The resistance to ammonia stress of Penaeus monodon Fabricius juvenile fed diets supplemented with astaxanthin

This study was aimed at determining if the increase of body astaxanthin content through dietary supplementation in tiger prawn Penaeus monodon juvenile could enhance its antioxidant defense capability and resistance to ammonia stress. Haemolymph total antioxidant status (TAS) and superoxide dismutase (SOD) were chosen as parameters of shrimp antioxidant capacity. Resistance to chemical stress was evaluated by shrimp survival rate, and haemolymph aspartate aminotransferase (AST) and alanine aminotransferase (ALT). P. monodon 5-day postlarvae were fed diets supplemented with 0 and 71.5 mg kg⁻¹ astaxanthin for 8 weeks. Shrimps were then subjected to 72-h exposure of ammonia at 0.02, 0.2, 2 and 20 mg l⁻¹. The survival rates of the astaxanthin-fed (AX) shrimp were higher than those of the control shrimp under all levels of ammonia except 20 mg l⁻¹, showing that the shrimp's resistance to ammonia stress had been improved by dietary astaxanthin. AX shrimp had higher TAS than control shrimp at ammonia levels higher than 0.02 mg l⁻¹ and lower SOD at all ammonia levels suggested that antioxidation capability had been greatly enhanced. AST in AX shrimp was lower than that in control shrimp under all levels of ammonia stress. ALT in AX shrimp was either lower than or equal to that in control shrimp under various levels of ammonia. Both AST and ALT reflected that shrimp hepatopancreatic function had been improved by dietary astaxanthin. Astaxanthin can become essential for P. monodon when the animal is under ammonia stress.     

Interspecific differences in carotenoid content and sensitivity to UVB radiation in three acanthocephalan parasites exploiting a common intermediate host

Few endoparasite species are pigmented. Acanthocephalans are an exception however, with several species being characterised by yellow to orange colouration both at the immature (cystacanth) and adult stages. However, the functional and adaptive significance of carotenoid-based colourations in acanthocephalans remains unclear. One possibility is that the carotenoid content of acanthocephalan cystacanths acts as a protective device against ultra-violet radiation (UVR) passing through the translucent cuticle of their crustacean hosts. Indeed, acanthocephalans often bring about behavioural changes in their aquatic intermediate hosts that can increase their exposure to light. Carotenoid composition and damage due to ultra-violet - B (UVB) radiation were investigated in three acanthocephalan parasite species that induce contrasting behavioural alterations in their common intermediate host, the crustacean amphipod Gammarus pulex. The fish acanthocephalans Pomphorhynchus laevis and Pomphorhynchus tereticollis both induce a positive phototaxis in gammarids, such that infected hosts spend more time out of shelters, while remaining benthic. The bird acanthocephalan Polymorphus minutus, on the other hand, induces a negative geotaxis, such that infected hosts typically swim close to the water surface, becoming more exposed to UV radiation. We show that differences in cystacanth colouration between acanthocephalan species directly reflect important differences in carotenoid content. The two fish parasites exhibit a contrasting pattern, with P. tereticollis harbouring a large diversity of carotenoid pigments, whereas P. laevis is characterised by a lower carotenoid content consisting mainly of lutein and astaxanthin. The highest carotenoid content is found in the bright orange P.minutus, with a predominance of esterified forms of astaxanthin. Exposure to UVB radiation revealed a higher susceptibility in P. laevis larvae compared with P. tereticollis and P. minutus, in terms of sublethality (decreased evagination rate) and of damage to DNA (increased cyclobutane pyrimidine dimers production). Although we found important and correlated interspecific differences in carotenoid composition and tolerance to high UVB radiation, our results do not fully support the hypothesis of adaptive carotenoid-based colourations in relation to UV protection. An alternative scenario for the evolution of carotenoid accumulation in acanthocephalan parasites is discussed.     

Nutritional value and intake of aquatic ferns (Azolla filiculoides Lam. and Salvinia molesta Mitchell.) in sows

Aquatic ferns (AFs) such as Azolla filiculoides and Salvinia molesta are grown on swine lagoons in the tropics and used in diets for pigs. The present work is aimed at evaluating their potential as feed ingredients for sows. When presented with ad libitum AFs, gilts weighing 110 ± 14 kg (mean ± SD), were able to ingest 9.1–9.7 kg fresh AF per day (from 597 to 630 g dry matter (DM) per day) and from 1240 to 1428 g DM per day when presented in a dry, ground form. A digestibility study was conducted, using sows weighing 213 ± 9 kg (mean ± SD), which were fed diets containing maize, soybean meal and 0, 150 or 300 g AF kg⁻¹ diet. The presence of AFs had a negative impact on the faecal digestibility of the crude protein, NDF and energy content of the whole diet (P<0.001) and on the ileal protein digestibility, especially with 300 g AFs kg⁻¹ diet. The level of AFs in the diet had no effect on stomach weight (P>0.05) but increased the weight of the rest of the gastrointestinal tract (P<0.001). The rate of AF fibre fermentation in the pig large intestine was measured using an in vitro gas test. The rates were much lower than tropical tree foliage, which can also be used in pig diets in the tropics. This could partly explain the low apparent digestibility of AFs in pigs. In conclusion, the inclusion level of AFs in rations for sows should be limited to 150 g AFs kg⁻¹ diet due to the low digestibility and energy density, as well as the negative impact on the digestibility of the whole diet.     

Effects of dietary astaxanthin supplementation on reproductive characteristics of rainbow trout (Oncorhynchus mykiss)

The effect of dietary astaxanthin supplementation on reproductive characteristics was investigated in five groups of female rainbow trout broodstock fed diets containing either 0.07, 12.46, 33.33, 65.06 or 92.91 mg astaxanthin kg^?1, respectively, and two groups of male rainbow trout broodstock fed diets supplemented with 0.07 and 33.33 mg astaxanthin kg^?1, respectively, for 6 months in an artificial photoperiod system until sexual maturation. The eggs from each group of female broodstock were divided into two equal batches. One batch was fertilized with homogenized sperm of four males fed diets with 0.07 mg astaxanthin kg^?1 and the other portion with sperm of four males fed diets with 33.3 mg astaxanthin kg^?1. The females produced eggs with astaxanthin concentrations ranging from 2.03 to 29.79 mg kg^?1. Dietary astaxanthin supplementation had positive effects on investigated reproductive traits. Significant differences in rate of fertilization, percentage of eyed and hatched eggs, and mortality of eyed eggs were found between treatments (P < 0.05), but no significant difference was found on percentage of mortality before hatching (P > 0.05). A significant difference (P < 0.05) in fertilization rate was found for male groups fed 0.07 and 33.3 mg astaxanthin kg^?1. The astaxanthin content in the eggs and fertilization rate, eyed‐egg percentage and percentage hatch were significantly correlated (P < 0.05). It is concluded that dietary supplements of astaxanthin are required for optimum reproduction in rainbow trout.     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Aerobic scaling and resting metabolism in oviferous and post-spawning Barents Sea capelin Mallotus villosus villosus (Müller, 1776)

As part of the quantitative investigations into the bioenergetic relationships of the Barents Sea capelin, Mallotus villosus villosus (Müller, 1776), resting metabolic rates were examined in the oviferous and post-spawning fish in order to provide insights to aerobic scaling and the basal energetic costs associated with reproduction. Aerobic scaling of the different categories of sexually mature fish (body weight, W=13-54 g) could be expressed as: Q_O2=0.106W^1.049 (oviferous fish; N=11), Q_O2=0.411W^0.430 (post-spawning females; N=9), and Q_O2=0.075W^1.012 (post-spawning males; N=14), where Q_O2 is the oxygen consumption (ml O₂ h⁻¹) per fish. The weight specific oxygen consumption of oviferous capelin was about 30% higher (∼125 ml O₂ kg⁻¹ h⁻¹) compared to those of the post-spawning fish (79-87 ml O₂ kg⁻¹ h⁻¹). The results are discussed in context with other empirical studies on the aerobic scaling and metabolic costs involved with the build-up of roe in the fish.     

Short term effects of hypoxia and bioturbation on solute fluxes, denitrification and buffering capacity in a shallow dystrophic pond

The effects of short term hypoxia on bioturbation activity and inherent solute fluxes are scarcely investigated even if increasing number of coastal areas are subjected to transient oxygen deficits. In this work dark fluxes of oxygen (O₂), dissolved inorganic carbon (TCO₂) and nutrients across the sediment-water interface, as well as rates of denitrification (isotope pairing), were measured in intact sediment cores collected from the dystrophic pond of Sali e Pauli (Sardinia, Italy). Sediments were incubated at 100, 70, 40 and 10% of O₂ saturation in the overlying water, with both natural benthic communities, dominated by the polychaete Polydora ciliata (11.100 ± 2.500  ind. m^− 2), and after the addition of individuals of the deep-burrower polychaete Hediste diversicolor. Below an uppermost oxic layer of ~ 1 mm, sediments were highly reduced, with up to 6 mM of S²⁻ in the 5 mm layer. Flux of S²⁻ and O₂ calculated from pore water gradients were 8.61 ± 1.12 and − 2.27 ± 0.56 mmol m^− 2 h^− 1, respectively. However, sediment oxygen demand (SOD) calculated from core incubation was − 10.52 ± 0.33 mmol m^− 2 h^− 1, suggesting a major contribution of P. ciliata to O₂-mediated sulphide oxidation. P. ciliata also strongly stimulated NH₄⁺ and PO₄³⁻ fluxes, with rates ~ 15 and ~ 30 folds higher, respectively, than those estimated from pore water gradients. P. ciliata activity was significantly reduced at 10% O₂ saturation, coupled to decreased rates of solutes transfer. The addition of H. diversicolor further stimulated SOD, NH₄⁺ efflux and SiO₂ mobilisation. Similarly to P. ciliata, the degree of stimulation of SOD and NH₄⁺ flux by H. diversicolor depended on the level of oxygen saturation. TCO₂ regeneration, respiratory quotients, PO₄³⁻ fluxes and denitrification of added ¹⁵NO₃⁻ were not affected by the addition of H. diversicolor, but depended upon the O₂ levels in the water column. Denitrification rates supported by water column ¹⁴NO₃⁻ and sedimentary nitrification were both negligible (< 0.5 µmol m^− 2 h^− 1). They were not significantly affected by oxygen saturation nor by bioturbation, probably due to the limited availability of NO₃⁻ in the water column (< 3 µM) and O₂ in the sediments. This study demonstrates for the first time the integrated short term effect of transient hypoxia and bioturbation on solute fluxes across the sediment-water interface within a simplified lagoonal benthic community.     

Impact of hypoxia on the survival, egg production and population dynamics of Acartia tonsa Dana

Concern for the increasing occurrence of coastal zone hypoxia has generally focused on the direct, short-term impact of reduced dissolved oxygen (DO) concentrations on the survival of commercially important species such as fish and crabs. Copepods, especially the naupliar stages, are important pelagic food web components, yet only a few studies have considered the effect of reduced DO concentrations on their survival and population dynamics. This study considers the impact of both lethal and sublethal DO concentrations on copepods. Acartia tonsa were reared at 25 °C at saturating DO (normoxic control) and reduced (hypoxic) DO concentrations of 1.5 or 0.7 ml l⁻¹. Oxygen concentrations were maintained in replicate flasks, by bubbling seawater with air (control), or mixtures of nitrogen and oxygen. Egg production, but not survival, was significantly higher in the controls compared to the 1.5 ml l⁻¹ DO treatment. Survival and egg production were significantly lower at 0.7 ml l⁻¹ DO compared to the control. The results suggest that the sublethal as well as the lethal effects of hypoxia may have important repercussions on population and community dynamics in coastal systems.     

Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb⁺) displayed higher biomass and YlLIP2 activity than control strains (VHb⁻). Compared with VHb⁻ cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb⁺ in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb⁻ cells than in VHb⁺ cells. The highest YlLIP2 activity of VHb⁺ cells was approximately 1.84-fold higher than that of VHb⁻ cells at lower DO levels. Moreover, the recombinant strain VHb⁺ exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb⁻ cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.     

Determination of the time transferring cells for astaxanthin production considering two-stage process of Haematococcus pluvialis cultivation

The two-stage culture system consisting of green vegetative growth and reddish inductive production stages has been widely accepted for the production of astaxanthin using Haematococcuspluvialis. However, little has been known about the appropriate cellular phase of H.pluvialis for transferring into the astaxanthin inductive conditions. In this study, we determined the optimal growth phase of H.pluvialis for transferring into the second production stage. Astaxanthin productivities were correlated with growth phases, as senescent green phases could increase more than 10-fold greater than juvenile green phases. Our results clearly demonstrated the appropriateness of the senescent vegetable cells for transferring into the production stage, due to the increased capacity to accumulate astaxanthin.     

Molecular cloning,characterization and mRNA expression of selenium-dependent glutathione peroxidase from abalone Haliotis discus hannai Ino in response to dietary selenium,zinc and iron

A novel selenium-dependent glutathione peroxidase (Se-GPX) was cloned from abalone Haliotis discus hannai Ino (HdhGPx) by homology cloning with degenerate primers and RACE techniques. The full length of HdhGPx cDNA was 963 bp with a 669 bp open reading frame (ORF) encoding 222 amino acids and a 101 bp eukaryotic selenocysteine insertion sequence (SECIS) in 3′ untranslated region (UTR). It was showed that HdhGPx has a characteristic codon at ²³⁵TGA²³⁷ that corresponds to selenocysteine (SeC) as U₇₂. Sequence characterization revealed that HdhGPx contains a characteristic GPx signature motif 2 (₉₆LGLPCNQF₁₀₃), an active site motif (₁₇₉WNFEKF₁₈₄). In addition, two potential N-glycosylation sites (₁₁₂NGTE₁₁₅ and ₁₃₂NLTQ₁₃₅) were identified in HdhGPx. 3D modeling analysis showed that the overall structure of HdhGPx monomer had more similarity to human GPx3 than human GPx1. Relatively higher-level mRNA expression was detected in hepatopancreas, mantle and gonad by real-time PCR assays. The relative expression levels of HdhGPx mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with nine different diets containing graded levels of selenium (0.15, 1.32 and 48.7 mg kg^− 1), zinc (6.69, 33.85 and 710.63 mg kg^− 1) and iron (29.17, 65.7 and 1267.2 mg kg^− 1) for 20 weeks, respectively. The results showed that the expressions of HdhGPx mRNA were statistically higher at adequate dietary selenium (1.32 mg kg^− 1), zinc (33.85 mg kg^− 1) and iron (65.7 mg kg^− 1) than those in low dietary minerals, respectively. But HdhGPx mRNA expression levels were down-regulated by high contents of dietary selenium (48.7 mg kg^− 1), zinc (710.63 mg kg^− 1) and iron (1267.2 mg kg^− 1), respectively. These results indicated that adequate dietary minerals could increase the mRNA expression of HdhGPx, and then to increase the total antioxidant capacities in abalone.     

Effects of dietary xanthophylls/astaxanthin ratios on the growth and skin pigmentation of large yellow croaker Larimichthys crocea (Richardson, 1846)

An 8‐week feeding trial was conducted to evaluate the effects of dietary xanthophylls/ astaxanthin ratio on the growth and skin color of large yellow croaker, Larimichthys crocea. Five pigment‐supplemented diets were formulated to contain 75/0, 50/25, 37.5/37.5, 25/50 and 0/75 mg kg^?1 of xanthophylls/astaxanthin. The xanthophylls contain 89.31% lutein and 6.12% zeaxanthin. A diet without pigment supplementation was used as the control. The large yellow croaker juveniles (13.80 ± 0.03 g) were randomly distributed in 18 sea cages (1.0 × 1.0 × 1.5 m) at a density of 45 fish per cage. Water temperature ranged from 21 to 31°C during the feeding trial. To obtain results, the survival rate, specific growth rate, feed conversion ratio, skin redness, skin yellowness, skin lightness, skin carotenoid content and skin melanin content were measured. The results showed that the survival rate, specific growth rate and feed conversion ratio were not significantly affected by dietary treatments (P > 0.05). The ventral skin lightness was also not affected by dietary treatments (P > 0.05); however, the dorsal skin lightness of fish fed with the control diet was significantly lower than those fed with pigment‐supplemented diets (P < 0.05). The lowest values of yellowness and carotenoid content both in the ventral skin and dorsal skin were found in the control group. Yellowness and carotenoid content increased with an increasing proportion of dietary xanthophylls in both the ventral and dorsal skin. Higher redness values were found in the compound pigment groups, either in the dorsal skin or ventral skin. Fish fed with the control diet showed a higher melanin content in the dorsal skin than those fed with pigment‐supplemented diets, although differences were not significant (P > 0.05). Lightness and yellowness were linearly related to skin carotenoid content. Meanwhile, skin yellowness and carotenoid content were linearly related to the proportion of xanthophylls in dietary pigments.     

Growth, reproductive and photosynthetic responses to copper in the yellow-horned poppy, Glaucium flavum Crantz.

Glaucium flavum Crantz. is found in an anthropized coastal grassland at the joint estuary of the Tinto and Odiel rivers (SW Spain), growing under the influence of high levels of copper contamination derived from nearby petrochemical industries, with no obvious adverse affects on the performance of the plant. In addition, this species exhibits a series of ecological characteristics which may render it appropriate for use in the phytoremediation of contaminated areas. Nonetheless, the response of G. flavum to elevated copper concentrations has not been studied. A greenhouse experiment was conducted to investigate the effects of a range of Cu concentrations (0 to 47 mmol l⁻¹) on the growth, reproduction and photosynthetic performance of G. flavum, by measuring relative growth rate, fruit and seed production, chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. We also determined total copper, nitrogen, phosphorous, sulphur, calcium and magnesium concentrations. G. flavum survived with concentrations of up to 730 mg Cu kg⁻¹ DW in the leaves, when treated with 30 mmol Cu l⁻¹ (2000 mg l⁻¹). Quantum efficiency of PSII, net photosynthesis rate, as well as leaf Ca and Mg concentrations were all negatively affected by Cu concentrations greater than 9 mmol l⁻¹ in the nutrient solution. Our results indicate that the reduction in photosynthetic performance may be attributed to the adverse effect of excess Cu on the photosynthetic apparatus of the plant, both directly, via a decrease in pigment concentrations, and indirectly, via interference of Cu with Ca ions of PSII. Growth and seed production were only slightly affected by leaf tissue concentrations as high as 230 mg Cu kg⁻¹ dry mass, which suggests that this species could play an important role in phytoremediation of Cu-contaminated soils.     

A microbial consortium involving the astaxanthin producer Xanthophyllomyces dendrorhous on freshly cut birch stumps in Germany

The astaxanthin-producing Xanthophyllomyces dendrorhous was the dominant pigmented yeast in yellow- and orange-coloured microbial mats formed on exudates from freshly cut birch stumps in Kaiserslautern (Germany) in April 2005. Other ubiquitous members of the consortium included Mucor hiemalis, two Fusarium spp., the yeast Hanseniaspora uvarum and two rod-shaped bacteria.     

Asexual propagation in the coral reef macroalga Halimeda (Chlorophyta, Bryopsidales): production, dispersal and attachment of small fragments

Siphonous, green macroalgae of the genus Halimeda are ubiquitous and ecologically important in tropical and subtropical marine environments. It has been hypothesized that the abundance of Halimeda on coral reefs is in part due to the ability of this genus to propagate asexually via vegetative fragmentation. However, vegetative fragmentation has only been documented for H. discoidea in a laboratory setting. To test the hypothesis that vegetative fragmentation contributes to field populations of Halimeda, we examined three aspects of fragmentation by H. tuna (Ellis and Solander) Lamouroux, H. opuntia (Linneaus) Lamouroux and H. goreaui Taylor on Conch Reef in the Florida Keys: (1) short-term (8 days) and long-term (14 weeks) fragment survival and rhizoid production in the laboratory and field (7 and 21 m), (2) size of the fragment pool and (3) influences of herbivory and water motion on production and dispersal of fragments. Although morphologically similar to H. discoidea, only a small percentage of H. tuna fragments survived. Fragments of H. opuntia and H. goreaui were more robust, and survival and rhizoid production were positively correlated with size in short-term trials. In 14-week field trials, one-third or fewer fragments of any species survived at 7 m, potentially because fragments were covered by large amounts of sediment. Survivors included some buried, seemingly dead individuals that turned green when exposed to light, highlighting the remarkable ability of this genus to survive disturbances. There was much less sediment accumulation at 21 m, where more fragments survived. Most (93%) eight-segment fragments of H. opuntia produced attachment rhizoids by the end of the 14-week trial. Overall, a range of 4.7-9.4 fragments of Halimeda m⁻² day⁻¹ were found on Conch Reef; most fragments were generated by H. goreaui. Fish bite marks were evident on 75-85% of the individuals of H. tuna and the number of bites per thallus ranged from 1 to 23. Herbivorous reef fish commonly fed on all three species of Halimeda. Some fish consumed the biomass, while others rejected most bites. For example, 83% of bites were rejected by the blue-striped grunt. Dispersal distances for rejected bites ranged from 0 to 31 m. Water motion was also responsible for fragment dispersal; experimentally produced fragments moved up to 48 cm day⁻¹. Results presented here suggest that asexual propagation of fragments of Halimeda is an important component of the life-history of this genus and vegetative fragmentation contributes to the abundance of this genus on coral reefs.     

Effect of meal size on the specific dynamic action of the juvenile snakehead (Channa argus)

The effect of meal size on the specific dynamic action (SDA) of the juvenile snakehead (Channa argus) was assessed at 25 °C. The fish were fed with test diets at meal sizes of 0.5, 1, 2, 3, 4, and 5% body mass and the postprandial oxygen consumption rate was determined at 1-h intervals until it returned to the pre-prandial level. The peak metabolic rate increased from 237.4 to 283.2 mg O₂ kg^− 1 h^− 1 as the relative meal size increased from 0.5% to 3% and leveled off at 4% and 5%. Factorial metabolic scope increased from 1.53 to 1.99 and SDA duration increased from 11.7 to 32.3 h as the relative meal size increased from 0.5% to 5%. The relationship between SDA duration (D) and relative meal size (M) was described as: D = 4.28 M + 10.62 (r² = 0.752, P < 0.05, n = 50). The energy expended on SDA increased while the SDA coefficient decreased with increasing meal size. The results of the present study suggest that the snakehead may adopt different feeding strategies when taking in different amounts of food.     

Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

Sorgoleone,a sorghum root exudate: Algicidal activity and acute toxicity to the ricefish Oryzias latipes

The discovery of natural and natural-based compounds has resulted in its application as an alternative to synthetic algicides to control harmful algae in aquatic systems. Of the many natural-product-based algicides, sorgoleone, a natural plant product from Sorghum bicolor root exudates has been investigated for its controlling effect on different algal species and its acute fish toxicity. Growth of the blue green algal species Microcystis aeruginosa Kützing was completely inhibited by the crude methanol extract of sorghum root at 20 μg mL⁻¹. The most noticeable inhibition was observed in the bioassay of n-hexane soluble extract, where 98% growth inhibition occurred in M. aeruginosa at the concentration of 1.25 μg mL⁻¹. Sorgoleone very effectively controlled blue green algae inhibiting 97% of M. aeruginosa at 0.5 μg mL⁻¹ and 99% of Anabaena affinis Lemmermann at 4 μg mL⁻¹. In contrast, inhibition of the green algae species Chlorella vulgaris Beijerinck and Scenedensmus spp. at 16 μg mL⁻¹ sorgoleone was 87 and 68%, respectively. There were no mortalities or adverse effects observed in any of the fish exposed to water control, solvent control, and a nominal concentration of 1 μg mL⁻¹ during the test period. The no observed effect concentration (NOEC) value was 1.5 μg mL⁻¹ for the tested fish (O. latipes). Sorgoleone can be considered as an effective and an ecologically and environmentally sustainable approach to controlling harmful algae.     

Effect of Coriolus versicolor supplemented diet on innate immune response and disease resistance in kelp grouper Epinephelus bruneus against Listonella anguillarum

The effect of Coriolus versicolor extract supplemented diets on innate immune response and disease resistance in kelp grouper, Epinephelus bruneus against Listonella anguillarum, is reported. Kelp grouper were divided into four groups of 25 each and fed with C. versicolor enriched diets at 0% (control), 0.01%, 0.1%, and 1.0% level. After 30 days of feeding, all fish were injected interaperitoneally (i.p.) with 50 μl of L. anguillarum (4.7 × 10⁷ CFU) to investigate the immune parameters at weeks 1, 2, and 4. The reactive oxygen species and reactive nitrogen species production were significantly enhanced in fish fed with 0.1% and 1.0% supplementation diets from weeks 1-4 when compared to the non enriched diet fed and infected control. The phagocytic activity significantly increased with 0.1% and 1.0% diets on weeks 2 and 4. The leucocyte myeloperoxidase content, lysozyme activity, and total protein level significantly increased when fed with 0.1% and 1.0% supplementation diets from weeks 1-4. The cumulative mortality was 35% and 45% in 1.0% and 0.1% enriched diet fed groups whereas it was 55% and 80% in 0.01% and 0% groups respectively. The present results suggest that diets enriched with C. versicolor at 0.1% or 1.0% level positively enhance the innate immune system and affords protection from L. anguillarum.