Age estimation of Antarctic minke whales <Emphasis Type="Italic">Balaenoptera bonaerensis</Emphasis> based on aspartic acid racemization technique

Counting of the growth layers in the earplugs is the most accepted technique for determining chronological age of Antarctic minke whales; however, unreadable growth layers form in some individuals, especially in young animals. In this study, aspartic acid racemization (AAR) technique was developed for estimating ages in this species with the aim of complementing the age estimated using earplugs. To validate the technique and to determine the specific coefficients for age estimation, the ratio of d and l-enantiomers of aspartic acid (Asp D/L) in lens of 18 whales and 20 fetuses were analyzed and compared with earplug-based age estimates. The equation for age estimation by AAR in this species was as follows: Log_e{[1 + (Asp D/L)_act]/[1 ? (Asp D/L)_act]} = 2.30 × 10^?3 × earplug age (year) + 0.0201 (p < 0.001, r ²  = 0.918). There is a strong correlation between the age estimates by AAR and earplugs. This study was successful in developing the AAR technique for the Antarctic minke whale, and the application of this technique can complement the age estimation of this species based on earplug readings, especially for young animals with unreadable earplugs.     

A study of the damage of the intestinal mucosa barrier structure and function of <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1β, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and d-lactic acid. The results indicated that the levels of EB and serum d-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase. Lower mRNA levels of occludin and lower Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.     

Effects of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced oxidative stress in grass carp ovary cells (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

This study was designed in vitro to investigate the effects of l-carnitine against H₂O₂-induced oxidative stress in a grass carp (Ctenopharyngodon idellus) ovary cell line (GCO). GCO cells were pre-treated with different concentrations of l-carnitine, followed by incubation with 2.5 mM H₂O₂ for 1 h to induce oxidative damage. The results indicated that adding l-carnitine at concentrations of 0.01–1 mM into the medium for 12 h significantly increased cell viability. Pre-treatment with l-carnitine at concentrations of 0.1–5 mM for 12 h significantly inhibited 2.5 mM H₂O₂-induced cell viability loss. The significant decreases in the level of reactive oxygen species and cell apoptosis were observed in 0.5 mM l-carnitine group compared to the H₂O₂ group. Malondialdehyde values of all of the l-carnitine groups were significantly lower than those of the H₂O₂ group, while total glutathione levels of all of the l-carnitine groups were significantly higher than of the H₂O₂ group. The activity of antioxidant enzymes, such as total superoxide dismutase (0.1 and 0.5 mM l-carnitine), catalase (0.5 mM l-carnitine) and γ-glutamyl cysteine synthetase (0.5 and 1 mM l-carnitine), was significantly increased. In addition, pre-treatment of l-carnitine in GCO cells exposed to 2.5 mM H₂O₂ significantly increased the mRNA expression of copper, zinc superoxide dismutase, catalase (0.5 mM l-carnitine), glutamate cysteine ligase catalytic subunit (0.1–1 mM) and glutathione peroxidase (0.1 mM l-carnitine). In conclusion, l-carnitine promotes GCO cell growth and improves antioxidant function, it plays a protective role against oxidative stress induced by H₂O₂ in GCO cells, and the appropriate supplemental amount of l-carnitine is 0.1–1 mM.     

Establishment of a model for chemoattraction of <Emphasis Type="Italic">Symbiodinium</Emphasis> and characterization of chemotactic compounds in <Emphasis Type="Italic">Acropora tenuis</Emphasis>

Corals harbor symbiotic dinoflagellates, Symbiodinium spp., acquired from surrounding environments. Because Symbiodinium are present at low densities in the water column, corals may attract these symbionts using chemotactic compounds. To examine whether corals contain chemotactic compounds, we established an assay to measure the chemotactic activity for Symbiodinium using an extract of the coral Acropora tenuis, a major reef-building coral in Japan. Our assay revealed that Symbiodinium strain NBRC102920 (clade A), which is taken up by juvenile A. tenuis polyps, is attracted to crude A. tenuis extracts. We found that the chemotactic compounds are water-soluble, heat-labile macromolecules and that the chemotactic activity was inhibited by N-acetyl-d-glucosamine (GlcNAc). We separated the GlcNAc-binding fraction (Fr-ActL) and identified it as the most plausible candidate for the chemoattractant, since the chemotactic activity of the crude A. tenuis extract appeared to be mainly attributable to the activity of Fr-ActL and was also inhibited by the addition of GlcNAc. These results indicate that chemoattraction is mediated via the binding of Symbiodinium to Fr-ActL.     

Assessing the efficacy of three methionine sources in low protein and low fish meal diet for Chinese soft-shelled turtle, <Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>

An 8-week feeding trial was conducted to evaluate the effects of three dietary methionine (Met) sources [dl-Met, coated-Met, and a methionine hydroxy analogue calcium salt (MHA-Ca)] for Chinese soft-shelled turtle (Pelodiscus sinensis). Triplicate groups of juvenile turtles (initial weight 3.48 ± 0.03 g) were fed twice per day at 3% of body weight with positive control diet (T1, 46% protein and 46% fish meal, FM), negative control diet (T2, 43% protein and 23% FM), or three other test diets supplemented with either 0.2% coated-Met (T3), 0.125% MHA-Ca (T4), or 0.1% dl-Met (T5) to the T2 basal formulation, respectively. The feeding trial was conducted in 15 350-L plastic containers with three replicates per dietary treatment. The results showed that the highest and lowest percentage weight gain (1023.5 ± 18.2 versus 882.1 ± 14.5%) and feed efficiency (87.2 ± 0.94 versus 81.4 ± 0.4%) were observed in turtles fed the T1 and T2 diet (P < 0.05), respectively. Significant improvement in weight gain was observed in turtles fed diets with Met supplementation, irrespective of source, compared with turtles fed the T2 diet. It was observed that dietary MHA-Ca had similar efficacy compared with dl-Met in terms of growth performance and feed utilization efficiency of the turtles (P > 0.05). Protein utilization efficiency was significantly higher in turtles fed T4 or T5 diet compared to the T2 diet. Whole body protein content (17.0 to 17.4% on a wet weight basis) in turtles fed with Met-added diets was comparable to those of turtles fed the T1 diet which were significantly higher compared to T2-fed turtles. Antioxidant defense system enzymes, superoxide dismutase, and glutathione peroxidase showed the highest activity, 658.8 ± 17.9 U/mL and 642.8 ± 17.5 μmol/L, respectively, in the serum of turtle fed the MHA-Ca supplemented diet and was significantly higher compared to turtles fed the T2 or T3 diet. The present results showed that P. sinensis are able to effectively use added MHA-Ca and dl-Met in low protein and low FM diets to enhance growth, feed utilization efficiency, nitrogen retention, and antioxidant defense system enzyme activities.     

Effects of glutamine and its precursors on the growth performance and relevant protein synthesis pathway of mirror carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

This study aimed to investigate the effects of Gln and its precursors on Gln anabolism and ammonia excretion to determine the role of Gln in protein synthesis in Cyprinus carpio. The growth performance, glutamine synthetase (GS) activity, blood ammonia level, and gene expression of GS, rhesus glycoprotein (Rhag, Rhbg and Rhcg), TOR and 4E-BP1 of fish were measured. Seven diet treatments including glucose (control), glutamine (Gln), glusate (Glu), α-ketoglutarate (AKG), l-ornithine-α-ketoglutarate (OKG), l-arginine-α-ketoglutarate (AAKG), and α-ketoglutarate sodium (2Na-AKG) were conducted. All were substituted for glucose at 1.5% of the dry diet. The results showed the feed conversion ratios (FCRs) of the AKG group and AAKG group were significantly lower (P < 0.05) than that of the control group. The expression levels of the Rhbg gene in the gills of the AKG, AAKG and 2Na-AKG groups were significantly higher than that in the control group (P < 0.05). The expression levels of the TOR gene in the gut of the fish in the AKG group and the Glu group were significantly higher than that in the control group (P < 0.05). Therefore, the addition of AAKG in feed can significantly reduce the FCR of Cyprinus carpio and significantly improve the weight gain rate (WGR) and protein efficiency of the fish. Gln can reduce ammonia release in gills, and AKG can effectively promote the excretion of ammonia. The addition of Gln, Glu, AKG and AAKG in diets can effectively promote protein synthesis. The Gln, Glu, AKG and AAKG can significantly up-regulate GS gene expression in the gut; however, the expression level of the GS gene is not significantly correlated with GS activity.     

Dietary ascorbic acid reduced micronucleus and nuclear abnormalities in <Emphasis Type="Italic">Clarias gariepinus</Emphasis> (Burchell 1822) exposed to hospital effluent

Hospital effluents contain myriad of mutagens and genotoxins capable of increasing DNA damage in aquatic biota. African mudfish, Clarias gariepinus, are exposed to genotoxins when cultured in swamps and derelict water bodies often contaminated by effluents. Moreover, its DNA is susceptible to xenobiotic-induced lesions since it lacks l-gulonolactone oxidase and hence cannot synthesize l-ascorbic acid. This study investigated 96-h acute toxicity and protective effects of dietary ascorbic acid (AA) against micronucleus (MN) and abnormal nuclear (NAs) formation in C. gariepinus exposed to sub-lethal concentrations of hospital effluent. Six concentrations (0.5–3.0%) of the effluent were selected to determine the 96-h acute toxicity of the effluent in C. gariepinus, after range finding test. Fish were exposed to sub-lethal concentrations (0.08–1.30%) of the 96 h LC₅₀. Two other groups were exposed to the 96 h LC₅₀ (1.30%) of the effluent +50 and +100 mg/kg of dietary ascorbic for 7 days, and MN and NAs assessed in peripheral erythrocytes. The 96 h LC₅₀ (1.30%) was 1.18 times more toxic than the 24 h LC₅₀ (1.54%), indicating that the toxicity of the effluent increased with exposure duration. MN, nuclear bud, enucleated, fragmented nucleus (apoptosis), and necrotic erythrocytes significantly increase in effluent treated fish. Dietary AA reduced MN from 6.35-fold (1.30% treated group) to 3.72-fold (1.30% + 50 mg AA) and 3.54-fold (1.30% + 100 mg AA). Also, AA reduced total NAs from 2.26-fold (1.30%) to 1.40-fold (1.30% + 50 mg AA) and 1.06-fold (1.30% + 100 mg AA) compared to the control. Heavy metals and physicochemical parameters analyzed in the tested effluent possibly induced the mortality and cytogenotoxicity in C. gariepinus, and this was ameliorated by dietary AA.     

The effects of starvation on fast-start escape and constant acceleration swimming performance in rose bitterling (<Emphasis Type="Italic">Rhodeus ocellatus</Emphasis>) at two acclimation temperatures

To investigate the effects of starvation and acclimation temperature on the escape ability of juvenile rose bitterling (Rhodeus ocellatus), we measured the fast-start escape and constant acceleration swimming performance of fish fasted for 0 (control), 1 and 2 weeks and half-lethal periods (6 or 4 weeks) at two temperatures (15 and 25 °C). Fish acclimated at a high temperature exhibited shorter response latency (R), higher maximum linear velocity (V _max) and longer escape distance during escape movement (D _120ms) than those at the low temperature. Starvation resulted in a significant decrease in V _max and D _120ms at either low or high temperature and a significant increase in R at only the high temperature in the half-lethal period groups (P < 0.05). The relationship between V _max (Y, m s^?1) and starvation time (X, week) was Y ₁₅ = ?0.062X + 1.568 (r = ?0.665, n = 36, P < 0.001) at low temperature and Y ₂₅ = ?0.091X + 1.755 (r = ?0.391, n = 40, P = 0.013) at high temperature. The relationship between U _cat (Y, cm s^?1) and starvation time (X, week) was Y ₁₅ = ?1.649X + 55.418 (r = ?0.398, n = 34, P = 0.020) at low temperature and Y ₂₅ = ?4.917X + 62.916 (r = ?0.793, n = 33, P < 0.001) at high temperature. The slopes of equations showed a significant difference between low and high temperature (F _1,63 = 9.688, P = 0.003), which may be due to the different energy substrate utilization when faced with food deprivation at different temperatures.     

Effects of dietary inclusion of <Emphasis Type="Italic">Moringa oleifera</Emphasis> leaves on growth and some systemic and mucosal immune parameters of seabream

The effect of the dietary incorporation of drumstick, Moringa oleifera, leaf meal (MOL; 0, 5, 10 and 15%) on the growth, feed utilization, some skin mucus and systemic immune parameters and intestinal immune-related gene expression in gilthead seabream (Sparus aurata) specimens. The experiment lasted 4 weeks. The results revealed that MOL can be incorporated in S. aurata diet up to 10% with no significant negative effect on growth and feed utilization. However, there was a significant decrease with MOL at a level of 15% after 2 weeks of feeding. The systemic immune status of fish fed with the different levels of MOL showed an improvement in head kidney leucocyte phagocytosis, respiratory burst and peroxidase activities. Also, serum humoral components, including protease, ACH₅₀ and lysozyme activities and IgM level, increased with MOL inclusion especially at the 5% level. MOL at 5% improved skin-mucosal immunity such as protease, antiprotease, peroxidase and lysozyme activities. Moreover, the feeding of MOL revealed an upregulation of the intestinal mucosal immunity genes (lyso and c3), tight junction proteins (occludin and zo-1) and anti-inflammatory cytokines (tgf-β) with a downregulation of pro-inflammatory cytokine (tnf-α). Therefore, it is recommended to incorporate MOL in S. aurata diets at a level of 5% for the best immune status or 10% for the high growth performance and acceptable immune surveillance.

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Rapid measurements of fat content in live and slaughtered common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

The objective of this study on common carp (Cyprinus carpio) was to obtain and predict the first cross-validated data of the fat content on market size carps using a non-invasive or non-destructive method in situ. The carps (1680 ± 388 g; n = 136) used were from a semi-intensive system and were on a different diet (cereal, pelleted and extruded diet). For the evaluation of the fat content, a Fish Fatmeter FM 692 from Distell.com. (FFM) and a manual measurement of back fat height using a digital calliper were used. For the prediction model, the following basic body measurements (variables) were used: total body length, body length, body height, the width of the body, and the circuit of the body. The body weight, weight of intestines, weight of gonads, weight of hepatopancreas, and fillet yield (%) were measured, and the Fulton coefficient was calculated. The study was focussed on evaluating the applicability of these methods and the accuracy of the obtained result, respectively. Results showed that all the rapid methods had a strong correlation. Multiple regression models with forward selection of variables were used throughout. The final prediction model between predicted and observed values for the fat content for FFM and calliper being adjusted index of determination is shown here (R _adj ²  = 0.88; 5 variables and R _adj ²  = 0.91; 7 variables), respectively.     

Molecular endocrine changes of Gh/Igf1 axis in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) exposed to different environmental salinities during larvae to post-larvae stages

The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.     

Molecular mobility of fish flesh measured by low-field nuclear magnetic resonance (LF-NMR) relaxation: effects of freeze–thaw cycles

In this study, the molecular mobility of fish flesh was measured by low field nuclear magnetic resonance (LF-NMR) relaxation. Sardine, tuna and mackerel were frozen at ?40 °C and stored for 1 day (24 h); and then these samples were thawed at room temperature (20 °C). The relaxation of water protons in fish flesh was measured for fresh (i.e., before freezing) and multi-cycle freeze–thaw samples (i.e., up to 12 times). Three domains from different pools of protons (i.e., low-mobile, medium-mobile and high-mobile) were identified from the relaxation curve. The T _2b (low-mobile), T ₂₁ (medium-mobile) and T ₂₂ (high-mobile) indicated the proton populations in the protein molecules, strongly bound water molecules, and weakly bound water molecules, respectively. In all cases, the relaxation time (T _2b: sardine r = 0.736 and p < 0.01, tuna r = 0.857 and p < 0.001, mackerel r = 0.904 and p < 0.001; and T ₂₂: sardine r = 0.956 and p < 0.0001, tuna r = 0.927 and p < 0.0001, mackerel r = 0.890 and p < 0.0001) increased with the freeze–thaw cycles and it reached a nearly constant value after 6 freeze–thaw cycles. The increased relaxation time (i.e., higher mobility) up to 6 freeze–thaw cycles could be due to the increase in proton mobility. However, relaxation time (T ₂₁: sardine r = ?0.510 and p > 0.05, tuna r = 0.162 and p > 0.5, mackerel r = 0.513 and p > 0.01) showed insignificant change with the increase of freeze–thaw cycles, which indicated minimal change in the medium-mobile protons. The results in this study revealed that the changes in proton mobility in the fish flesh during freeze–thaw cycles could be identified using T _2b and T ₂₂ relaxation of LF-NMR.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Effect of the early temperature on the growth of larvae and postlarvae turbot, <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> L.: muscle structural and ultrastructural study

Turbot specimens were kept at three temperatures (T _s ): warm (W) (21–22 °C), ambient (A) (17–18 °C) and cold (C) (13–14 °C) during the larval and early postlarval stages. At 90 days posthatching (dph), all of them were transferred to ambient T until 190 dph. At 2–3 dph, the specimens showed a monolayer of red muscle and immature white fibres; external or dermomyotome cells (presumptive myogenic cells) were observed on the surface of the red muscle. In the following stages, many myogenic cells and presumptive myogenic precursors were observed within the myotome, presumably derived of the dermomyotome. When comparing the growth at the same age (2, 10, 25, 37 dph), the body length and the muscle growth were positively influenced by the warm T, being the hyperplasia the muscle parameter more significantly influenced. The development rate was also positively correlated with the high T: the beginning of the metamorphosis took place at 15, 23 and 25 dph at W, A and C temperatures, respectively, with the highest body length values at ambient temperature. The metamorphosis finished at 25, 30 and 37 dph at W, A and C temperatures, respectively, with the highest body length values at warm temperature. However, the muscle cellularity was similar in all the groups at the end of the metamorphosis. At 90 and 190 dph, the largest body length was observed at W temperature. However, the muscle cellularity was similar between A and W; the number of fibres was similar in all the groups at 190 dph, which shows the beginning of a compensatory muscle growth in A and C, mainly in A.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis> transferrin gene and effects of exposure to iron and infection with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5′ and a 204 bp 3′ untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl₃-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl₃-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.     

Immunomodulatory effects of <Emphasis Type="Italic">Rhodomyrtus tomentosa</Emphasis> leaf extract and its derivative compound,rhodomyrtone, on head kidney macrophages of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10 μg mL^?1 of R. tomentosa and 1 μg mL^?1 of rhodomyrtone for 4 and 24 h. R. tomentosa and rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1β, il8, and tnfα), anti-inflammatory cytokines (il10 and tgfβ), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1β, il8, tnfα, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1β, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.