Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes—Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.     

刺五加鲨烯合酶基因的表达及其对皂苷含量的影响

为了了解刺五加鲨烯合酶基因(squalene synthase,SS)的表达特点及其对刺五加中皂苷含量的影响情况,以GAPDH基因为内参照基因,采用实时定量PCR技术,分析了SS基因在刺五加不同生长发育时期不同器官中的表达量及茉莉酸甲酯对其表达的影响情况;还利用SPSS 17.0软件分析了SS基因的表达量与刺五加中皂苷总含量的相关性。结果表明,在刺五加的整个生长期中,SS基因的表达量呈现出"低—高—低—高—低"的变化趋势:萌芽期(4月26日)该基因的表达量最低,而盛花期(6月26日)该基因的表达量最高,为萌芽期的3.11倍;SS基因在刺五加根中的表达量显著高于茎、叶和叶柄中的表达量(P<0.05),其在茎中的表达量最低,仅为根中表达量的51.88%。茉莉酸甲酯可提高SS基因的表达量,但其与对照组的差异未达到显著水平。刺五加中的皂苷总含量与SS基因的表达量间呈现出同升同降的变化趋势,存在极显著的正相关关系(P<0.01)。文中初步判定,鲨烯合酶是刺五加皂苷生物合成的关键酶。     

杧果短截结果母枝玉米素核苷含量的动态变化

【目的】测定杧果花芽分化期间结果母枝各器官内玉米素核苷(ZR)含量,探索ZR在杧果花芽分化中的作用。【方法】利用杧果花枝短截后剪口芽在当年还能再次花芽分化并开花结果的现象,应用酶联免疫吸附分析方法(ELISA)定期测定剪口叶、附近韧皮部及芽内ZR含量。【结果】短截结果母枝后,剪口叶、附近韧皮部和剪口芽内ZR含量分别于2月25、20日和3月2日降至最低,为1.73、1.52、2.0μg·g-1FW;叶片、韧皮部及剪口芽于3月12日达最大值,为6.39、8.47、10.32μg·g-1FW。2月25日剪口芽芽眼中心颜色逐渐呈蜡黄,并向芽体四周扩散;3月2～7日,芽体逐渐膨大,蜡黄色加深,芽眼呈半透明状;3月7日后绿色逐渐增加,半透明状消失。【结论】ZR在杧果花芽分化的不同阶段具有不同作用。花芽分化临界期前,ZR含量低,生长点细胞活性下降,有利于杧果植株从叶芽生理状态转化为花芽生理状态;花芽临界期后,相对高的ZR含量有利于杧果植株生长点进行花芽分化。     

Development and evaluation of a fish‐based index to assess biological integrity of Mediterranean streams

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Decapsulated Artemia Cysts: A Suitable Dietary Supplement for Juvenile Tench (Tinca tinca L.)

There are no specifically formulated dry foods for tench (Tinca tinca L.), which forces farmers to use diets formulated for other fish species. This has major drawbacks, such as high mortality, slow growth, and body deformities. A 120 day experiment was performed with five-month-old juvenile tench (initial mean weight: 0.388 g; total length: 31.78 mm) to evaluate decapsulated Artemia cysts as a supplement to a dry diet for other fish species. Three treatments, differing in the daily supplement, were tested: 1,800 freshly hatched nauplii, 1,800 cysts, and 300 cysts per g of tench biomass. Final survival ranged between 95.3% and 97.9%. Juvenile tench that received the supplement of 1,800 decapsulated Artemia cysts had a specific growth rate (1.28), weight (1.83 g), and total length (52.30 mm) significantly higher than those with the same amount of nauplii. The lowest supplement (300 cysts/g of fish biomass) allowed significantly lower growth and higher condition coefficient (1.40) than the rest. Animals with body deformities (1.06%) were only recorded in the groups that received the lowest cyst supplement. Results showed Artemia cysts are a suitable dietary supplement for juvenile tench, being an advantageous alternative to live nauplii.     

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land‐based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real‐time RT‐PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re‐isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real‐time RT‐PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.     

First results of embryonic development,spawning and larval rearing of the Mediterranean spider crab Maja squinado (Herbst ) under laboratory conditions,a candidate species for a restocking program

The Mediterranean spider crab, Maja squinado, is depleted due to overfishing. The crab has virtually disappeared from areas where it was abundant, such as the Balearic Islands and the Catalan coast. Maja squinado, is economically and ecologically very valuable, and it is essential to obtain information on its biology and rearing conditions to attempt to repopulate the damaged stocks of the species in the Mediterranean basin. Herein, we describe the first successful rearing of M. squinado under laboratory conditions. Our results show that M. squinado is an excellent candidate for restocking using cultured juveniles. Two consecutive broods with a 1–4 day interbrood period were observed in the laboratory in wild‐caught females, the maximum observed duration of embryonic development of the egg mass being 32 days at 18.4 ± 0.9°C, and went through four different stages. The complete larval and first juvenile development was studied in laboratory cultures fed enriched Artemia metanauplius. At 19.6 ± 0.6°C, development from hatching to first crab moult took 17 days, and it comprised two zoeae stages and one megalopa stage. The survival rate at the different stages was monitored, and 7.13 ± 2.3% was achieved at the first crab instar.     

Litopenaeus vannamei (Boone) post-larval survival related to age, temperature, pH and ammonium concentration

Transport of post‐larvae shrimp used in aquaculture is an important element of successful cultivation because of the potential for stress during stocking procedures. To find optimum transport conditions, several bioassays were performed in the laboratory to evaluate survival of whiteleg shrimp Litopenaeus vannamei 5–30‐day‐old postlarvae under conditions similar to those encountered during transport from the hatchery to nursery and shrimp ponds. Postlarvae were exposed for 4 h to different temperatures and pH levels ammonia concentrations. Survival was significantly reduced after a 4 h exposure to pH 9 and was inversely related to temperature with or without 7 mg L^?1 of ammonia. The 15‐ and 20‐day‐old postlarvae had higher survival rates than other ages. The lowest survival occurred in alkali conditions (pH 9), with 7 mg L^?1ammonia at 30 and 32°C. To assure optimal survival of postlarvae during transfer from the hatchery to the nursery and shrimp ponds, we recommend temperatures below 28°C, pH no higher than 8, no ammonia and post‐larval age at least 15 days.     

Regulation of growth, fatty acid composition and delta 6 desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aurata)

The Δ6 and Δ5 desaturases and elongases show only very limited activity in marine fish, and little is known of the possibility of enhancing Δ6 desaturase gene expression in these fish. The use of plant oils in marine fish diets is limited by their lack of n−3 highly unsaturated fatty acids (HUFA) despite an abundant content of the 18C fatty acid precursor linoleic and α-linolenic acids. The objective of the present study was to determine the ability of larval gilthead seabream to utilize vegetable oils and assess the nutritional regulation of Δ6 desaturase gene expression. Seventeen-day-old gilthead seabream larvae were fed during a 17-day period with one of four different microdiets formulated with either sardine fish oil (FO), soybean, rapeseed or linseed oils, respectively, or a fifth diet containing defatted squid meal and linseed oil. Good larval survival and growth, both in terms of total length and body weight, were obtained by feeding the larvae either rapeseed, soybean or linseed oils. The presence of vegetable oils in the diet increased the levels of 20:2n−9 and 20:2n−6, 18:2n−9, 18:3n−6, 20:3n−6 and 20:4n−6, in larvae fed rapeseed and soybean oils in comparison to those fed FO. In addition, a sixfold increase in the relative expression of Δ6 desaturase-like gene was found in larvae fed rapeseed and soybean oils, denoting the nutritional regulation of desaturase activity through its gene expression in this fish species. However, feeding linseed oil did not increase the expression of the Δ6 desaturase gene to such a high extent.     

A genetic tool for evaluating male‐mediated stock introgression in Atlantic salmon

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