Skin abrasions often occur in farmed fish following handling by labourers, injury by farm facilities, cannibalism and ectoparasites. Vibrio spp. are opportunistic pathogens that can invade host fish through damaged tissues and cause outbreaks of vibriosis. This study describes the effect of skin abrasions on the infectivity of V. harveyi using Asian seabass Lates calcarifer (Bloch, 1790) fingerlings as a case example and compares bacterial load and fish survival following immersion challenge with different doses. In total, 315 fish (6.67 ± 1.8 g) were divided into 3 treatments: skin abrasion followed by immersion infection, immersion infection only and an uninfected, uninjured control. Fish in the infection treatments were divided into 3 subgroups and exposed in triplicate to a 7 d immersion challenge with 106, 107 and 108 CFU ml-1 of live V. harveyi. No mortalities were observed in the control and immersion infection groups. However, fish in the skin abrasion treatment group that were infected with 108 CFU ml-1 of live V. harveyi showed signs of progressing disease throughout the experiment, which resulted in mortalities. Significantly higher bacterial loads (p < 0.05) were recorded in the intestine, liver and gills of the fish in this group. Fish in the skin abrasion treatment that were exposed to 107 and 108 CFU ml-1 of V. harveyi showed 100% mortality by Days 5 and 4, respectively. These findings confirm that skin injuries increase the susceptibility of seabass fingerlings to V. harveyi infection.
Rising of temperature in conjunction with acidification due to the anthropogenic climates has tremendously affected all aquatic life. Small changes in the surrounding environment could lead to physiological constraint in the individual. Therefore, this study was designed to investigate the effects of warm water temperature (32 °C) and low pH (pH 6) on physiological responses and growth of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles for 25 days. Growth performance was significantly affected under warm water temperature and low-pH conditions. Surprisingly, the positive effect on growth was observed under the interactive effects of warm water and low pH exposure. Hybrid grouper exposed to the interactive stressor of warm temperature and low pH exhibited higher living cost, where HSI content was greatly depleted to about 2.3-folds than in normal circumstances. Overall, challenge to warm temperature and low pH induced protein mobilization as an energy source followed by glycogen and lipid to support basal metabolic needs.
In this study, we aimed to isolate, identify and characterize lactic acid bacteria (LAB) from the intestine of juvenile seabass (Lates calcarifer) as a new potential probiotic. Four strains of LABs were isolated from the intestines of ten healthy seabass juveniles. In the in vitro screening process using spot lawn assay, one isolate labeled as LAB3 showed inhibitory activity against Vibrio harveyi (ATCC 35,084). Strain LAB3 was determined to belong to the gram positive bacteria group with cocci shape and was identified as Enterococcus hirae using 16S rDNA analysis. This bacterium was able to grow at pH ranging from pH 2 to 10 with the best growth at pH 7. This strain was also able to grow at 0-4% NaCl after 24 h incubation and grew best at 1.5% NaCl. Enterococcus hirae strain LAB3 of the present study is worthy to be further characterized as a potential probiotic for use in seabass culture.
Feed efficiency (FE) is the amount of body weight gain for a given feed intake. Improving FE through selective breeding is key for sustainable finfish aquaculture but its evaluation at individual level is technically challenging. We therefore investigated whether individual routine metabolic rate (RMR) was a predictor of individual FE in the European sea bass Dicentrarchus labrax, a major species in European mariculture. The European sea bass has three genetically distinct populations across its geographical range, namely Atlantic (AT), West Mediterranean (WM), and East Mediterranean (EM). We compared FE and RMR of fish from these three populations at 18 or 24 °C. We held 200 fish (62 AT, 66 WM, and 72 EM) in individual aquaria and fed them from ad libitum down to fasting. FI was assessed for an ad libitum feeding rate and for a fixed restricted ration (1% of metabolic body weight·day-1, with metabolic body weight = body weight0.8). After being refed 12 wk in a common tank, individual RMR was measured over 36 h by intermittent flow respirometry. There was a significant effect of temperature whereby fish at 18 °C had greater mean FE (P < 0.05) and lower RMR (P < 0.001). There was also a significant effect of population, where AT fish had lower FE (P < 0.05) and greater RMR (P < 0.001) than WM and EM, at both temperatures. Despite these differences in temperature and population means, individual FE and RMR were not significantly correlated (P > 0.05). Therefore, although the results provide evidence of an association between metabolic rate and FE, RMR was not a predictor of individual FE, for reasons that require further investigation.
Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 +/- 0.71 (range: 6-19), and the expected heterozygosity averaged 0.76 +/- 0.02 (range: 0.63-1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM-T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3-5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 x 10 and 20 x 20) demonstrated a high power of these markers for revealing parent-sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.
Some of Vibrio species is well known as pathogenic bacteria in aquaculture and the marine industry. Its infection is able to generate a massive outbreak and affect the fish population, especially for net caged fish such as seabass. This study was conducted to investigate the prevalence of Vibrio spp. isolated from seabass (Lates calcarifer) in Sri Tujuh Lagoon, Tumpat, Kelantan. Then, to determine the antibiotic resistance in Vibrio isolates. Polymerase chain reaction (PCR) was used to detect Vibrio species using specific primer VR169 and VR744 with estimation base pair size band, 597 bp and further identified by sequencing. On the other hand, antibiotic susceptibility tests were continued by using 13 types of antibiotics; kanamycin (K30), chloramphenicol (C30), neomycin (N10), ampicillin (AMP10), nitrofurantoin (F300), tetracycline (TE30), streptomycin (S10), norfloxacin (NOR10), ciprofloxacin (CIP5), nalidixic acid (NA30), gentamicin (CN10), doxycycline (DO30) and sulfamethoxazole (SXT100). As a result, 14 Vibrio isolates were identified, including Vibrio fluvialis (n=6), Vibrio parahaemolyticus (n=3), Vibrio harveyi (n=2) and each isolate for Vibrio vulnificus, Vibrio alginolyticus and Vibrio spp. The results showed that all isolates were sensitive to most antibiotics except ampicillin, neomycin and streptomycin. The MAR index value was ranging from 0 to 0.31. This study demonstrates the prevalence of Vibrio spp. in seabass and the report on multidrug resistance strains that could be of concern to the fish farmers. In addition, data from this study can be further used in fish disease management plans.
Grouper culture has been expanding in Malaysia due to the huge demand locally and globally. However, due to infectious diseases such as vibriosis, the fish mortality rate increased, which has affected the production of grouper. Therefore, this study focuses on the metabolic profiling of surviving infected grouper fed with different formulations of fatty acid diets that acted as immunostimulants for the fish to achieve desirable growth and health performance. After a six-week feeding trial and one-week post-bacterial challenge, the surviving infected grouper was sampled for GC-MS analysis. For metabolite extraction, a methanol/chloroform/water (2:2:1.8) extraction method was applied to the immune organs (spleen and liver) of surviving infected grouper. The distribution patterns of metabolites between experimental groups were then analyzed using a metabolomics platform. A total of 50 and 81 metabolites were putatively identified from the spleen and liver samples, respectively. Our further analysis identified glycine, serine, and threonine metabolism, and alanine, aspartate and glutamate metabolism had the most impacted pathways, respectively, in spleen and liver samples from surviving infected grouper. The metabolites that were highly abundant in the spleen found in these pathways were glycine (20.9%), l-threonine (1.0%) and l-serine (0.8%). Meanwhile, in the liver l-glutamine (1.8%) and aspartic acid (0.6%) were found to be highly abundant. Interestingly, among the fish diet groups, grouper fed with oleic acid diet produced more metabolites with a higher percent area compared to the control diets. The results obtained from this study elucidate the use of oleic acid as an immunostimulant in fish feed formulation affects more various immune-related metabolites than other formulated feed diets for vibriosis infected grouper.
Fish epidermal mucus is an important reservoir of antipathogenic compounds which serves as the first line of the immune defence. Despite its significant role in the physiology and health of fish, detailed profiling of fish epidermal mucus has yet to be explored. Therefore, this study investigates a label-free colloidal surface-enhanced Raman spectroscopic (SERS) method for profiling grouper mucus. Gold nanoparticles were first synthesised using the standard citrate reduction and characterised using ultraviolet-visible spectroscopy, transmission electron microscopy and dynamic light scattering. The influence of acidified sodium sulphate (Na2SO4) at pH 3 as the aggregating agent on the enhancement of the SERS spectrum of different analyte samples including rhodamine 6G (R6G) dye, lysozyme solution and hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) mucus was observed. Based on the results, an optimal Na2SO4 concentration of 1 M was recorded to achieve the highest enhancement of the SERS signal for R6G and grouper mucus, while the optimal concentration for lysozyme was 0.1 M. The results indicated a higher degree of aggregation induced by lysozyme than R6G and grouper mucus. A few overlapping peaks of the SERS spectra of lysozyme and grouper mucus made it possible to confirm the presence of lysozyme as potential biomarkers.
Vibriosis disease by Vibrio spp. greatly reduced productivity of aquaculture, such as brown-marbled grouper (Epinephelus fuscoguttatus), which is an economically important fish species in Malaysia. Preventive measures and immediate treatment are critical to reduce the mortality of E. fuscoguttatus from vibriosis. To investigate the molecular mechanisms associated with immune response and host-bacteria interaction, a transcriptomic analysis was performed to compare between healthy and Vibrio-infected groupers. This permits the discovery of immune-related genes, specifically the resistance genes upon infection. Herein, we provide the raw transcriptome data from Illumina HiSeq. 4000 that have been deposited into NCBI SRA database with the BioProject accession number PRJNA396437. A total of 493,403,076 raw sequences of 74.5 Gb were obtained. Trimming of the raw data produced 437,186,232 clean reads of ~58 Gb. These datasets will be useful to elucidate the defence mechanisms of E. fuscoguttatus against Vibrio vulnificus infection for future development of effective prevention and treatment of vibriosis.
The grow out of Asian seabass Lates calcarifer in marine net-cages is a popular aquaculture activity in Malaysia. Production of this species is greatly affected by the occurrence of vibriosis, which causes heavy mortality. Generally, young fish are more susceptible; they exhibit anorexia and skin darkening, followed by heavy mortality. The acutely affected older fish may also exhibit bloody lesions around the anus and the base of the fins. Twenty-one bacterial isolates obtained from internal organs (kidney, heart, spleen and liver) of the affected specimens were subjected to phenotypic characterization, testing for antibiotic susceptibility, and 16S ribosomal DNA sequencing. The sequencing result showed that all of the bacterial isolates belonged to Vibrio harveyi. The phenotypic characterization, however, identified 4 of the bacterial isolates as V. harveyi, 16 as V. parahaemolyticus, and 1 as V. alginolyticus. These findings suggest that biochemical features alone cannot be reliably used to identify bacterial pathogens, including V. harveyi, in aquaculture. Antibiotic susceptibility assays showed that some antibiotics, including oxytetracycline, nitrofurantoin, furazolidone, streptomycin, sulfamethoxazole, chloramphenicol, nalidixic acid, and oxolinic acid were effective against V. harveyi. Considering the side effects of these antibiotics, however, their use is not recommended in the aquaculture of Asian seabass.
A new sanguinicolid blood fluke, Parasanguinicola vastispina, is described from sea bass Lates calcarifer cultured in Malaysia. It is distinguished by its massive armature and widely spaced genital pores, the female pore being pre-ovarian. P. vastispina inhabits the branchial arteries, dorsal aorta, mesenteric venules and renal artery of its host. No pathological effect was observed in infected fish.
This study focuses in investigating the fatty acid contents of surviving infected hybrid grouper fed with oleic acid immunostimulant. After a 6-week feeding trial, Epinephelus fuscoguttatus × Epinephelus lanceolatus fingerlings were infected with Vibrio vulnificus. One week after bacterial challenge, fish oil was extracted from body tissue of surviving infected fingerlings using the Soxhlet extraction method. The extracted samples were then sent for GC-MS analysis. The raw GC-MS data were analyzed using software programs and databases (i.e., MetaboAnalyst, SIMCA-P, NIST Library, and KEGG). A total of 39 metabolites were putatively identified, with 18 metabolites derived from the fatty acid group. Our further analysis revealed that most metabolites were highly abundant in the oleic acid dietary samples, including oleic acid (4.56%), 5,8,11-eicosatrienoic acid (3.45%), n-hexadecenoic acid (3.34%), cis-erucic acid (2.76%), and 9-octadecenoic acid (2.5%). Worthy of note, we observed a greater abundance of α-linoleic acid (15.57%) in the control diet samples than in the oleic acid diet samples (14.59%) with no significant difference in their results. The results obtained from this study revealed that surviving infected hybrid grouper expressed more immune-related fatty acids due to the effect of oleic acid immunostimulant. Therefore, in this study, we propose oleic acid as a potential immunostimulant in enhancing fish immunity in aquaculture industry.
In the present study, we employed microsatellite DNA markers to analyze the genetic diversity and differentiation between and within cultured stocks and wild populations of the orange-spotted grouper originating from the South China Sea and Southeast Asia. Compared to wild populations, genetic changes including reduced genetic diversity and significant differentiation have taken place in cultured grouper stocks, as shown by allele richness and heterozygosity studies, pairwise F(st), structure, molecular variance analysis, as well as multidimensional scaling analysis. Although two geographically adjacent orange-spotted grouper populations in China showed negligible genetic divergence, significant population differentiation was observed in wild grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia. However, the Mantel test rejected the isolation-by-distance model of genetic structure, which indicated the genetic differentiation among the populations could result from the co-effects of various factors, such as historical dispersal, local environment, ocean currents, river flows and island blocks. Our results demonstrated that microsatellite markers could be suitable not only for genetic monitoring cultured stocks but also for revealing the population structuring of wild orange-spotted grouper populations. Meanwhile, our study provided important information for breeding programs, management of cultured stocks and conservation of wild populations of the orange-spotted grouper.
A 25-week feeding trial was conducted to assess the growth performance, organoleptic quality, and to estimate the viability of nourishing hybrid grouper (Epinephelus fuscoguttatus x Epinephelus lanceolatus) with low-cost fish (LCF) and commercially compound feed (CCF). A group of 3600 juvenile fish (182g) were released in four sea cages and fed with either LCF or CCF in duplicate. At the end of the trial, the hybrid grouper provided LCF attained a significantly higher (P0.05). Although technicalities of fish fed with LCF suggest that LCF is more efficient than CCF, feeding LCF to high-value fish is an unsustainable practice as LCF is usually obtained through trawling – a destructive fishing method for the marine ecosystem. Therefore, feeding with CCF without the use of LCF as the source of protein for its fishmeal will contribute to sustainable aquaculture. In order to convince the local farmers in Sabah to adopt the practice of feeding CCF, future research should focus on completing the species-specific diet formulation to promote optimum growth, and find ways to reduce the CCF local selling price.
Gelatins from the skin of four local marine fish, namely “kerapu” (Epinephelus sexfasciatus), “jenahak” (Lutjianus argentimaculatus), “kembung” (Rastrelliger kanagurta), and “kerisi” (Pristipomodes typus) have been successfully extracted by acid extraction. Results characterization showed that the fish gelatins were comparable to the fish gelatins from other fish species previously reported. They appeared snowy white in color with crystal-like and light texture. The gelatine extracted from “kerapu” had the strongest fishy odor, followed by the gelatines derived from “jenahak”, “kembung” and “kerisi”. In terms of bloom strength, the gelatin extracted from “kerapu” was found to be the strongest one compared to others, with the bloom value of more than 2000 g. The gelatins developed in this study contained almost all essential amino acids, with glycine being the most predominant one.
The otoliths of sea bass (Lates calcarifer) were sampled from 23 locations in Peninsular Malaysia in order to analyse the Sr/Ca, Mg/Ca and Ba/Ca ratios. This study found that these elements/Ca ratios in sea bass otoliths were correlated with the salinity zone (thalassic series) compared with elements/Ca ratios in ambient water. The contradictory pattern of Sr/ Ca ratios and Ba/Ca ratios in otoliths was found according to salinity zone variations. Thus, oligohaline waters showed the highest Ba/Ca ratios while the highest Sr/Ca ratios were recorded for saline waters. The terrigenous enrichment of Ba in water also affects the Ba content in otoliths. The presence of Mg in otoliths acts as a rival to Ba, thus affecting Mg and Ba deposition. Meanwhile, Ba is the tracer for salinity fluctuations based on the partition coefficient (D) and it shows significant changes. However, the elements in otoliths were found to originate indirectly from the water column. The sequence of the elements/Ca ratios in otoliths and ambient water was Sr/Ca>Mg/Ca>Ba/Ca and Sr/Ca>Ba/Ca>Mg/ Ca, respectively.
The effects of dietary protein level on the growth performance and ammonia excretion of the leopard coral grouper,
Plectropomus leopardus were investigated for eight weeks. Fish were fed diets with 40, 45, 50, 55 and 60% crude protein
levels in separate recirculating systems. Fish fed with the 50% crude protein containing diet showed the best ingestion
rate, which was significantly higher than that found in the other groups. As the dietary protein level increased, the
specific growth rate increased significantly and it reached the highest level at 50% crude protein containing diet. Based
on the results of all measured parameters 50% protein containing diet was the best among all test diets. The regression
equation for dietary protein level versus ammonia excretion indicated that the optimal dietary protein level with the
least ammonia excretion was 53.14%. More research is still needed to elucidate the effects of 53.14% crude protein
containing diet on the specific growth rate, feed conversion ratio, protein efficiency ratio and ingestion rate of leopard
coral grouper before recommending this level. Until then, 50% protein containing diet can be recommended for leopard
coral grouper culture in the recirculation system.
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.
Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture. In this study, we evaluated if the recombinant sea bass ferritin-H could stimulate the innate immune system of gnotobiotic European sea bass larvae resulting in protection against a V. anguillarum challenge. We also evaluated the effect of a V. anguillarum infection on the transcription of immune-related genes in gnotobiotic European sea bass larvae. Recombinant sea bass ferritin-H was produced, encapsulated in calcium alginate microparticles and orally delivered to sea bass larvae at seven days after hatching. Our results showed V. anguillarum caused an acute infection, resulting in high mortality. The infection significantly upregulated the expression of tlr3, tlr5, cas1, il1β, tnfα, mif, il10, cc1, cxcl8 at 18, 24 and 36 h post infection, but not of the chemokine receptor genes cxcr4 and ccr9. There was no protective effect of ferritin-H. Remarkably, ferritin-H caused significantly higher transcript levels for cxcr4 and ccr9. Sea bass ferritin-H was more likely involved in immune-suppression and results point in the direction of a negative regulation of CXCR4 resulting in inhibition of cell proliferation, differentiation and migration which is detrimental to innate immunity and might explain the non-protective effect of ferritin-H in fish larvae.