Osteological development and anomalies in larval stage of hatchery‐reared yellow catfish Pelteobagrus fulvidraco

Comprehensive knowledge of osteological development of fish not only provides means for understanding its functional development, but also allows early detection of skeletal anomalies. The present study was conducted to determine osteological development and occurrence of anomalies from the first to the 40th day after hatching (DAH) for yellow catfish Pelteobagrus fulvidraco, a commercially important species in China. Vertebral ontogeny started with formation of anterior neural arches at 3 DAH, and completed at 6 DAH. Vertebral centra started to develop at 4 DAH and ossification in all centra was visible at 15 DAH. Caudal fin appeared at 2 DAH and ossification was visible at 26 DAH. The onset of dorsal and anal fins appeared at 4 DAH and their ossifications were visible at 20 DAH. Pectoral fins were present before first feeding and formed entirely at 9 DAH, and their ossifications were visible at 21 DAH. Pelvic fins appeared at 9 DAH, and formed completely at 21 DAH. The ossification of pelvic fins was not finished until the end of the experiment (40 DAH). 24 types of skeletal anomalies were observed. About 20% of individuals showed at least one anomaly. Haemal vertebrae anomalies occurred at the highest frequency, followed by pre‐haemal, caudal vertebrae and anal fin anomalies, and caudal fin had the lowest anomalies frequency. For the first time, our study determined osteological development and anomalies incidence in larval yellow catfish, which help further investigations into rearing conditions leading to appearance of these anomalies to prevent their incidence.     

Skeletal development and deformities in cultured larval and juvenile seven-band grouper, Epinephelus septemfasciatus (Thunberg)

The seven‐band grouper, Epinephelus septemfasciatus (Thunberg), is currently recognized as a potential new species for aquaculture in Japan. This study describes normal and abnormal skeletal development of the jaw and vertebrae in cultured larvae and early juveniles of E. septemfasciatus. The ontogenetic stages at which skeletal deformities of jaw and vertebra developed were also described for this species. Osteological observations were made using a clearing and staining method for larvae and soft X‐ray photographs for juveniles. A high incidence of skeletal deformities was observed in the jaws and vertebral column during the larval and juvenile stages. Most of the jaw deformities were explained by an abnormal maxilla curvature. Jaw deformities were visually evident from flexion stage after ossification of the deformed elements. Deformities in the vertebral column (mostly lordosis) were observed from the post‐larval stage and became more evident as growth proceeded. The lordosis generally occurred on the positions of the 8–11th vertebra. These types of deformities are compared with those of other species, and possible causative factors of the skeletal deformities are discussed.
     

Skeletal development and abnormalities of the vertebral column and fins in larval stage of hatchery‐reared American shad,Alosa sapidissima

The present study describes the skeletal development and the occurrence of deformities in American shad, Alosa sapidissima, larvae from hatching to 45 days after hatching (DAH). The ontogeny of the vertebral column started at 16 DAH (days after hatching), with the formation of the posterior neural and haemal arches, and was complete at 28 DAH. In comparison, the vertebral centra started to form at 16 DAH, with ossification being visible in all centra at 38 DAH. The pectorals were the only fins that formed before the onset of feeding (at 2 DAH), with ossification being complete at 45 DAH. The caudal fin formed at 5 DAH, with ossification being complete at 40 DAH. Dorsal and pelvic fin development began at 6 DAH and 20 DAH respectively. The ossification of both the dorsal and pelvic fins was visible at 45 DAH. The anal fin began forming at 14 DAH, and was complete at 30 DAH. The ossification of the anal fin was complete at 45 DAH. Overall, 22 types of skeletal deformities were detected in about 41% of individuals. Most anomalies were detected in the haemal region, while the fewest anomalies were detected in the anal fin. In addition, the frequency of deformities gradually increased with fish age at the different developmental stages. Our results are expected to contribute baseline information on how rearing conditions impact skeletal development, in addition to identifying potential causative factors of skeletal deformities.     

Early development and allometric growth in hatchery‐reared Eurasian perch,Perca fluviatilis L.

The morphological development and allometric growth patterns of Eurasian perch, Perca fluviatilis L., a highly valued commercial species, were studied under intensive rearing conditions from hatching up to 50 DPH (Days Post Hatch). Based on the external morphology, four different phases during early development of Eurasian perch were identified: pre‐flexion larva 0–20 DPH (5.70–10.16 mm TL); flexion larva 22–30 DPH (11.09–15.14 mm TL) and post‐flexion larva/juvenile 32–50 DPH (18.00–24.75 mm TL). The results indicate that growth period when final replacement of all temporary (larval) structures and most important changes in the shape of P. fluviatilis occurred (between 13.95 and 24.06 mm TL, during flexion and post‐flexion phase) can be considered as a transitional period between the larva and juvenile. All body segments, except trunk length and tail length showed fast growth (positive allometry) throughout the entire studied period or up to the respective inflexion point with a common tendency to isometry. In addition, the specific behaviours (e.g. pelagic way of life) of Eurasian perch larvae resulted in some characteristic allometric growth patterns in the posterior region, different from the majority of other teleosts. The results are discussed with respect to the ontogeny of the functional morphology under both ecological and aquaculture considerations.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

Exophthalmia in wild‐caught cod (Gadus morhua L.): development of a secondary barotrauma effect in captivity

Capture‐based aquaculture (CBA) of Atlantic cod (Gadus morhua) has become increasingly important in recent years, and increased attention is being paid to animal welfare issues linked to these activities. Earlier studies showed that some cod develop secondary exophthalmia in captivity. This study investigated the development of secondary exophthalmia in two groups of wild‐caught cod, one of which was exposed to rapid decompression causing acute barotrauma (treatment group) while the other was not (control group). Photographs and radiographs before and up to 33 days after barotrauma revealed a significant increase in overall eye protrusion caused by an accumulation of gas in the orbita in the treatment group, first observed on day 9 after decompression, while no protrusions were observed in the control group. Barotrauma was thus identified as an important trigger for the development of secondary uni‐ or bilateral exophthalmia of wild‐caught cod. Two underlying mechanisms are suggested, where the more likely is residual swim bladder gas taking the route of least resistance, while the less likely is the exsolution of gas from the blood. Our results have implications for a wide range of contexts in which cod are rapidly brought to the surface from great depth.     

Promotion of initial swim bladder inflation and effect of initial inflation failure on skeletal malformation in cultured seven‐band grouper Hyporthodus septemfasciatus (Thunberg)

The effects of the removal of the oily surface film on initial swim bladder inflation and survival in cultured seven‐band grouper Hyporthodus septemfasciatus were investigated during the larval stage. Additionally, the relationship between swim bladder inflation failure and malformations in later stages was examined. To explore promotion conditions for inflation, duplicate experiments were conducted under three different water surface conditions: removal of the oily surface film (ROF group), covering the water surface with an oil film (COF group), and sealing the water surface with liquid paraffin (SLP group). Larval swim bladder inflation was first observed 14 days after hatching (DAH) in the ROF group; thereafter, swim bladder inflation rates increased to 11.1% and 38.7% until the end of the experiments. This indicates that initial swim bladder inflation occurs at the pre‐flexion stage in larval development. Conversely, bladder inflation was rarely observed in the COF or SLP groups, indicating the promotional effect of surface film removal on larval swim bladder inflation and suggesting that gulping atmospheric air is necessary for initial inflation. Although larval water surface death occurred frequently in the ROF group, no significant difference existed in any group between the survival rates at 10 DAH and at the end of the experiment. The incidence rate of spinal curvature and deformation of the centra was significantly higher in individuals without initial swim bladder inflation, and these malformations mainly occurred in the caudal vertebra. Therefore, the removal of the oily surface film promotes initial swim bladder inflation and reduces larval malformations.     

Assessment of temperature or salinity effects on larval development by catecholamine‐induced metamorphosis of hatchery‐reared flat oyster,Ostrea angasi (Sowerby 1871) larvae

A need to improve larval rearing techniques led to the development of protocols for catecholamine‐induced settlement of flat oyster, Ostrea angasi, larvae. To further refine these techniques and optimize settlement percentages, the influence of salinity or temperature on development of O. angasi larvae was assessed using epinephrine‐induced metamorphosis. Larvae were reared between salinities of 15–35 and temperatures between 14.5 and 31°C. The greatest percentage survival, growth, development occurred when larvae were reared between 26 and 29°C and between salinities of 30 and 35. Larvae reared outside this salinity and temperature range exhibited reduced growth, survival and/or delayed development. Short‐term (1 h) reduction in larval rearing temperature from 26°C to 23.5°C significantly increased larval metamorphosis without affecting larval survival. Short‐term (1 h) increase in larval rearing temperature from 26°C to 29 and 31°C decreased larval survival and metamorphosis. To ensure repeatability in outcomes, tests showed that larvae sourced from different estuaries did not vary significantly in their metamorphic response to short‐term temperature manipulation and epinephrine‐induced metamorphosis.     

Parasites of cultured and wild brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) in Lampung Bay,Indonesia

A total of 210 Epinephelus fuscoguttatus (brown‐marbled grouper) was examined for parasites. During three consecutive seasons (two rainy and one dry season from 2002 to 2004), 35 specimens each taken from floating net cages of the National Sea Farming Development Centre (Balai Budidaya Laut) and from wild catches in Lampung Bay, South Sumatra, Indonesia were studied. Twenty‐five (cultured grouper) and 30 (wild grouper) parasite species/taxa were identified, with an infracommunity ranging from one to nine (cultured) and three to 14 parasite species (wild), demonstrating a species‐rich parasite fauna even in the cultured fish. Protozoans (1 species), microsporeans (1), myxozoans (1), digeneans (8), monogeneans (5), cestodes (3), nematodes (8), acanthocephalans (2) and crustaceans (6) were found. The most abundant parasites were the monogeneans Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus lantauensis for both, cultured and wild grouper during all seasons. For the cultured fish, the prevalence of monoxenous ectoparasites (e.g. P. epinepheli, P. lantauensis, Capsalidae gen. et sp. indet., Benedenia epinepheli) was in most cases higher than that of heteroxenous endoparasites. This contrasts the wild grouper, where heteroxenous parasites such as Allopodocotyle epinepheli and Raphidascaris sp. occurred at a similar prevalence compared with the fairly abundant Pseudorhabdosynochus spp. No seasonality of infestation was observed for both cultured and wild fish. The high levels of infestation of potentially pathogenic monogeneans throughout the year could result in significant parasite outbreaks at the locality studied.     

Soy protein concentrate as an alternative in replacement of fish meal in the feeds of hybrid grouper,brown‐marbled grouper (Epinephelus fuscoguttatus) × giant grouper (E. lanceolatus) juvenile

Hybrid grouper juveniles (body weight, 6.1 ± 0.7 g) (brown‐marbled grouper, Epinephelus fuscoguttatus × giant grouper, E. lanceolatus) were fed with six isoproteic (50% crude protein) and isolipidic (12% crude lipid) feeds containing different levels of soy protein concentrate (SPC) in replacement of fish meal (SPC at 20%, 30%, 40%, 50% and 60% protein) and control feed (SPC0) for 6 weeks. Hybrid grouper juveniles were cultured in 100‐L fibreglass tank equipped with flow‐through water system and fed twice a day to apparent satiation level. The highest and lowest growth was recorded in fish fed SPC20 and SPC60 respectively. However, growth of SPC20 was not significantly higher than those fed SPC0, SPC30, SPC40 and SPC50 (p > .05). A decreasing growth trend was observed with the increasing level of SPC from feed SPC40 to SPC60. A noticeable better feed utilization was also observed in fish fed SPC0, SPC20, SPC30, SPC40 and SPC50 compared to fish fed SPC60 (p < .05). The fish condition factor, hepatosomatic index, viscerosomatic index and whole body proximate content of the fish were not affected by the graded levels of SPC. However, the body lipid content was significantly lower in fish fed SPC40 to SPC60 (p < .05). The apparent digestibility coefficient (ADC) of protein and lipid was significantly higher in fish fed SPC0 and SPC20 compared to other dietary treatments (p < .05). Based on the regression analysis on specific growth rate, the study suggests that the hybrid grouper grow best at 21.4% and can utilize up to 50% inclusion level of SPC in protein without significantly affect their growth and its body condition.     

Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.     

Effect of temperature,hypoxia and disinfection with ozonated seawater during somitogenesis on muscular development of the trunk in larval seven‐band grouper,Epinephelus septemfasciatus (Thunberg)

The cause of abnormal musculature on both sides of the same myotome in artificially cultured seven‐band grouper Epinephelus septemfasciatus (Thunberg) larvae remains unclear. We evaluated the effect of three factors: changes in temperature (between 22 and 24°C), disinfection with ozonated seawater (0.3 mg TROs L^?1, 1 min), and hypoxia (22 or 14% saturation) during the early (20 h post‐fertilization, hpf) and mid‐ (24 hpf) somite stages on trunk muscular development in larval seven‐band grouper. The thermal regimes and disinfection with ozonated seawater had no effect on muscle development (P > 0.05, G‐test). In contrast, exposure to hypoxia during both the early and mid‐somite stages increased the incidence of abnormal musculature in the trunk (P < 0.01, G‐test). These larvae had abnormal musculature on both sides of the trunk. In addition, the number of abnormal myotomes differed among these larvae. Exposure in the early and mid‐somite stages resulted in the highest frequency of abnormalities in the 11th and 13th myotome respectively. Our results suggest that hypoxia during somitogenesis can lead to abnormal muscle development in the trunk of artificially cultured seven‐band grouper larva.     

Pharmacokinetics of florfenicol and behaviour of its metabolite florfenicol amine in orange‐spotted grouper (Epinephelus coioides) after oral administration

Pharmacokinetics and elimination of florfenicol and florfenicol amine in grouper held in sea water at 23.3 ± 0.8 °C were studied using HPLC method after they were given a single peroral dose of florfenicol at 24 mg kg^?1 body weight. Florfenicol was rapidly absorbed from intestine and distributed extensively to all the tissues examined. The maximum concentrations (C_max, μg g^?1 or μg mL^?1) in plasma and tissues were observed at 2–6 h (the time to reach maximum concentration, T_max) except for bile (T_max = 24 h) and were in the order of intestine (52.02 ± 25.07) > bile (49.41 ± 28.16) > gill (45.12 ± 11.10) > plasma (28.28 ± 5.43) > liver (21.97 ± 12.08) > muscle (21.63 ± 6.12) > kidney (20.88 ± 11.28) > skin (19.10 ± 5.88). The drug distribution level was higher in plasma than in extravascular tissues except for bile, based on the ratios of the area under concentration–time curve between tissue and plasma (AUC_{tissue/plasma}). The elimination of florfenicol was rapid in fish, and the corresponding half‐lives (T_1/2β) in the order of magnitude were bile (13.92 h) > muscle or liver (12.31 h) > skin (11.77 h) > plasma (11.57) > gill (11.04 h) > intestine (10.55 h) > kidney (10.05 h). The delayed T_max, lower C_max and longer T_1/2β for florfenicol amine compared with florfenicol were measured in grouper.     

Experimental infection of brown‐marbled grouper,Epinephelus fuscoguttatus (Forskal), with Vibrio parahaemolyticus identifies parvalbumin beta‐2 subunit I,alpha‐2‐macroglobulin,nattectin and immunoglobulin light chain,differentially expressed in resistant grouper

The mechanisms through which brown‐marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown‐marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two‐dimensional gel electrophoresis (2‐DE). The results showed that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.     

Analysis of growth and first reproduction of hatchery‐reared juvenile Cortez oyster (Crassostrea corteziensis) in northwestern Mexico: proposal of a minimal fishing size

This study presents complimentary data of early growth and age/size of first reproduction of hatchery‐reared Crassostrea corteziensis juveniles cultivated in the field for 9 months. Samples of oysters and gonads were collected monthly to determine absolute growth and growth rates, as well as overall gonad development. At the end, adult oysters ranging 75.6 ± 0.62 mm shell height (SH) yielded a mean growth rate of 0.308 mm day⁻¹, which is among the highest reported for the species. Gametogenesis began in March–April and spawning occurred during August and September. Males outnumbered females at smaller sizes, but female were more abundant at larger sizes. From planting time in the field, the age/size of first maturity was 3.25 months for males (42 mm SH) and 4.5 months for females (54 mm SH), with a mean population age of 4.75 months for males (size range 55–59 mm) and 5.25 months for females (60–64 mm). Based on these results, and the time needed for hatchery care prior planting juveniles in the field (2.25 months), we propose limiting fishing of wild oysters to sizes >65 mm height to allow them to breed at least once and contribute to restoring wild populations.     

In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Mouse monoclonal antibodies (MAbs) were produced by using yellow grouper nervous necrosis virus (YGNNV) as an immunogen, isolated from infected yellow grouper, Epinephelus awoara (Temminck & Schlegel), and propagated in GB cells. In enzyme linked immunosorbent assay (ELISA), 43 hybridoma clones secreting MAbs strongly reacted with the purified virus. Ten of them showed a higher neutralization index (NI) value between 6.5 and 4.5 (log₁₀ NI) than the other 33 MAbs against YGNNV infection in cell culture. All 10 MAbs belonged to the IgG isotype with a κ light chain and recognized the 42 kDa coat protein of YGNNV by Western blot analysis. Immunohistochemical results demonstrated that the viral signals co-located with pathological lesions observed in retina, brain and spinal cord. These results indicate that the MAbs are useful for confirmative diagnosis of YGNNV infection.     

Comparative efficacy of trash fish versus compounded commercial feeds in cage aquaculture of Asian seabass (Lates calcarifer) (Bloch) and tiger grouper (Epinephelus fuscoguttatus) (Forsskål)

Trash fish/low valued fish is still the most commonly used feed in marine cage culture in Asia. Use of trash fish is controversial with regard to the sustainability of the farming practices, and related issues on environmental degradation. In this study, the results of farm based trials on Asian seabass (Lates calcarifer) and tiger grouper (Epinephelus fuscoguttatus) to evaluate the efficacies of the use of commercial pellet feed in comparison to trash fish/low valued fish in marine cages in Thailand are presented. In spite of the variations observed in each of the farm sites, the results indicate that the overall growth performances and fish survival rates between the two feed types were similar, for both species. However, better cost‐benefit and resource use were recorded for fish reared on trash fish/low valued fish. No significant differences were observed in water quality parameters, dissolved oxygen, transparency and ammonia, between sites where pellet or trash fish were used, inside and outside the cages and for both species. The results of the present study are discussed in the light of the current controversies on the use of trash fish/low valued fish in marine cage culture.     

Aeration rate adjustment at night to prevent sinking syndrome‐related death in the tiger grouper Epinephelus fuscoguttatus (Perciformes:Serranidae) larvae

The effects of different aeration rates at night to prevent sinking syndrome‐related death (SSRD) of the tiger grouper, Epinephelus fuscoguttatus were examined. The aeration rates were fixed at 300 mL min^?1 at daytime (07:00–19:00 hours) and regulated to 0, 300 and 900 mL min^?1 at night (19:00–07:00 hours). Larval survival, growth, feeding intake, sinking velocity, distribution and behaviour, stress level, surface tension‐related death (STRD) and flow velocity distribution were assessed. The occurrence of SSRD in the tiger grouper was observed through the accelerated sinking velocity (Vl) (from 0.15 ± 0.09 cm s^?1 at 4 days AH to 0.41 ± 0.09 cm s^?1 at 12 days AH) coupled with larval passive swimming behaviour at night‐time. On the final day of experiment (15 days AH), larvae reared in 900 mL min^?1 at night had attained significantly higher (P < 0.05) survival (34.4 ± 5.5%), growth (5.8 ± 0.5 mm) and feeding intake (60.46 ± 6.98 ind. larva^?1). A favourable flow field for the tiger grouper was produced in 900 mL min^?1 at night‐time, in which larvae were transported 15–25 cm above the tank bottom and 1.0 cm beneath the water surface. Under these night‐time rearing conditions, larval stress level and number of STRD reared in 900 mL min^?1 compared with those observed in 300 mL min^?1 remained insignificant, indicating that strong turbulence of flow velocity was not detrimental for larvae. Our findings recommend aeration at 900 mL min^?1 at night as this could improve larval survival by reducing SSRD.