Characterization and expression pattern of a novel β-defensin in common carp (Cyprinus carpio L.): implications for its role in mucosal immunity

β-defensins are a group of cysteine-rich cationic antimicrobial peptides that play antibacterial and antiviral roles in immune systems of vertebrates. Here, we report the cloning and identification of a β-defensin 3 cDNA sequence from the common carp (Cyprinus carpio L.). Sequence alignment and phylogenetic analysis indicated that this β-defensin 3 belonged to the BD-2 group of fish. Real-time PCR showed that the β-defensin 3 mRNA was expressed in all the tissues of normal common carp that we examined and was highly expressed in the spleen and gills. When challenged with Vibrio anguillarum, the expression level of common carp β-defensin 3 mRNA was quickly upregulated in various tissues. Our results indicate that the β-defensin 3 showed markedly high constitutive expression in the gills, and significantly upregulated expression in the hindgut of the common carp after infection, suggesting it plays an important role in the innate and mucosal immunity of common carp.     

Proximate Composition of Freshwater Fish — Grass Carp,Puntius gonionotus and Tilapia

Summary The proximate analyses of freshwater fish — Grass carp (Ctenopharyngodon idellus), Puntius gonionotus and Tilapia (male hybrid) are compared. Dried meals from eviscerated freshwater fish contained 57.36–75.92% protein; 4.48–31.84% fat and 10.68–22.88% ash. The results indicate that Tilapia had a higher fat content than gonionotus and grass carp, the latter having the lowest fat content. The reverse is true for protein, ash and minerals like sodium, phosphorus and potassium. The composition of grass carp feeding on different diets, such as Hydrilla verticillata, Napier grass (Pennisetum purpureum) and tapioca leaves (Manihot utilissins) indicated only a slight difference in protein content but a significant difference in fat and ash. The tabulated results show that grass carp and goniotus were of higher nutritive value when compared to the Tilapia.     

A beta-defensin 1-like antimicrobial peptide from the tree shrew, Tupaia belangeri

A novel beta-defensin 1-like antimicrobial peptide (β-defensin 1TB) containing 36 amino acid residues was purified and characterized from the serum of the tree shrew, Tupaia belangeri. Its amino acid sequence was determined as DHYLCVKNEGICLYSSCPSYTKIEGTCYGGKAKCCK, by Edman degradation, mass spectrometry analysis, and cDNA cloning. Evolution analysis indicated that β-defensin 1TB showed maximal similarity to the β-defensin 1 identified from cotton-top tamarin, Saguinus oedipus. β-defensin 1TB exerted potential antimicrobial activities against wide spectrum of microorganisms including Gram-negative and -positive bacteria and fungi. It showed little hemolitic activity to human or rabbit red cells. To the best of our knowledge, this is the first report of antimicrobial peptide from Tupaiidae.     

Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in ¹H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.     

Diet spectrum of great cormorants (<Emphasis Type="Italic">Phalacrocorax carbo sinensis</Emphasis> L.) at the Donji Miholjac carp fishponds in eastern Croatia

This paper reports the results of a diet analysis from the digestive tracts of 203 great cormorants shot at the Donji Miholjac fishponds in eastern Croatia, in the period 2000–2002. Eight fish species were determined. The dominant species was common carp (Cyprinus carpio) with a relative frequency of 73.4%, followed by grass carp (Ctenopharingodon idella) (11.6%), bighead carp (Aristichthys nobilis) (7.2%), pikeperch (Sander lucioperca) (2.0%), wels catfish (Silurus glanis) (1.7%), pike (Esox lucius) (1.5%), topmouth gudgeon (Pseudorasbora parva) (1.5%) and Prussian carp (Carassius auratus gibelio) (1.1%). The diet spectrum of the great cormorants from the Donji Miholjac fishponds was in accordance with the structure of the fish population in the fishponds. The average stomach weight per bird was 244 g. When birds shot with an empty stomach were excluded, the average stomach weight increased to 286 g. The length of consumed fish ranged from 40–335 mm, with 47% of the fish belonging to the length category 100–149 mm. The study revealed no significant relationship between the weight of the consumed fish and the body weight of male and female cormorants.     

Effects of contrasting omnivorous fish on submerged macrophyte biomass in temperate lakes: a mesocosm experiment

1. Freshwater fish can affect aquatic vegetation directly by consuming macrophytes or indirectly by changing water quality. However, most fish in the temperate climate zone have an omnivorous diet. The impact of fish as aquatic herbivores in temperate climates therefore remains unclear and depends on their dietary flexibility. 2. We tested the effects of a flexible omnivore and an herbivore on aquatic vegetation by comparing the effects of rudd (Scardinius erythrophthalmus, the most herbivorous fish in temperate climates) with grass carp (Ctenopharyngodon idella) in a mesocosm pond study. Exclosures distinguished herbivorous effects of fish on submerged macrophytes from indirect effects through changes in water quality, whereas stable isotope food‐web analysis provided information on fish diets. 3. We hypothesised that rudd, with its flexible diet and preference for animal food items, would only indirectly affect macrophytes, whereas grass carp, with its inflexible herbivorous diet, would directly affect macrophyte biomass. 4. Only grass carp significantly reduced macrophyte biomass through consumption. Rudd had no effect. Food‐web analysis indicated that rudd predominantly consumed animal prey, whereas grass carp included more plants in their diet, although they also consumed animal prey. Grass carp significantly affected water quality, resulting in lowered pH and increased N‐NH₄ concentrations, whereas more periphyton growth was observed in the presence of rudd. However, the indirect non‐herbivorous effects of both fish species had no effect on macrophyte biomass. 5. Both fish species should be considered as omnivores. Despite the fact that rudd is the most herbivorous fish in the western European climate zone, its effect on submerged macrophyte biomass is not substantial at natural densities and current temperatures.     

Effect of probiotic Bacillus subtilis Ch9 for grass carp,Ctenopharyngodon idella (Valenciennes, 1844), on growth performance,digestive enzyme activities and intestinal microflora

In the present study, Bacillus subtilis Ch9 was evaluated as a probiotic in grass carp, Ctenopharyngodon idella (Valenciennes, 1844). For 56 days the grass carp (50 ± 2.5 g) were given a feed containing B. subtilis Ch9 in three concentrations: 1.0 × 10⁹ (T₁), 3.0 × 10⁹ (T₂) and 5.0 × 10⁹ (T₃) CFU kg^?1 feed in triplicate treatments. The control group (T₀) was given feed without B. subtilis Ch9 for the same period. Determined were the specific growth rate (SGR), feed conversion ratio (FCR), and digestive enzyme activities in the intestine and hepatopancreas as well as the intestinal microflora. After 56 days, fish receiving the diets supplemented with B. subtilis Ch9 showed significantly higher SGR and lower FCR (P < 0.05) than those fed the control diet. There was no significant different in SGR and FCR among T₁, T₂ and T₃ nor was the survival rate affected (P > 0.05) by the dietary treatments. From days 14 to 56 of the experiment, higher protease, amylase and lipase activities in the foregut, midgut hindgut and hepatopancreas were observed in T₁, T₂ and T₃ (P < 0.05) compared with the control over a short‐term (14–28 days). Enzyme activity did not increase after long‐term feeding with B. subtilis Ch9 (56 days), but was still higher than that of control fish (P < 0.05). Fish fed the probiotic had an increase in trend of total aerobic and facultative anaerobic bacterial quantity (P > 0.05), but the ratio of Bacillus was significantly higher (P < 0.05) than in control fish. The total anaerobic bacterial quantity, Bifidobacterium and Lactobacillus were significantly higher (P < 0.05) in fish fed B. subtilis Ch9 compared with fish fed control feed. In conclusion, an optimum dose of B. subtilis Ch9 could modulate intestinal microflora, induce digestive enzyme activity and potentially promote the digestion and absorption of nutrients, as well as improve the growth performance of grass carp significantly.     

草鱼β-防御素1的趋化活性及免疫佐剂效应研究

为了探究草鱼防御素的免疫调节作用, 研究通过Clustal Omega多序列比对和I-TASSER二级结构预测, 发现草鱼β-防御素1(Ctenopharyngodon idella β-defensin 1, CiBD1)是一类具有两亲性的富含半胱氨酸的小分子阳离子肽, 其结构在硬骨鱼类中高度保守; 通过构建pET-32a-CiBD1原核表达载体, IPTG诱导表达后经过Ni2+亲和层析法纯化和肠激酶酶切获得CiBD1重组蛋白, 通过CFU平板法验证了CiBD1的抑菌活性, 当蛋白浓度达到200 μg/mL时, 对革兰氏阴性菌(G+)及革兰氏阳性菌(G–)都具有显著的抑制活性; 趋化实验表明CiBD1在50 ng/mL时对草鱼原代白细胞趋化活性最佳; 通过个体实验探究了CiBD1重组蛋白对嗜水气单胞菌灭活疫苗的免疫佐剂效应, 发现添加 CiBD1佐剂组死亡率显著低于单独疫苗组, 且该组IgM和MHC Ⅱ转录水平及血清中补体3(C3)和溶菌酶含量都显著高于对照组。研究表明CiBD1重组蛋白在细菌抗感染免疫中发挥着重要作用, 在水产养殖中具有一定的应用前景。     

Relationship between meteorological conditions and natural reproduction behavior of the four famous Chinese carps

Known as the four famous carps in China, black carp (Mylopharyngodon piceus), grass carp (Ctenopharyngodon idella), silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) are commercially important fish species with a high production in China. To reveal the relationships between meteorological conditions and the natural spawning behavior of these fishes, we considered six meteorological factors (including wind, rainfall, temperature, air pressure, sunshine hours and humidity) and the weather conditions in 80 spawning events. The results showed that the spawning activities were more likely to be activated in consecutive rainy days or days when weather changed drastically, while the spawning activities showed no tendency for the weather type except consecutive rainy days. Our analyses also showed that the average rainfall in the initial spawning days is higher than that in the spawning time windows (the time from the earliest spawning activity to the latest spawning activity), while other meteorological elements remained at more or less the same values in both time periods; spawning activity tended to happen in days when the average air pressure was going down or the average temperature going up, while the other meteorological elements showed no consistent tendency.     

Experimental induction of Sphaerospora renicola (Myxosporea) infection in common carp (Cyprinus carpio) by transmission of SB-protozoans

The authors have experimental evidence that the protozoa causing the swimbladder inflammation (SBI) of the common carp (Cyprinus carpio) are indentical with presporogonic stages of Sphaerospora renicola Dyková et Lorn, 1982 parasitizing the renal tubules. Homogenates prepared from the thickened and inflamed swimbladder of naturally infected common carp, when injected into the abdominal cavity of fish, produced renal sphaerosporosis in the infection-free common carp if the homogenates contained the parasites described by Kovács -Gayer et al. (8). By intraperitoneal injection, the Unidentified Blood Organisms (UBOs) living in the blood of the common carp were transmissible to common carp, from the blood of which they were demonstrable for a long time. However, they were not transformed into Sphaerospora. To other cyprinids (gibel carp, silver carp, grass carp, tench, roach) neither the blood stages nor the swimbladder stages were transmissible from the common carp.     

Complete mitogenome sequence of black carp (<Emphasis Type="Italic">Mylopharyngodon piceus</Emphasis>) and its use for molecular phylogeny of leuciscine fishes

The black carp Mylopharyngodon piceus (Cyprinidae), native to eastern Asian, is a large, commercially important fish, and has been introduced to many other countries for variable reasons. In this study, the complete mitochondrial genome sequences from three specimens of black carp were first determined and were used to evaluate the sister relationship between black carp and grass carp (Ctenopharyngodon idellus). Two individuals had a mitogenome of 16,609 bp, while the other was 16,611 bp in length. Similar to most vertebrates, the black carp contains the same gene order and an identical number of genes or regions, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 putative control region. Phylogenetic analyses using three different computational algorithms (Maximum Parsimony, Maximum Likelihood, and Bayesian analysis) revealed two distinct clades in subfamily Leuciscinae. However, the sister taxonomic relationship of black carp and grass carp was not observed using sequences of nearly complete mitochondrial genomes, which suggests more nuclear gene markers are needed to resolve the phylogenetic relationship between black carp and grass carp.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

Effect of meal size on excess post-exercise oxygen consumption in fishes with different locomotive and digestive performance

Effects of feeding on pre-exercise VO₂ and excess post-exercise oxygen consumption (EPOC) after exhaustive exercise were investigated in sedentary southern catfish, active herbivorous grass carp, omnivorous crucian carp, and sluggish omnivorous darkbarbel catfish to test whether feeding had different effects on EPOC and to compare EPOC in fishes with different ecological habits. For fasting fish, the pre-exercise and peak post-exercise VO₂ were higher and recovery rates were faster in crucian carp and grass carp compared to those of darkbarbel catfish and southern catfish. EPOC magnitudes of grass carp and southern catfish were significantly larger than those of crucian carp and darkbarbel catfish. Feeding had no significant effect on peak post-exercise VO₂, recovery rate, and EPOC magnitude in grass carp. Both the pre-exercise and peak post-exercise VO₂ increased with meal size, while the EPOC magnitude and duration decreased significantly in the larger meal size groups of crucian carp and southern catfish. In darkbarbel catfish, both the pre-exercise and peak post-exercise VO₂ increased with meal size, but the VO₂ increment elicited by exercise was larger in feeding groups compared with the fasting group. These results suggest that (1) the characteristics of the post-exercise VO₂ profile, such as peak post-exercise VO₂ and recovery rate, were closely related to the activity of fishes, whereas the EPOC magnitude was not and (2) the effects of feeding on EPOC were more closely related to the postprandial increase in VO₂.     

The effects of argulosis-saprolehniasis on the growth and production ofCyprinus carpoi

Growth profiles of maleCyprinus carpio, 12 uninfected (healthy) and 12 infected with the crustaceanArgulus indicus and associated fungusSaprolegnia sp. are presented. The fish were maintained under controlled conditions and provided withad libitum food for 8 weeks. The instantaneous (G) and relative (h) rates of growth, and production (P) in uninfected carp (0.003 to 0.036 g.d^–1, 0.29 to 3.68%, 0.25 to 3.23 g respectively) were much higher than those of infected carp ( – 0.007 to 0.021 g.d^–1, 0.71 to 2.17%, 0.59 to 1.76 g). The condition factor (K) for uninfected carp was 4.5 to 4.8 compared to 2.6 to 3.0 for infected carp. During each of the 8 wk the weekly change in weight of infected carp was about 4% less than that of uninfected carp. These data show thatArgulus-Saprolegnia infections significantly suppress carp growth and indicate that comparative growth rates can be used as indicators of parasite stress.     

中国淡水渔业碳汇强度估算

【背景】碳汇是指从大气中消除二氧化碳的过程、活动或机制,我国最先提出碳汇渔业概念。【方法】捕捞鱼类的碳均来自天然饵料,故以其平均碳含量估算碳移出量。而养殖鱼类中,一般假定不考虑施肥养鱼的碳输入;鲢和鳙是滤食性鱼类,主要摄食浮游生物,鳜属鱼类以其他种鱼类为食物,而这些鱼类主要摄食天然饵料,故可以认为其碳均来自天然饵料。此外,假设草鱼、鲫和鲤等产量的20%来自天然饵料,而河蟹产量的50%来自天然饵料。基于渔业统计年鉴(2011—2015年),估算了我国近5年来淡水渔业碳汇强度。【结果】2010—2014年,全国淡水养殖碳移出量逐年稳步增长,分别为136.2万、140.5万、146.0万、153.0万和164.5万t,平均每年的碳移出量为148.0万t。2010—2014年全国淡水捕捞碳移出量分别为29.3万、28.7万、29.6万、29.7万和29.6万t,平均每年的碳移出量为29.4万t。【结论与意义】在自然资源日益减少的情况下,淡水养殖渔业碳汇的发展必然会成为淡水渔业经济发展的主体。     

Evidence for pituitary adenylate cyclase-activating peptide as a direct immunoregulator in teleost head kidney

In mammals, pituitary adenylate cyclase activating polypeptide (PACAP) is a potent anti-inflammatory factor, showing that it inhibits the expression and release of proinflammatory cytokines and enhances the production of anti-inflammatory factors. However, whether fish PACAP plays similar regulatory roles as seen in mammals remains unclear. In the present study, expression of PACAP-specific receptor PAC1-R was shown in grass carp head kidney and spleen, supporting that PACAP may have a direct effect on fish immune cells. To test this hypothesis, the immunoregulatory role of grass carp PACAP (gcPACAP) was examined in head kidney leucocytes (HKLs). Results showed that gcPACAP inhibited basal and further attenuated lipopolysaccharide (LPS)-stimulated cell viability of HKLs, indicating that gcPACAP may possess similar inhibitory property at cellular level as seen in mammals. Curiously, in vitro and in vivo studies revealed that gcPACAP stimulated proinflammatory factors (IL-1β and TNF-α) but not IL-10 mRNA expression in HKLs and head kidney. Moreover, bacterial infection and LPS enhanced IL-1β, TNF-α and IL-10 mRNA expression in grass carp head kidney and HKLs, respectively, and these stimulatory effects were not influenced by gcPACAP. These findings suggest that PACAP plays distinct roles, at least does not function as an anti-inflammatory factor, in fish compared with that in mammals.     

Impact of Juvenile Stocking Size on the Major Carp Production in a Minor Reservoir,Bibinagar, India

The present study deals with the impact of juvenile stocking sizes of the major carp on production in a minor reservoir, Bibinagar, Nalgonda, India. Reservoir surface area equaled over 23.8 ha and water from the reservoir was used for irrigation and fisheries. Four experiments were conducted for 4 years from 2000–2001 to 2003–2004. The experiments were planned in such a way that every year juvenile stocking size was held constant and subsequent fish production analyzed. During the first year’s experiment (2000–2001) a stocking size of 25–30 mm (fry) was maintained. Similarly 50–55 (advanced fry), 75–80 mm (fingerling) and 100–105 mm (advanced fingerling) were stocked during 2001–2002, 2002–2003 and 2003–2004 respectively. Uniform yearly stocking densities (2000/ha) were established in each experiment in the month of July and fish were harvested in June of the subsequent year. Major carp production was enhanced with larger stocking size. Yearly productions equaled 144.00, 231.48, 632.91 and 1005.03 kg/ha/year with the above stocking sizes. Variation in stocking sizes had significant (P<0.05) effects on fish production. The number of fish per kg production decreased gradually with increases in stocking size with an average value of 3.97. Reservoir production of catla was the most abundant species, followed by rohu, common carp, mrigal and grass carp. These results show that stocking size has a great impact on fish production and the stocking of advanced fingerlings will provide maximum carp production in minor reservoirs in India.