunk vertebrae and 25–28 tail vertebrae. The number of spinal vertebrae had an important influence on biological characteristics like size, length, and height [22]. This data can provide a theoretical basis for the study of fish traits and biological classification through the analysis of developmental characteristics of fish vertebrae [23].


## 4.2. Reasons of Vertebral Column Deformities

The skeletal deformities can not only affect the movement, and external characteristics of fish but also their economic value [24]. The research development on skeletal deformities of larval and juvenile fish in recent years have been summarized by a recent review [25]. The results showed that skeletal deformities may cause poor food intake, poor growth, weak physical ability, and low survival, which may ultimately lead to large economic losses. Generally, common skeletal deformities in fish include lordosis, kyphosis, vertebrae ectopic, and neural and haemal spines deformities, among which the rate of vertebral deformity is the highest. The influencing factors of skeletal deformities include heredity, environment, and nutrition. Many studies have confirmed that proteins, lipids, and vitamins can affect the development of larval and juvenile fish to a certain extent. Notably, vitamins have considerable effects on skeletal development. For example, vitamin D can promote cartilage formation. Further, vitamin C deficiency has been shown to increase the probability of developmental deformities such as lordosis and kyphosis in larvae and juvenile fish [26]. Several skeletal abnormal phenomena such as lordosis, kyphosis, neural and haemal spines deformities, and vertebrae split were observed in our study, among which the vertebrae split was relatively uncommon. These skeletal deformities may be related to the feed nutrients provided during the artificial cultivation of larval and juvenile M. miiuy. However, other factors such as environment and disease can not be excluded. The relationship between skeletal deformities and heredity has not been investigated in this study due to the lack of observation and analysis on parent skeleton of M. miiuy.



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*Source: 4767850-2023-02-03.xml*