Skeletal Ontogeny and Anomalies in Larval and Juvenile Crimson Snapper, Lutjanus erythropterus Bloch, 1790

Dachuan Cheng1,2, Md Mahbubul Hassan3, Zhenhua Ma1, 2, 3,*, Qibin Yang1 and Jian G. Qin3

1Tropical Aquaculture Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Sanya 572018, China

2Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, Guangzhou 510300, China

3School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia

ABSTRACT

Skeletal anomalies in farmed fish affect animal welfare and economic return in aquaculture but very limited information exists on skeletal ontogeny and anomalies among species of the family Lutjanidae. This study describes the skeletal ontogeny and anomalies of crimson snapper Lutjanus erythropterus larvae and juveniles from hatching to 36 day-post hatching (DPH). Mandible, ceratobranchial, cleithrum and gill arches were the initial skeletal structures appeared at 3 DPH that supported the vital life functions such as feeding and respiration. Ossification of premaxilla and maxilla and dentary started at 3.21 ± 0.25 mm (9 DPH), and completed at 5.91 ± 0.34 mm (18 DPH). The head skeleton formation completed at 22.35 ± 2.26 mm (31 DPH). The axial skeleton development started with the formation of neural arches at 3.64 ± 0.07 mm (10 DPH) and ossification of axial skeleton completed at 11.01 ± 0.88 mm (24 DPH). The fins developed sequentially and the ossification of fins completed at 30.57 ± 2.44 mm (36 DPH). A total of 39.5% fish exhibited anomalies in the present study and the anomalies were: lordosis, vertebral fusion, neural spines bifurcation, connection of adjacent pterygiophores, haemal spine anomaly, neural spines anomaly, anomaly in pterygiophores and supernumerary neural spines. Results from this study add new knowledge to functional morphology of crimson snapper that would be useful to larval aquaculture of marine teleosts.

Article Information

Received 16 July 2017

Revised 30 August 2017

Accepted 01 November 2017

Available online 5 April 2018

Authors’ Contribution

DC, ZM and JGQ designed this study. DC, QY and ZM conducted the field work and analyzed the sample. DC, MMH, ZM and JGQ drafted this manuscript.

Key words

Crimson snapper Lutjanus erythropterus, Skeleton, Ontogeny, Ossification, Malformations.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.3.799.807

* Corresponding author: zhenhua.ma@hotmail.com

0030-9923/2018/0003-0799 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

 

Skeletal abnormality of farmed fishes is a major drawback in aquaculture. Fish with abnormal mouth, vertebrate or fin shows low feeding or swimming performance, and disease susceptibility (Wittenrich et al., 2009; Isaac et al., 2017). Skeletal anomalies cause significant problems for mechanical fish filleting as machines are designed for normal shaped fishes, thus require extra trimming and manual handing. Abnormal fishes intrinsically represent low market value than normal shaped fishes. Some farmers and traders cull out abnormal shaped fishes during marketing to maintain reputation of their business. The economic loss due to body deformity of farmed fish in the European aquaculture industry is estimated to be over €50,000,000 (Boglione et al., 2013a).

Since the first publication on body shape anomaly of rainbow trout in 1971 (Aulstad and Kittelsen, 1971), significant progress has been achieved in development of skeletal biology in fish (Boglione et al., 2013a, b; Babbucci et al., 2016; Azevedo et al., 2016). Skeletal anomalies (such as jaw malformation, skull malformation, spine malformation) have been described in many farmed fish species, including European sea bass Dicentrarchus labrax (Abdel et al., 2004; Georgakopoulou et al., 2007), gilthead sea bream Sparus aurata (Andrades et al., 1996; Georgakopoulou et al., 2010), Senegal sole Solea senegalensis (Gavaia et al., 2002), red sea bream (Kihara et al., 2002), yellowtail kingfish Seriola lalandi (Cobcroft et al., 2004), and golden pompano Trachinotus ovatus (Zheng et al., 2014). However, very limited information exists on skeletal development and anomalies in the species of the family Lutjanidae except the study on skeletal ontogeny of red snapper (Potthoff et al., 1988). A survey in Australian finfish hatcheries has identified skeletal anomalies among snappers and warranted species-specific research (Cobcroft and Battaglene, 2013).

The types of skeletal anomalies vary among species, life stages and rearing conditions (Andrades et al., 1996; Boglione et al., 2013b). The onset of anomalies mostly occurs at the larval and early juvenile stage (Boglione et al., 2013a; Estivals et al., 2015; Huang et al., 2016). In addition, the types of anomalies are extremely diverse and many typologies are difficult to define at the onset of anomalies (Boglione et al., 2013b). Setting a reference point for normal skeletal developments is an important step to recognize early stages of skeletal anomalies. Since no information exists on the skeletal ontogeny and anomalies of crimson snappers, the descriptions of skeletal development would minimize this knowledge gap.

According to FishBase (http://www.fishbase.org), a total of 67 species of snappers are distributed in tropical and subtropical oceans, and many species are important aquaculture candidate. The crimson snapper Lutjanus erythropterus, distributed in the tropical Indo-Pacific (from Gulf of Oman to Southeast Asia, northward to southern Japan and southward to northern Australia), is an important aquaculture candidate in Asia and Australia. Skeletal ontogeny and anomalies provide information on the functional morphology of a species that is important for basic biological perspective and larval aquaculture. Therefore, the objective of this study is to describe the skeletal ontogeny and anomalies of crimson snapper.

 

Materials and methods

 

Fishes were maintained according to the recommendation of Chinese Academy of Fishery Sciences Animal Welfare Committee. The protocol, species and number of animals used in this study were approved by the South China Sea Fisheries Research Institute Animal Welfare Committee (Approved Number: 2014YJ01).

Larvae acquisition and rearing

Fertilized eggs were received from Shenzhen Longqizhuang Aquaculture Hatchery, Guangdong Province, P.R. China, and were transported to South China Sea Fisheries Research Institute. Upon arrival, the eggs were hatched in 500-L fiberglass incubators at 27.5oC. On 2 days post-hatch (DPH), larvae were reared into three 2500-L tanks at a density of 60 fish L-1. Each rearing tank was supplied with filtered seawater (5-µm pores) with a daily water exchange rate of 200% of the tank volume. Daily photoperiod of 14 h light and 10 h dark with 2000 lux light intensity was maintained at the water surface. The water temperature and salinity was maintained at 29.0 ± 1.0oC and 33 ± 0.8 ppt, respectively.

Fish were fed with rotifers (Brachionus rotundiformis) from 2 DPH to 10 DPH at a density of 10-20 rotifers mL-1. On 9 DPH, Artemia nauplii were introduced at 0.1 nauplii mL-1, and increased daily 5 nauplii mL-1 until 18 DPH. Afterwards, Artemia nauplii were gradually replaced from 19 DPH by inert diets. Fish were fed with inert diets Otohime A1 (~250 µm, Marubeni Nisshin Feed Co. Ltd., Tokyo, Japan) and Huacheng No.5 (850-1100 µm, Haikang Aquatic Biotechnology Co. Ltd., Yantai, China), and the amount of feed was adjusted to apparent satiation. Rotifers and Artemia nauplii were enriched with the DHA protein Selco (INVE Aquaculture, Salt Lake City, UT, USA) before adding into the larval rearing tanks. Fish age specific feeding protocols are illustrated in Figure 1.

 

 

Staining and visualization

A total of 20 larvae were collected from rearing tanks daily and anaesthetized with Aqui-S (AQUI-S New Zealand Ltd., Lower Hutt, New Zealand). The larvae were initially fixed in 10% neutral buffered formalin, and then stained with alcian blue and alizarin red followed by Taylor and Van Dyke (1985). After staining, samples were photographed under stereomicroscope (Olympus SZ40) equipped with a digital camera (Oneplus A2001). The terminologies of the skeletal elements were adopted from Kihara et al. (2002) and Sfakuanakis et al. (2004). A total of 450 larvae and juveniles were examined to identify skeletal anomalies. The incidence of anomalies was calculated using the following equation: Incidence of anomalies = (number of larvae with skeletal anomaly/total number of larvae) ×100%.

 

Results

Head skeleton

In crimson snapper larvae, the ceratobranchial, basibranchial and hypobranchial cartilages were attached to the gill arch. The first four gill arches formed at 2.83 ± 0.01 mm (standard length ± SD, 3 DPH, Fig. 2A). In front of the anterior end of the hypobranchial cartilage, the hypohyal cartilage formed, and symmetrically connected to a pair of ceratohyal-epihyal cartilage. The Meckel’s cartilage stretched out forward, surpassing the ventral area of the eyes. The head of quadrate cartilages bound to the Meckel’s cartilage and the hyomandibular-sympletic cartilage. The neurocranium, trabeculae cartilage and ethmoid cartilage formed at this stage (Fig. 2B). The tranvecula cartilages, connecting with the posterior end of the ethmoid cartilages, stretched through the midcourt line of the two eyes. Figure 2B, C, D and E shows formation of different jaw elements. Ossification of jaw elements such as premaxilla and maxilla and dentary started at 3.21 ± 0.25 mm (9 DPH), and completed at 5.91 ± 0.34 mm (18 DPH, Fig. 2D). The skull formation completed at 22.35 ± 2.26 mm (31 DPH, Fig. 2F).

 

 

Vertebral column and fins

The axial skeleton consisted of the vertebrae and epineurals, and the median fin supports, including the proximal and distal pterygiophores. Notochord was the only axial structure at the lengths between 2.23 mm to 3.21 mm (1-9 DPH, Fig. 3A). The neural arches were the first element of the vertebral column that developed at 3.64 ± 0.07 mm (10 DPH, Fig. 3B). The haemal arches developed subsequently at 3.78 ± 0.08 mm (11-12 DPH, Fig. 3C). The buds elongated ventrally and joined together forming the haemal arch and then the spine appeared and elongated ventrally. The neural arches to ossified at 5.28 ± 0.57 mm (15 DPH), and the haemal arches ossified at 6.66 ± 0.31mm (20 DPH). Ossifications of neural arches, neural spines, haemal arches and haemal spines completed at 11.01 ± 0.88 mm (24 DPH, Fig. 3F).

Vertebral column developed at 5.35 ± 0.67 mm (16 DPH, Fig. 3D). Vertebral centra (V1-V8) ossified at 6.66 ± 0.31 mm (20 DPH), and the ossification of centrum proceeded from cephalic to caudal region (Fig. 3E). Ossification of vertebral centra completed at 12.93 ± 1.58 mm (27 DPH, Fig. 3G). Parapophysies, the process of plural rib formation, started at 11.01 ± 0.88 mm (24 DPH, Fig. 3F) with the formation of first thoracic rib. The ossification of thoracic ribs completed between 12.92 mm to 19.09 mm (27-30 DPH).

 

 

The formation of anterior dorsal pterygiophores started at 3.64 ± 0.07 mm (10 DPH, Fig. 4A) toward the caudal end. The spines of anterior dorsal pterygiophores formed at 3.84 ± 0.11 mm (12 DPH, Fig. 4B). Sharp hooks of the dorsal fin formed at 3.97 ± 0.13 mm (13 DPH, Fig. 4C). First five hard spines and 6th cartilaginous spine developed at 4.13 ± 0.12 mm (14 DPH, Fig. 4D). Ossification of anterior dorsal pterygiophores completed at 5.12 ± 0.92 mm (17 DPH, Fig. 4E). The dorsal fin rays developed at 5.34 ± 0.96 mm (16 DPH). The dorsal pterygiophores, hard spines and fin rays ossified completely between 26.48 and 30.7 mm (34-36 DPH, Fig. 4F).

Cleithrum in pectoral fin started to develop at 2.62 ± 0.09 mm (3 DPH, Fig. 5A), and clearly visible at 3.25 ± 0.12 (10 DPH, Fig. 5B). Pectoral fin plate and the fin rays developed at 6.22 ± 0.28 mm (19 DPH, Fig. 5C). The proximal pterygiophore developed at 11.45 ± 0.42 mm (25 DPH, Fig. 5D). The coracoid, scapula, proximal pterygiophore developed at 22.48 ± 0.94 mm (32 DPH, Fig 5E). Pectoral fin rays started to ossify at 11.01± 0.88 mm (24 DPH), and ossification completed at 30.47 ± 2.14 mm (36 DPH, Fig. 5F).

 

 

The basipterygium appeared at 3.61 to 3.64 mm (9-11 DPH) as a small cartilaginous element that gradually elongated to 3.84 mm (11-12 DPH, Fig. 6A, B). The hard spine developed at 3.99 ± 0.28 mm (13 DPH, Fig. 6C). Pelvic fin rays developed at 5.91 ± 0.34 mm (18 DPH, Fig. 6D). The hard spines ossified at 11.01 ± 0.88 mm (24 DPH, Fig. 6E), and the fin rays ossified at 30.57 ± 2.44 mm (36 DPH, Fig. 6F).

 

 

The anal proximal pterygiophore developed as small cartilages between 3.99 mm to 4.02 mm (13-14 DPH, Fig. 7A, B). Subsequently, a hard spine formed before anal fin rays between 5.12 mm to 5.35 mm (15-16 DPH) from the cephalic to caudal direction (Fig. 7C). Pterygiophores, hard spine and fin rays started to ossify at 11.01 ± 0.88 mm (22-24 DPH, Fig. 7D, E) and ossification completed at 26.48 ± 1.04 mm (34 DPH, Fig. 7F).

Formation and ossification of caudal complex

The caudal complex in crimson snapper consisted of the epurals, hypurals, preural arches, modified spines, urostyle, preural centra and caudal fin rays.

Hypurals 1-3 developed first as a small cartilage at 4.20 ± 0.01 mm (14 DPH, Fig. 8A). Afterwards hypurals 3-5, the neural spines, haemal, epural and caudal fin rays developed between 5.34 mm to 5.91 mm (16-18 DPH, Fig. 8B, C). Upward flexion of the urostyle developed at 11.45 ± 0.42 mm (25 DPH, Fig. 8D). The urostyle and fin rays ossified between 18.35-21.21 mm (29-30 DPH, Fig. 8E, F). The hypurals, modified neural spines and modified haemal spines ossified at 26.48 ± 1.04 mm (34 DPH, Fig. 8G). Except epural, ossification of the caudal complex completed at 30.57 ± 2.44 mm (36 DPH, Fig. 8H).

 

 

Skeletal anomalies

In the present study, skeletal anomalies were observed from the yolk sac stage to the juvenile stage. At 36 DPH, a total of 39.5% juvenile had anomalies of which 12.5% had one type of anomaly while 27% had multiple anomalies. The rate of each type of anomaly was: lordosis (10.5%) characterized by a V-shaped dorsoventral curvature of the vertebral trunk (Fig. 9A, B), vertebral fusion (2%, Fig. 9D), bifurcated neural spines (1%, Fig. 9E), connection of adjacent pterygiophores (4%, Fig. 9A), malformed haemal spines (32%, Fig. 9C), malformed neural spines (32%, Fig. 9B), malformed pterygiophores (2%, Fig. 9C) and supernumerary neural spines (6%, Fig. 9F). However, no anomalies observed in the skull, dorsal fins, pectoral fins, pelvic fins and anal fins during larval development up to 36 DPH.

 

 

Discussion

 

This is the first description of the skeletal ontogeny and anomalies of the larval and juvenile crimson snapper L. erythropterus. This study adds new knowledge to skeletal ontogeny and anomalies in crimson snapper that would be useful from basic biological perspective and larval aquaculture of marine teleosts.

Marine teleosts have a very shorter embryonic period (compared to freshwater species), and the larvae hatch out without the development fins, mouth and anus. The initial anatomical regions with osteological development were mandible, pectoral fin (cleithrum), ceratobranchial and gill arches at 3 DPH in crimson snapper. The Mandible directly involved in feeding and cleithrum supports the sternohyoideus muscle that is involved in mouth movement (Wagemans et al., 1998), therefore development of mandible and cleithrum supported feeding during early larval stages. The development of ceratobranchial and gill arches supports respiration. A similar early osteological development to support feeding and respiration has been observed in numerous marine fish species (Gluckmann et al., 1999; Koumoundouros et al., 2000, 2001; Çoban et al., 2009).

In crimson snapper, the fins developed sequentially as - pectoral, anal, dorsal, caudal and pelvic. The cartilaginous pterygiophore and basipterygium developed before fin rays. The ossification of fins completed at different lengths, but ossification completed at a similar rate in all the fins. This pattern of development and ossification rate of pterygiophore and basipterygium among different fins are similar among other species in Perciformes (Matsuoka, 1985; Faustino and Power, 1998). However, the sequence of fins development was different in L. erythropterus from Diplodus puntazzo although both species belongs to Perciformes. In Diplodus puntazzo larvae, the fins development sequence was-pectoral, caudal, dorsal, anal and pelvic fin (Sfakianaki et al., 2005).

In addition to the variability of the developmental sequence, the most remarkable variability exists is developmental duration of skeletal structures. This variability is mainly attributed by the environmental and physiological conditions of each species. For example, fins development completed at 16.0 mm (SL) in Sparus aurata (Faustino and Power, 1998), and 15.5 mm TL in Pagrus pagrus (Coban et al., 2009), whereas fins development completed at a much higher lengths of 30.57 mm (SL) in this species. In fact, it is extremely difficult to compare the developmental duration of different species since the culture conditions of different species are not similar.

Certain skeletal structures are species specific among marine teleosts. For example, most species have five hypurals in the caudal region (Gavaia et al., 2002; Sfakianakis et al., 2004; Wang et al., 2010) whereas other species have six hypurals (Chen et al., 2011). Variations also exist in the number of epurals among different species. For example, majority of species have two or more epurals (Kohno, 1997; Laggis et al., 2014) whereas a few species have only one epurals in the caudal complex (Koumoundouros et al., 1999). We observed five hypurals and three epurals in crimson snapper which is conserved in most species.

Osteological development in fish larvae begins with cartilage formation prior to ossification (Faustino and Power, 1998). In this study, vertebral column ossification started at the cephalic region and proceeded in the caudal region. In addition, the ontogeny of the vertebral centra proceeds from the front to the end. Among the species in Lutjanidae, ossification of the vertebral centra proceeds caudally up to the preural centra (Potthoff et al., 1988), whereas the first centrum ossify after the formation of the second centrum in Sparidae (Faustino and Power, 1998; Koumoundouros et al., 1999, 2001; Sfakianakis et al., 2004).

The development of skeletal anomalies is linked to both environmental and biotic factors. However, the specific aetiologies for skeletal anomalies in crimson snapper could not be confirmed. In this study, vertebral anomalies particularly malformed neural spine and malformed haemal spines were most frequently observed. Similar to this species vertebral anomalies were the most common type in many other species, such as gilthead sea bream Sparus aurata (Boglione et al., 2001), Pandora Pagellus erythrinus (Sfakianakis et al., 2004), zebrafish Danio rerio (Ferreri et al., 2000), European sea bass Dicentrarchus labrax (Barahona-Fernandes, 1982) and striped trumpeter Latris lineata (Negm et al., 2014).

 

Conclusions

 

A comprehensive understanding of the skeletal development facilitates hatchery management and larval rearing of a species. This study has explored the skeletal ontogeny and identified some skeletal anomalies in crimson snapper. Results from the present study would be useful to understand functional morphology and larval aquaculture of marine teleosts.

 

Acknowledgments

 

This project was funded by Nature Science Foundation of Hainan Province (317289) and Special Scientific Research Funds for Central Non-profit Institutes, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (2014YJ01).

 

Statement of conflict of Interest

The authors declare that there is no conflict of interests regarding the publication of this article.

 

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