Giant Grouper Epinephelus Lanceolatus Research Articles

Swim bladder inflation failure and associated spinal deformities in giant grouper Epinephelus lanceolatus

Giant grouper (Epinephelus lanceolatus, GG) is a valuable aquaculture species in Southeast Asia. Its farming is challenging, and the occurrence of spinal deformities is one of the bottlenecks found in aquaculture production. This study investigated the development of the swim bladder (SB) in GG and a possible causative relationship between failing to inflate the SB and spinal deviations in juvenile fish. The study of the ontogeny of the SB in GG revealed that the organ is connected through the pneumatic duct to the digestive tube from day 4 to day 7 post hatching (DPH). Repeated observations of larvae over this period showed that GG SB inflation period commences on day 5 DPH and lasts until the end of day 7 DPH. However, a proportion of day 7 DPH larvae failed to inflate their SB showing unorganised acidophilic tissue in the lumen of the organ. Assessment of skeletal anomalies in post metamorphic fish (42 DPH) revealed a relationship between the lack of an inflated SB and anomalies in the caudal region; more specifically, the urostyle (p < 0.05). Morphological assessment of juvenile fish showed a direct relationship between the lack of an inflated SB and lordotic/kyphotic anomalies. This was confirmed by X-ray of fish with and without an inflated SB following separation by hypersaline flotation. These findings show that SB inflation failure is the major contributing factor to spinal anomalies in GG. Also, this study described the timing of the SB inflation period in GG and its development.

Isolation and Characterization of a Ranavirus Associated with Disease Outbreaks in Cultured Hybrid Grouper (♀Tiger Grouper Epinephelus fuscoguttatus×♂Giant Grouper E.lanceolatus) in Guangxi, China.

An outbreak of suspected iridovirus disease in cultured hybrid grouper (♀Tiger Grouper Epinephelus fuscoguttatus×♂Giant Grouper Epinephelus lanceolatus) occurred in the Guangxi Province in July, 2018. In this study, grouper iridovirus Guangxi (SGIV-Gx) was isolated from diseased hybrid grouper that were collected from Guangxi. Cytopathic effects were observed and identified in grouper spleen cells that were incubated with diseased tissue homogenates after 24h, and the effects increased at 48h postinfection. The transmission electron microscopy results showed that viral particles that were about 200nm in diameter with hexagonal profiles were present in the cell cytoplasm of suspected virus-infected cells. The presence of SGIV-Gx (accession number: MK107821) was identified by polymerase chain reaction (PCR) and amplicon sequencing, which showed that this strain was most closely related to Singapore grouper iridovirus (AY521625.1). The detection of SGIV-Gx infection was further supported by novel aptamer (Q2c)-based detection technology. The effects of temperature and pH on viral infectivity were analyzed by using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and cell culture. The results indicated that SGIV-Gx was resistant to exposure to pH levels 5, 7, and 7.5 for 1h, but its infectivity was remarkably lower at pH levels 3 and 10 after 1h. The analyses showed that SGIV-Gx was stable for 1h at 4°C and 25°C but was inactivated after 1h at 40, 50, and 60°C.

A transcriptomic investigation of appetite‐regulation and digestive processes in giant grouper Epinephelus lanceolatus during early larval development

The giant grouper Epinephelus lanceolatus is an ecologically vulnerable species with high market demand. However, efforts to improve larval husbandry are hindered by a lack of knowledge surrounding larval developmental physiology. To address this shortfall, a transcriptomic approach was applied to larvae between 1 and 14 days post hatch (dph) to characterise the molecular ontogenesis of genes that influence appetite and digestion. Appetite regulating factors were detected from 1 dph, including neuropeptide Y, nesfatin-1, cocaine and amphetamine regulated transcript, cholecystokinin and pituitary adenylate cyclase activating peptide and the expression level of several genes changed sharply with the onset of exogenous feeding. The level of expression for proteases, chitinases, lipases and amylases typically followed one of two expression patterns, a general increase as development progressed, or an inverted U-shape with maximal expression at c. 6 dph. Similarly, the tendency among both expression patterns was for the level of expression to increase around the time of mouth-opening. There was also evidence to suggest the presence of putative isoforms for several digestion-related genes. We have provided an insight into appetite-regulation and digestive processes in groupers during early larval development and have developed a transcriptomic database that will aid future efforts to rear this species in an aquaculture setting.

Patterns of gene expression in larvae of the giant grouper.

The giant grouper Epinephelus lanceolatus (Bloch 1790) is the largest bony fish found on coral reefs, achieving lengths of over 2 m. It has delicious flesh that is highly prized by the live reef fish trade. Consequently, declining wild populations have been listed as Vulnerable by IUCN. There is now aquaculture of E. lanceolatus but, as is often the case for marine fishes, larval feeding is a major production bottleneck. In particular, knowing how the digestive system develops and how this might determine the feed that larvae can digest to grow satisfactorily. In this issue, Anderson et al. (2018) used a transcriptomic approach to explore molecular processes that underpin development of appetite, feeding and digestion in E. lanceolatus larvae. They focussed on the first two weeks of life, a critical period for larval husbandry during which the larvae first open their mouth and start exogenous feeding. Because transcriptomics typically yield a very large quantity of information, the challenge is to reveal patterns. The authors used Self Organising Maps to hone in on genes that contribute to specific developmental milestones. They developed a conceptual model to show patterns of expression of transcripts in relation to major developmental stages, such as mouth-opening, onset of exogenous feeding, swim bladder development. There were clear trends for digestive enzyme transcripts, which tended to increase their expression in concert with mouth-opening and onset of exogenous feeding. Gene expression for various appetite-regulating factors was highly variable although, in some cases, this could be linked to important developmental milestones. Although such studies are inductive, they provide an essential knowledge base for development of targeted hypothesis-driven research. A major objective would be to find means of improving growth and survival of larvae without having to rely on feeding with copepods, which are expensive to produce in large quantities. Such improvements in methods for larval husbandry, in the farming of this highly-prized species, will take pressure off vulnerable wild stocks. The authors commented that Self Organising Maps can be applied to any project generating high volumes of multi-dimensional data, and are especially useful for time-course studies and pattern visualisation. As such, fish biologists are able to use this approach to more easily hone in on genes that contribute to specific developmental milestones, phenotype, mortality event, whose expression changes in response to particular stimuli.

Isolation of a Ranavirus-type grouper iridovirus in mainland China and comparison of its pathogenicity with that of a Megalocytivirus-type grouper iridovirus

Megalocytivirus- and Ranavirus-type grouper iridoviruses (GIV-M and GIV-R, respectively) are causative agents in cultured groupers. GIV-R is recoded in the grouper industry in Chinese Taiwan and Singapore but rarely found in other regions or countries, including mainland China. In this study, a novel GIV-R, designated as GIV-R-SY1301, was isolated and identified from diseased juvenile hybrid groupers (tiger grouper Epinephelus fuscoguttatus ♀×giant grouper Epinephelus lanceolatus ♂) in Sanya, Hainan Island of China through PCR detection, cell culture, and transmission electron microscopy observation. Multiple alignment analysis based on the full-length major capsid protein (MCP) genes (1392bp) showed that twenty-three and four nucleotide substitutions were observed among GIV-R-SY1301 and two early GIV-R isolates from Taiwan and Singapore, respectively. At the protein level, there are one or two amino acid substitutions in MCP among these isolates. Artificial challenge with GIV-R-SY1301 and a GIV-M isolate (OSGIV-HN11) indicated that both OSGIV-NH11 and GIV-R-SY1301 were highly virulent to infected tiger groupers. Cumulative mortalities of 97.5% (39/40) and 85% (34/40) were obtained in OSGIV-HN11- and GIV-R-SY1301-challenged fish, respectively. Histopathological analysis showed numerous abnormally enlarged cells in OSGIV-HN11-infected grouper spleen and kidney tissues but not in GIV-R-SY1301-infected grouper tissues. Abnormally enlarged cells in infected fish tissues could be a notable histopathological marker to distinguish GIV-M infection from GIV-R infection. Taken together, this study is the first to isolate and characterize GIV-R iridovirus in mainland China. GIV-R would be an alarming agent to grouper industry in mainland China, as well as GIV-M has been influencing. Statements of relevance1.First isolation and characterization of a Ranavirus-type grouper iridovirus in mainland China;2.Comparative pathogenicity of Ranavirus- and Megalocytivirus-type GIV was firstly studied; both Ranavirus- and Megalocytivirus-type GIV showed highly virulent to infected tiger groupers under artificial infection of the same viral dosages. By contrast, GIV-M showed a higher virulence than GIV-R, but the latter showed a more rapid lethality;3.The differential histopathology caused by GIV-R and GIV-M was also studied. Abnormally enlarged cells in infected fish tissues could be a notable histopathological marker to distinguish GIV-M infection from GIV-R infection. All these findings contributed important information for grouper aquaculture worldwide.

Sperm cryopreservation in different grouper subspecies and application in interspecific hybridization

The present study established and confirmed an efficient technology for groupers: giant grouper Epinephelus lanceolatus, orange-spotted grouper E. coioides, seven-band grouper, E. septemfasciatus, and kelp grouper E. moara sperm cryopreservation and successfully applied the cryopreserved E. lanceolatus sperm into interspecific hybridization with E. coioides. For both E. lanceolatus and E. coioides, the best motility of postthaw sperm were achieved using 6% to 10% DMSO, 6% to 16% propylene glycol, and 6% ethylene glycol as cryoprotectants. Furthermore, we have successfully applied this method into the other two species of E. septemfasciatus (74.56 ± 5.45%) and E. moara (71.67 ± 5.10%) sperm cryopreservation and obtained high motility, respectively. Computer-assisted sperm motion analysis showed that the postthaw sperm of the four species of grouper could keep 30 to 35 minutes motile state in nature seawater. And the freezing–thawing process decreased the sperm motility, speed, and longevity but did not significantly change the sperm movement pattern, and the progressive linear motion still was the dominant movement pattern. For the four species of grouper, the ultrastructural analysis showed 70% to 80% of the spermatozoa had intact morphology with a little of swelling; 20% to 30% were damaged, such as swelling or rupture of head, midpiece, and tail region; and 10% to 20% were severely damaged. Whereas, by the microscopic observation, more than 90% of the postthaw sperm presented normal morphology. In the artificial insemination and hybridization experiment, high fertilization rates and hatching rates were achieved when using 10% DMSO (88.7 ± 5.3%, 85.3 ± 7.4%) and 10% propylene glycol (86.8 ± 3.3%, 83.1 ± 6.6%), with no significant difference in comparison with control (92.2 ± 1.4%, 87.9 ± 4.2%). In addition, we found the embryos from postthaw sperm of E. lanceolatus and E. coioides eggs developed and grew normally as reported in previous study on hybridization of groupers (E. coioides × E. lanceolatus) using cryopreserved sperm. The results of the present study further validated the safety of the cryopreserved sperm in breeding production by assessing the fertilization capacity, embryo development, and larval growth.

Research Report Development and characterization of 32 microsatellite loci in the giant grouper Epinephelus lanceolatus (Serranidae)

An economically important marine fish species, the giant grouper Epinephelus lanceolatus (Serranidae) is widely cultured in Taiwan and costal areas of China. We isolated and characterized 32 polymorphic microsatellite loci from a CA-enriched genomic library of giant grouper. The number of alleles per locus ranged from 3 to 7, with a mean of 4.69. Observed and expected heterozygosities per locus varied from 0.387 to 1.000 and from 0.377 to 0.843, respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium. After sequential Bonferroni's correction, only two loci showed deviation from Hardy-Weinberg equilibrium, and no linkage disequilibrium was found between any pair of loci. These microsatellites can be useful tools for the study of population genetics in the giant grouper.