New isolate of Loma psittaca (Microsporidia: Glugeidae) infecting the stomach wall of cultured hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) in South China

Loma psittaca, previously described as inhabiting the intestinal mucosa of an anadromous fish, Colomesus pisttacus, from the Amazon Basin, is reported as being found for the first time in a marine fish, the hybrid grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀), from Lingshui city, Hainan Province, China, expanding the geographical distribution and host range of this parasite. Numerous whitish xenomas (0.5–0.7 mm in diameter) of this new isolate of L. psittaca were found distinctly in the muscle layer of the host stomach wall. Electron microscopic observations showed a monokaryotic nucleus in all developmental stages. Round or elongated multinucleate merogonial plasmodia surrounded by numerous mitochondria were observed initially, subsequently transforming into uninucleate sporonts through multiple fissions. Sporonts, each with a large centrally positioned nucleus, further developed into sporoblasts. Each sporoblast mother cell gave rise to two uninucleate sporoblasts by binary fission. Mature spores were ellipsoidal, measuring 4.0 ± 0.3 (3.7–4.3) μm in length and 2.2 ± 0.2 (2.1–2.5) μm in width. Spores possessed a mushroom-like anchoring disk, a bipartite polarplast, isofilar polar filaments arranged in 12–14 turns in one row, and a trilaminar spore wall. The obtained partial SSU rRNA gene sequence of the new isolate was 1330 bp in length and showed 99.4% sequence similarity with an estuary isolate of L. psittaca previously reported in South America. SSU rRNA gene-based phylogenetic analyses demonstrated that the two L. psittaca isolates first clustered together and then formed a dichotomy that included the digestive-tract-infecting Loma species, L. acerinae, with high support values within group I.     

Evaluation of the effects of different stocking densities on growth and stress responses of juvenile hybrid grouper ♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus in recirculating aquaculture systems

This study was aimed at evaluating the physiological and metabolic responses of juvenile hybrid grouper ♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus to stocking density. Hybrid grouper juveniles (mean ± SE = 25.43 ± 2.36 g live mass) were stocked for 22 weeks in a recirculating aquaculture system (RAS) under four different densities: low stocking density (LD; 1.03 kg m⁻³), medium stocking density (MD; 2.06 kg m⁻³), high stocking density (HD; 3.09 kg m⁻³) and extra-high stocking density (EHD; 4.11 kg m⁻³). Biometric variables were recorded and plasma, liver, intestine and stomach samples were taken for biochemical analysis at the end of the experimental period. Final stocking densities were 6.27, 16.04, 23.77 and 28.32 kg m⁻³, respectively, with significant differences in growth performance. Our results showed that the best growth rates and feed utilisation occurred in the MD group. Higher plasma cortisol and glucose levels and lower triglyceride levels reflected the stress responses in the EHD group. Moreover, the activity of aspartate and alanine transaminases was elevated in the HD and EHD groups due to enhanced gluconeogenesis. The activity of the digestive enzyme pepsin significantly increased in the MD group. We found that 2.06–3.09 kg m⁻³ is the most suitable starting density for culturing juvenile hybrid grouper in recirculating aquaculture systems.     

Identification,characterization, and expressions profile analysis of growth hormone receptors (GHR1 and GHR2) in Hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus polyphekadion ♂)

Growth hormone is an essential hormone that plays essential roles in growth, metabolism, cellular differentiation, immunity and reproduction in fish, by means of the growth hormone receptors. The encoding cDNA growth hormone receptors (GHR1 and GHR2) were cloned and characterized from Hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus polyphekadion♂). Sequence analysis of the cloned GHR1 was observed as containing 2176, which comprised an ORF of 1842 bp, 5 UTR of 6 bp and 3 UTR of 328 bp, with 612 amino acids encoding proteins, while GHR2 was observed as containing 1824 bp that encompassed an ORF of 708 bp, 5 UTR of 48 bp and 3 UTR of 1068 bp with 235 amino acids encoding proteins. Relative mRNA expression of GHR1 and GHR2 in the liver and muscle was found to be highest respectively. Our findings provide vital statistics of GHRs likely to play a significant role in the growth of the fish.     

Genetic characteristics and growth patterns of the hybrid grouper derived from the hybridization of Epinephelus fuscoguttatus (female) × Epinephelus polyphekadion (male)

Hybridization is one of the primary methods used to cultivate farmed grouper species. The hybrid grouper derived from crossing Epinephelus fuscoguttatus (♀) and E. polyphekadion (♂) exhibits growth superiority over its parents. The genetic characteristics and growth patterns of the hybrid grouper have not yet been defined. This study confirms the ploidy level of the hybrid grouper (2n = 48) using chromosome count analysis and flow cytometry. The 5S rDNA family was used to evaluate genetic diversity. Only one 5S class (~400 bp) was detected in the hybrid grouper, which could be used to distinguish between two different types based on nucleotide sequences, likely representing homologous unit classes from the female and male parental species. Growth patterns of 5–8-month-old hybrid groupers were also monitored. In this phase, a positive allometric growth pattern in body mass with total length was found. Body height and body mass were significantly correlated based on correlation and path coefficient, suggesting that body height could serve as an excellent index to increase body mass. These results aid our understanding of the genetic evolution of the hybrid grouper and inform the development of improved rearing techniques.     

Morphogenesis of sense organs and behavioural changes in larvae of the brown-marbled grouper Epinephelus fuscoguttatus (Forsskål)

The morphogenesis of sense organs and related behavioural changes in the hatchery-reared brown-marbled grouper Epinephelus fuscoguttatus larvae were examined to gain better understanding of its early life history because ecological field observations for grouper species is difficult. The newly hatched larvae (2.1 mm total length) had developing eyes and otic vesicles, a pair of free neuromast on the head and ciliated olfactory epithelium. At 3 days post hatching (dph), the eyes became fully pigmented with pure-cone retinae, the semicircular canals formed in the inner ear, and the larvae (2.8 mm) were able to swim horizontally, preying on rotifers. Retinal rods and the intra-oral taste buds at pharyngeal appeared next. The olfactory lamellae and the head lateral line system then formed, and the inner ears developed completely in the larvae during the metamorphosis period (15–40 dph; 5.1–18.1 mm). At settlement (50 dph; 32.8 mm), the fish possessed taste buds in the mouth entrance region, and the lateral line system developed completely. The sensory development correlates well with the known aspects of its life history at sea whereby the larvae can feed early and avoid predators during the passive drift, are able to swim shoreward to search nursery ground along the metamorphosis stage and survive in seagrass beds at settlement.     

Effects of a Potential Host Gut-Derived Probiotic,Bacillus subtilis 6-3-1, on the Growth,Non-specific Immune Response and Disease Resistance of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)

Probiotics and Antimicrobial Proteins - A potential host-derived probiotic, Bacillus subtilis 6-3-1, was successfully screened from 768 isolates from the intestines of healthy hybrid grouper...     

Parasite diversity as an indicator of environmental change? An example from tropical grouper (Epinephelus fuscoguttatus) mariculture in Indonesia

Fish parasites are used to monitor long-term change in finfish grouper mariculture in Indonesia. A total of 210 Epinephelus fuscoguttatus were sampled in six consecutive years between 2003/04 and 2008/09 and examined for parasites. The fish were obtained from floating net cages of a commercially run mariculture facility that opened in 2001. The fauna was species rich, consisting of ten ecto- and 18 endoparasite species. The ectoparasite diversity and composition was relatively stable, with the monogeneans Pseudorhabdosynochus spp. (83-100% prevalence, Berger-Parker Index of 0·82-0·97) being the predominant taxon. Tetraphyllidean larvae Scolex pleuronectis and the nematodes Terranova sp. and Raphidascaris sp. 1 were highly abundant in 2003/04-2005/06 (max. prevalence S. pleuronectis 40%, Terranova sp. 57%, Raphidascaris sp. 1 100%), and drastically reduced until 2008/09. These parasites together with the prevalence of Trichodina spp., ecto-/endoparasite ratio and endoparasite diversity illustrate a significant change in holding conditions over the years. This can be either referred to a definite change in management methods such as feed use and fish treatment, or a possible transition of a relatively undisturbed marine environment into a more affected habitat. By visualizing all parameters within a single diagram, we demonstrate that fish parasites are useful bioindicators to monitor long-term change in Indonesian grouper mariculture. This also indicates that groupers can be used to monitor environmental change in the wild. Further taxonomic and systematic efforts in less sampled regions significantly contributes to this new application, supporting fish culture and environmental impact monitoring also in other tropical marine habitats.     

Antiviral effects of β-defensin derived from orange-spotted grouper (Epinephelus coioides)

Defensins are a group of small antimicrobial peptides playing an important role in innate host defense. In this study, a β-defensin cloned from liver of orange-spotted grouper, Epinephelus coioides, EcDefensin, showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (VNNV). The expression profiles of EcDefensin were significantly (P < 0.001) up-regulated after challenging with Lipopolysaccharide (LPS), SGIV and Polyriboinosinic Polyribocytidylic Acid (polyI:C) in vivo. Immunofluorescence staining observed its intracellular innate immune response to viral infection of SGIV and VNNV. EcDefensin was found to possess dual antiviral activity, inhibiting the infection and replication of SGIV and VNNV and inducting a type I interferon-related response in vitro. Synthetic peptide of EcDefensin (Ec-defensin) incubated with virus or cells before infection reduced the viral infectivity. Ec-defensin drastically decreased SGIV and VNNV titers, viral gene expression and structural protein accumulation. Grouper spleen cells over-expressing EcDefensin (GS/pcDNA-EcDefensin) support the inhibition of viral infection and the upregulation of the expression of host immune-related genes, such as antiviral protein Mx and pro-inflammatory cytokine IL-1β. EcDefensin activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. Reporter genes of IFN-Luc and ISRE-Luc were significantly up-regulated in cells transfected with pcDNA-EcDefenisn after infection with SGIV and VNNV. These results suggest that EcDefensin is importantly involved in host immune responses to invasion of viral pathogens, and open the new avenues for design of antiviral agents in fisheries industry.     

Inactivation of nervous necrosis virus infecting grouper (Epinephelus coioides) by epinecidin-1 and hepcidin 1–5 antimicrobial peptides,and downregulation of Mx2 and Mx3 gene expressions

Betanodaviruses are one of the serious pathogens in nervous necrosis viral (NNV) disease that brings about mortality in the larval stage of grouper (Epinephelus coioides). In this study, the efficacy of pretreatment, co-treatment, and posttreatment with the antimicrobial epinecidin-1 and hepcidin 1–5 peptides against a betanodavirus was evaluated by intraperitoneal inoculation in grouper. The results showed that co-treatment of epinecidin-1 or hepcidin 1–5 with the virus was effective in promoting a significant decrease in grouper mortality. Re-challenge with virus again after 30 day in co-treated grouper groups showed high survival suggesting that epinecidin-1 and hepcidin 1–5 enhanced fish survival. However, grouper inoculated with NNV and then inoculated with epinecidin-1 8 h later showed significantly different survival from the group inoculated with virus alone, suggesting that epinecidin-1 can be used as a drug to rescue infected grouper. Infection after pretreatment, co-treatment, and posttreatment with epinecidin-1 or hepcidin 1–5 was verified by RT-PCR which showed downregulation of Mx2 and Mx3 gene expressions. All these data strongly suggest that epinecidin-1 and hepcidin 1–5 are effective peptides for protecting grouper larvae by reducing NNV infection.     

Reproductive movement,residency and fisheries vulnerability of brown-marbled grouper, <Emphasis Type="Italic">Epinephelus fuscoguttatus</Emphasis> (Forsskål, 1775)

The brown-marbled grouper, Epinephelus fuscoguttatus, is a long-lived, late-maturing protogynous species listed as Near Threatened by the IUCN. In Pohnpei, Micronesia, reproductively active brown-marbled grouper were tagged with acoustic and spaghetti-type tags at a multi-species fish spawning aggregation (FSA) site to establish patterns of movement, residency and seasonality. Telemetry confirmed the use of common reproductive migratory corridors and significant sex-specific variations in residency at the FSA. Combined underwater visual census and telemetry data verified a 3-month peak aggregation period, with aggregations forming and persisting over ca. 12 days prior to full moon between January and May. FSA formation coincided with seasonally low and relatively stable seawater temperatures. Some males frequented the FSA site during each aggregation month over two consecutive years. Conversely, most females were present at the FSA during only a single aggregation period, with the month of visitation consistent among years. Nearly two-thirds of tagged fish were relocated or recaptured within 11 km of the aggregation site, with a maximum detected distance of 26 km and a minimum estimated catchment area of 100–175 km². Findings highlight the need for a combined approach to management that prohibits the capture and sale of reproductive adults and protects both spawning sites and common reproductive migratory corridors during aggregation periods.     

How is a giant sperm ejaculator formed? Development of the Zenker organ after the last moult in Pseudocandona marchica (Crustacea,Ostracoda, Candonidae)

‘Giant sperm’, in terms of exceptionally long spermatozoa, occur in various taxa in the animal kingdom, particularly in arthropods. In freshwater ostracods (Cypridoidea) reproducing with giant sperm, males are equipped with a pair of specialized ejaculators, or Zenker organs, located in the distal part of vas deferens.