The purpose of this study was to characterize major allergens of Indian scad (Decapterus russelli) which is among the most commonly consumed fish in Malaysia. Raw and cooked extracts of the fish were prepared. Protein profiles and IgE binding patterns were produced by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from subjects with fish allergy. The major allergens of the fish were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry of the peptide digests. The SDS-PAGE of the raw extract revealed 27 protein fractions over a wide molecular weight range, while the cooked extract demonstrated only six protein fractions. The 1-DE immunoblotting detected 14 IgE-binding proteins, with a molecular weight range from 90 to < 6.5 kDa. Three protein fractions with molecular weights of approximately 51, 46 and 12 kDa were identified as the major allergens of this fish. The approximately 12 kDa band was a heat-resistant protein while the approximately 51 and 46 kDa proteins were sensitive to heat. The 2-DE gel profile of the raw extract demonstrated > 100 distinct protein spots and immunoblotting detected at least 10 different major IgE reactive spots with molecular masses as expected and isoelectric point (pI) values ranging from 4.0 to 7.0. A comparison of the major allergenic spot sequences of the 12 kDa proteins with known protein sequences in databases revealed extensive similarity with fish parvalbumin. In conclusion, this study demonstrated that a parvalbumin which is similar to Gad c 1 is the major allergen of Indian scad. Interestingly, we also detected heat-sensitive proteins as major allergenic components in our fish allergy patients.
Topical emulsions stabilized with non-ionic emulsifiers have been an attractive alternative as vehicles for drug delivery, particularly for the patients suffering from dermatological problems. Haruan (a natural wound healer) creams were formulated with different types of emulsifiers (Tween 80 and Span 80) using different grades of Malaysian Palm-oleins (DFPL 56, 60, 62 and 65). The stability (at room temperature and accelerated stability testing) of the various creams was evaluated at different temperatures (5, 25 and 45 degrees C) for a period of 6 months by measuring changes in droplet size, viscosity and percentage oil separation. The emulsifier type and concentration showed pronounced effect on the physicochemical properties of the cream, whereas storage time did not. This study suggested that the choice of emulsifiers and concentration of haruan extract are the most important factors in the stability of the haruan creams.
Periophthalmodon schlosseri is an amphibious and obligatory air-breathing teleost, which is extremely tolerant to environmental ammonia. It actively excretes NH(4)(+) in ammonia loading conditions. For such a mechanism to operate efficaciously the fish must be able to prevent back flux of NH(3). P. schlosseri could lower the pH of 50 volumes (w/v) of 50% seawater in an artificial burrow from pH 8.2 to pH 7.4 in 1 day, and established an ambient ammonia concentration of 10 mmol l(-1) in 8 days. It could alter the rate of titratable acid efflux in response to ambient pH. The rate of net acid efflux (H(+) excretion) in P. schlosseri was pH-dependent, increasing in the order pH 6.0<7.0<8.0<8.5. Net acid flux in neutral or alkaline pH conditions was partially inhibited by bafilomycin, indicating the possible involvement of a V-type H(+)-ATPase. P. schlosseri could also increase the rate of H(+) excretion in response to the presence of ammonia in a neutral (pH 7.0) external medium. Increased H(+) excretion in P. schlosseri occurred in the head region where active excretion of NH(4)(+) took place. This would result in high concentrations of H(+) in the boundary water layer and prevent the dissociation of NH(4)(+), thus preventing a back flux of NH(3) through the branchial epithelia. P. schlosseri probably developed such an 'environmental ammonia detoxification' capability because of its unique behavior of burrow building in the mudflats and living therein in a limited volume of water. In addition, the skin of P. schlosseri had low permeability to NH(3). Using an Ussing-type apparatus with 10 mmol l(-1) NH(4)Cl and a 1 unit pH gradient (pH 8.0 to 7.0), the skin supported only a very small flux of NH(3) (0.0095 micromol cm(-2) min(-1)). Cholesterol content (4.5 micromol g(-1)) in the skin was high, which suggests low membrane fluidity. Phosphatidylcholine, which has a stabilizing effect on membranes, constituted almost 50% of the skin phospholipids, with phosphatidyleserine and phsophatidylethanolamine contributing only 13% and 15%, respectively. More importantly, P. schlosseri increased the cholesterol level (to 5.5 micromol g(-1)) and altered the fatty acid composition (increased total saturated fatty acid content) in its skin lipid after exposure to ammonia (30 mmol l(-1) at pH 7.0) for 6 days. These changes might lead to an even lower permeability to NH(3) in the skin, and reduced back diffusion of the actively excreted NH(4)(+) as NH(3) or the net influx of exogenous NH(3), under such conditions.
Endocrine disrupting compound (EDC) contamination in food is a global concern. Concerning potential environmental and human health exposed to EDCs via food intake, an experiment was conducted on the selected EDCs concentration in the mariculture fish, Trachinotus blochii (golden pomfret), Lutjanus campechanus (snapper), and Lates calcarifer (sea bass) at Pulau Kukup, Johor. Mariculture activity at Pulau Kukup involves active export of fishes to Singapore and Indonesia. The recovery of BPA (bisphenol A), 4OP (4-octylphenol), and 4NP (4-nonylphenol) were 61.54%-93.00%, 16.79%-17.13%, and 61.24%-71.49%, respectively. Relatively high concentration of BPA was recorded in T. blochii (0.322ng/g), followed by L. calcarifer (0.124ng/g) and L. campechanus (0.023ng/g). Furthermore, 4OP and 4NP were detected only in T. blochii at concentrations of 0.084ng/g and 0.078ng/g, respectively. The results of the present study provide insights on monitoring and managing mariculture activity in relation to environmental protection and food safety.
Mariculture fish contains a rich source of protein, but some species may bioaccumulate high levels of heavy metals, making them unsafe for consumption. This study aims to identify heavy metal concentration in Lates calcarifer (Barramudi), Lutjanus campechanus (Red snapper) and Lutjanus griseus (Grey snapper). Three species of mariculture fish, namely, L. calcarifer, L. campechanus and L. griseus were collected for analyses of heavy metals. The concentration of heavy metal (As, Cd, Cu, Cr, Fe, Pb, Mn, Ni, Se, and Zn) was determined using inductive coupled plasma mass spectrometry (ICP-MS). The distribution of heavy metals mean concentration in muscle is Zn > Fe > As > Se > Cr > Cu > Mn > Pb > Ni > Cd for L. calcarifer, Fe > Zn > Cr > As > Ni > Mn > Se > Cu > Pb > Cd for L. campechanus and Fe > Zn > Cr > Ni > Se > Cu > As > Mn > Pb > Cd for L. griseus. Among all of the species under investigation, the highest concentration of Fe was found in the muscle tissue of L. campechanus (19.985 ± 1.773 mg kg-1) and liver tissue of L. griseus (58.248 ± 8.736 mg kg-1). Meanwhile, L. calcarifer has the lowest concentration of Cd in both muscle (0.007 ± 0.004 mg kg-1) and liver tissue (0.027 ± 0.016 mg kg-1). The heavy metal concentration in muscle tissue is below the permissible limit guidelines stipulated by the Food & Agriculture Organization, 1983 and Malaysia Food Act, 1983. The concentration of heavy metals varies significantly among fish species and tissues. L. campechanus was found to have a higher ability to accumulate heavy metals as compared to the other two species (p
Thirty snakehead fish, Channa micropeltes (Cuvier, 1831) were collected at Lake Kenyir, Malaysia. Muscle, liver, intestine and kidney tissues were removed from each fish and the intestine was opened to reveal cestodes. In order to assess the concentration of heavy metal in the environment, samples of water in the surface layer and sediment were also collected. Tissues were digested and the concentrations of manganese (Mn), zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) were analysed by using inductively-coupled plasma mass-spectrometry (ICP-MS) equipment. The results demonstrated that the cestode Senga parva (Fernando and Furtado, 1964) from fish hosts accumulated some heavy metals to a greater extent than the water and some fish tissues, but less than the sediment. In three (Pb, Zn and Mn) of the five elements measured, cestodes accumulated the highest metal concentrations, and in remaining two (Cu and Cd), the second highest metal accumulation was recorded in the cestodes when compared to host tissues. Therefore, the present study indicated that Senga parva accumulated metals and might have potential as a bioindicator of heavy-metal pollution.
Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species.
The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA), a process to convert C18 polyunsaturated fatty acids into eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or arachidonic acid (ARA), requires the concerted activities of two enzymes, the fatty acyl desaturase (Fads) and elongase (Elovl). This study highlights the cloning, functional characterisation and tissue expression pattern of a Fads and an Elovl from the Boddart's goggle-eyed goby (Boleophthalmus boddarti), a mudskipper species widely distributed in the Indo-Pacific region. Phylogenetic analysis revealed that the cloned fads and elovl are clustered with other teleost orthologs, respectively. The investigation of the genome of several mudskipper species, namely Boleophthalmus pectinirostris, Periophthalmus schlosseri and Periophthalmus magnuspinnatus, revealed a single Fads2 and two elongases, Elovl5 and Elovl4 for each respective species. A heterologous yeast assay indicated that the B. boddarti Fads2 possessed low desaturation activity on C18 PUFA and no desaturation on C20 and C22 PUFA substrates. In comparison, the Elovl5 showed a wide range of substrate specificity, with a capacity to elongate C18, C20 and C22 PUFA substrates. An amino acid residue that affects the capacity to elongate C22:5n-3 was identified in the B. boddarti Elovl5. Both genes are highly expressed in brain tissue. Among all tissues, DHA is highly concentrated in neuron-rich tissues, whereas EPA is highly deposited in gills. Taken together, the results showed that due to the inability to perform desaturation steps, B. boddarti is unable to biosynthesise LC-PUFA, relying on dietary intake to acquire these nutrients.
The objective of this study was to elucidate if chronic and acute ammonia intoxication in mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti, were associated with high levels of ammonia and/or glutamine in their brains, and if acute ammonia intoxication could be prevented by the administration of methionine sulfoximine [MSO; an inhibitor of glutamine synthetase (GS)] or MK801 [an antagonist of N-methyl D-aspartate type glutamate (NMDA) receptors]. For P. schlosseri and B. boddaerti exposed to sublethal concentrations (100 and 8 mmol l(-1) NH4Cl, respectively, at pH 7.0) of environmental ammonia for 4 days, brain ammonia contents increased drastically during the first 24 h, and they reached 18 and 14.5 micromol g(-1), respectively, at hour 96. Simultaneously, there were increases in brain glutamine contents, but brain glutamate contents were unchanged. Because glutamine accumulated to exceptionally high levels in brains of P. schlosseri (29.8 micromol g(-1)) and B. boddaerti (12.1 micromol g(-1)) without causing death, it can be concluded that these two mudskippers could ameliorate those problems associated with glutamine synthesis and accumulation as observed in patients suffering from hyperammonemia. P. schlosseri and B. boddaerti could tolerate high doses of ammonium acetate (CH3COONH4) injected into their peritoneal cavities, with 24 h LC50 of 15.6 and 12.3 micromol g(-1) fish, respectively. After the injection with a sublethal dose of CH3COONH4 (8 micromol g(-1) fish), there were significant increases in ammonia (5.11 and 8.36 micromol g(-1), respectively) and glutamine (4.22 and 3.54 micromol g(-1), respectively) levels in their brains at hour 0.5, but these levels returned to normal at hour 24. By contrast, for P. schlosseri and B. boddaerti that succumbed within 15-50 min to a dose of CH3COONH4 (15 and 12 micromol g(-1) fish, respectively) close to the LC50 values, the ammonia contents in the brains reached much higher levels (12.8 and 14.9 micromol g(-1), respectively), while the glutamine level remained relatively low (3.93 and 2.67 micromol g(-1), respectively). Thus, glutamine synthesis and accumulation in the brain was not the major cause of death in these two mudskippers confronted with acute ammonia toxicity. Indeed, MSO, at a dosage (100 microg g(-1) fish) protective for rats, did not protect B. boddaerti against acute ammonia toxicity, although it was an inhibitor of GS activities from the brains of both mudskippers. In the case of P. schlosseri, MSO only prolonged the time to death but did not reduce the mortality rate (100%). In addition, MK801 (2 microg g(-1) fish) had no protective effect on P. schlosseri and B. boddaerti injected with a lethal dose of CH3COONH4, indicating that activation of NMDA receptors was not the major cause of death during acute ammonia intoxication. Thus, it can be concluded that there are major differences in mechanisms of chronic and acute ammonia toxicity between brains of these two mudskippers and mammalian brains.
There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.
In February 2013, forty-seven Notched threadfin bream, the Nemipterus peronii, were sampled from the eastern coastal waters of the South China Sea. The concentration of various elements, namely cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), strontium (Sr), manganese (Mn), selenium (Se), Lead (Pb), nickel (Ni), aluminum (Al), arsenic (As), iron (Fe), and Zinc (Zn) were analyzed in the liver, muscle, and kidney organs of the host, as well as in their parasites Hysterothalycium reliquens (nematode) and the Paraphilometroides nemipteri (nematode), using inductively coupled plasma mass spectrometry (ICP-MS). The former group of parasites showed highest accumulation capacity for Cr, Cu, Fe, Mn, Se, Ni, and Zn while the latter group had high accumulation potential of As, Hg, Cd, Al, Pb, and Sr. The divergence in heavy-metal accumulation profiles of both nematodes is linked with the specificity of microhabitats, cuticle morphology, and interspecific competition. The outcome of this study indicates that both parasite models can be used for biomonitoring of metal pollution in marine ecosystems.
A new proteomics technology has been implemented to study the protein repertoires of developing oocytes of giant grouper (Epinephelus lanceolatus). Knowledge of the chemical composition and physiochemical properties of vitellogenin (Vtg) is necessary to interpret the functional and biological properties attributed during ovulation. Vtg, as a biomarker indicator in sex determination, has been analyzed to determine the sex and maturational status of fish in the absence of the gonad tissue. A male giant grouper was induced by 2 mg/kg of 17ß-estradiol (E2), and blood was sampled at days 0, 1, 3, 5, and 10. SDS-PAGE 1D electrophoresis was used to analyze Vtg protein, and Vtg identification was done with 4800 Plus MALDI TOF/TOF™ mass spectrophotometer (Applied Biosystems/MDS SCIEX, USA). Meanwhile, MS/MS de novo sequencing identified the proteins by matching sequences of tryptic peptides to the known sequences of other species. Vtg was confirmed by MASCOT at 95% significant level, and molecular mass was 187 kDa. Protein resolved on SDS-PAGE as a double band of approximately the same mass as determined with MALDI-TOF. The N-terminal sequences and identification of Vtg were also determined. The potential of using MS methods to understand the structure and function of Vtg is discussed.
There is a lack of understanding on how the environment and trophic niche affect the capability of long-chain polyunsaturated fatty acids (LC-PUFA) in freshwater carnivorous teleost. In this present study, we isolated and functionally characterised a fatty acyl desaturase (Fads) from the striped snakehead Channa striata. Sequence comparison and phylogenetic analysis suggested a Fads2 protein that is closely related to previously characterised Fads2 proteins from freshwater carnivorous and marine herbivorous fish species. We further demonstrated the capacity of Δ6 and Δ5 desaturation activities for this particular desaturase, with highest activities towards the conversion of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Low Δ4 desaturation activity was also detected, although the significance of this at a physiological level remains to be studied. The expression of this striped snakehead Δ6/Δ5 fads2 gene was highest in brain, followed by liver and intestine. In liver, diet fortified with high LC-PUFA concentration impeded the expression of Δ6/Δ5 fads2 gene compared to vegetable oil (VO) based diets. The discovery of Δ6/Δ5 Fads2 desaturase here complements the previous discovery of a Δ4 Fads2 desaturase and an Elovl5 elongase, lending proof to the existence of all the required enzymatic machinery to biosynthesise LC-PUFA from C18 PUFA in a freshwater carnivorous species.
The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.