Raw frozen Antarctic krill (Euphausia superba) as an alternative feed source for cuttlefish Sepiella japonica in artificial breeding systems

The study aims to evaluate the feasibility of completely replacing raw frozen shrimp Palaemon gravieri diets with raw frozen Antarctic krill (Euphausia superba) in the diets of cuttlefish Sepiella japonica. To address the knowledge gap, we conducted a 60‐day feeding trial. At the end of the experiment (day 60), the cuttlefish Sepiella japonica eating Palaemon gravieri (SJP) grew significantly faster than those eating Euphausia superba (SJE), with the specific growth rate (SGR)_SJP (7.92%) > (SGR)_SJE (7.09%). Approximately 33.3% and 20.0% mortality was observed in the SJE and SJP, during the course of the experiment respectively. Some important fatty acids (i.e. n‐3 and n‐6 PUFAs) were elevated in SJE with respect to SJP. Replacement of Antarctic krill increased the diversity of the gut microbiome composition in the SJE group. Fluoride accumulated in the ink sac and cuttlebone of cuttlefish in SJE. Overall, these findings imply that PUFA‐rich Antarctic krill could replace P. gravieri shrimp for feeding cuttlefish S. japonica.     

Revealing the compositions of the intestinal microbiota of three Anguillid eel species using 16S rDNA sequencing

Anguilla japonica, A. marmorata and A. bicolor pacifica are important eel species for aquaculture in Taiwan. In the past, seldom studies focused on the intestinal microbiome of them or the probiotics used in their cultivation. Probiotics are microbes that can improve the health of organisms, and the microbes isolated from the intestines of organisms might be the potential probiotics. Therefore, the aim of this study was to collect the intestinal mucus samples of these three eel species for investigating the autochthonous microbiome using 16S rDNA sequencing. The diversity, abundance of the intestinal microbiome and compositions of all the libraries were identified. Moreover, the composition of intestinal microbiome of eel was suggested that it would be affected by the waters and the characteristics of different eel species. Furthermore, most of the identified predominant genera of the intestinal microbiome of these three eel species, such as Cetobacterium, Clostridium, Shewanella, Acinetobacter and Bacteroides, seemed to have great potential as the probiotics. In the future, more studies shall be conducted to promote the practical application of these candidate probiotics for improving the techniques of eel farming.     

Analysis of bacterial diversity in the intestine of grass carp (Ctenopharyngodon idellus) based on 16S rDNA gene sequences

In the current study, we assessed bacterial diversity in the gut content of pond-reared grass carp (Ctenopharyngodon idellus), in the associated habitat environments (pond water and sediment) and in the ingested food (commercial feed and the reed Phragmites australis) by analysing 16S rDNA sequences from clone libraries. The highest bacterial diversity was observed in the gut content and was determined by the total number of operational taxonomic units, Shannon diversity index (H), Shannon equitability index (E_H), Coverage (C_good) and rarefaction curves calculated from the 16S rDNA gene libraries. Our data indicated that allochthonous gut microbes of grass carp were distinctively different from the corresponding environmental microbes. The pairwise similarity coefficient (C_s) for microbe communities between gut content and ingested food was higher than for those between the gut content and habitats, indicating that the allochthonous microbiota identified in the intestines of grass carp were phylogenetically closer to those in the ingested food than to those in the habitat. Based on our study and previous research, we suggest that the digesta of grass carp harbours a microbiota phylogenetic core of Proteobacteria and Firmicutes and this observation deserves further investigations with respect to a potential pool of probiotics to grass carp.     

基于COI和16S rRNA的库页岛马珂蛤遗传多样性和分子系统进化研究

本研究以库页岛马珂蛤(Pseudocardium sachalinense)为研究对象,讨论COI和16S rRNA两种DNA条形码在贝类的遗传多样性、分子进化和种类鉴定的适用性,并利用两种条形码评估库页岛马珂蛤的遗传多样性。本文在获得库页岛马珂蛤线粒体全基因组的基础上,测序获得库页岛马珂蛤群体的COI和16S rRNA序列,发现COI基因核苷酸多样性为0.00195,高于16S rRNA核苷酸多样性(0.00073)。基于COI基因的单倍型多样性为0.76,大于16S rRNA的单倍型多样性(0.318)。其次,用全线粒体基因组构建8种贝类的系统进化树为参考,发现基于COI和16S rRNA的系统进化树与参考一致,提示这两种条形码片段可用于推断贝类的分子进化关系。最后,分别对马珂蛤科和帘蛤科15属17种贝类的COI基因和16Sr DNA进行序列比较,发现COI基因和16S rRNA的种间遗传距离均是种内距离的62倍。以上结果说明,16S rRNA与COI基因一样,能有效地构建马珂蛤科和帘蛤科的系统发育关系和物种鉴定,但在分析库页岛马珂蛤的遗传多样性时利用COI基因比16S rRNA能发现更多的遗传变异。     

中国南海斑节对虾5个地理群体线粒体18S rRNA基因序列比较分析

摘要：本文对中国南海海域5个斑节对虾野生群体（三亚群体（SY）、深圳群体（SZ）、阳江群体（YJ）、湛江群体（ZJ）、北海群体（BH））100个样品的 16SrRNA 序列进行PCR扩增,并对扩增产物进行了测序。用Clustal_X排序软件对测序所得的100个16S rRNA序列进行比对。通过ARLEQUIN软件对所得100个mtDNA16S rRNA序列进行比较分析,共检测出28个变异位点,19种单倍型。三亚、深圳、阳江、湛江以及北海等5个野生群体的核苷酸多样性（π）依次分别为0.00435,0.00586,0.01050,0.01081,0.01168。三亚、深圳、阳江、湛江以及北海等5个野生群体的单倍型多样性（H）依次分别为0.6895, 0.5211, 0.5737, 0.6000, 0.7211。对5个野生群体的16S rRNA序列进行FST分析,结果表明湛江、北海群体分别与三亚、深圳两个群体有显著的遗传差异,其余群体之间的遗传差异不显著。对5个群体进行AMOVA分析,结果表明5个群体间存在显著性遗传差异。对5个群体构建分子系统树,结果表明：三亚和深圳群体之间的亲缘关系最近;北海、湛江群体与三亚、深圳群体的亲缘关系很远。以上结果表明,中国南海海域5个斑节对虾野生群体可以为斑节对虾的选择育种提供两个基础群体：一个是三亚、深圳野生原种基础群体;另一个是湛江和北海野生原种基础群体。     

Genetic diversity in the Indian population of Penaeus monodon (Fabricius, 1798) as revealed by mtDNA sequence analysis

Genetic diversity in the Indian population of the tiger shrimp Penaeus monodon was determined by using partial sequence data of the mitochondrial DNA (mtDNA) control region (D‐loop) and the 16S rRNA gene. Eight populations from different geographical locations (Mumbai, Kochi, Mangalore, Kakinada, Gopalpur, Chilika, Paradeep and Andaman) were collected and analysed. The amplified polymerase chain reaction products of size 577 bp for the control region and 472 bp for 16S rRNA were sequenced in both directions and data were analysed through clustal , arlequin , mega and phylip . A significant genetic structure was found among the Indian populations. The mtDNA control region proved to be a powerful marker in comparison with 16S rRNA for population studies of this species. The east coast population was more genetically diverse than the west coast. The Andaman population was found to be the most diverse among all the populations. The populations on the west coast were found to be genetically more structured and differentiated than the populations on the east coast. The results revealed a high level of genetic diversity and also distinct population structuring of P. monodon, suggesting great possibilities of genetic improvement for growth and other economic traits.     

Genetic diversity and structure of Northern Sheatfish (Silurus soldatovi) assessed by newly developed and cross‐species microsatellites

Twenty‐five polymorphic microsatellite markers, including 13 newly isolated markers and 12 existing cross‐species markers, were used to assess the genetic variation of 141 individuals from three cultured stocks of northern sheatfish, Silurus soldatovi, in China. Samples were collected from Heilongjiang (H), Tongerpu (T) and Liaoning (L). Allelic richness, expected and observed heterozygosity among 25 loci were calculated, and the mean A_e, H_o and H_e were estimated in three stocks. In low polymorphic locus HLJcf19 and HLJcf38, most individuals in all three stocks were homozygous and had only one same allele, while only few heterozygous individuals had the other allele in one certain stock. And all the three stocks showed excess of heterozygotes, because of significant negative F_IS values. Genetic distance, UPGMA dendrogram, as well as structure analysis, all indicated that Stock H and L were closer among three stocks. The results revealed that though all stocks had no reduction in recent time, their effective population size were significantly lower to the ideal value.     

PCR扩增特异性16SrDNA和溶血素基因检测致病性嗜水气单胞菌

根据已发表的气单胞菌16S rDNA基因序列及气单胞菌气溶素(aerolysin)基因序列，设计了2对引物，建立了检测致病性嗜水气单胞菌的PCR方法。通过对12株气单胞菌的检测，发现16S rDNA引物具有高度的特异性，仅对嗜水气单胞菌扩增阳性。而Aero基因引物检测结果与采用生物学方法(鲜血平板法)检测的结果符合率为97．2％，且具有高度的敏感性，可检测最低1fg的模板。将16S rDNA与Aero基因结合PCR方法检测致病性嗜水气单胞菌与用致病性嗜水气单胞菌检测试剂盒的符合率为94．4％。该方法的建立为致病性嗜水气单胞菌的检测提供了一种简便、快速的途径，是一种比较实用的致病性嗜水气单胞菌的检测方法。     

Genetic diversity in three redclaw crayfish (Cherax quadricarinatus,von Martens) lines developed in culture in China

The redclaw crayfish Cherax quadricarinatus (von Martens) accounts for the entire commercial production of freshwater crayfish in Australia. Two forms have been recognized, an ‘Eastern’ form in northern Queensland and a ‘Western’ form in the Northern Territory and far northern Western Australia. To date, only the Eastern form has been exported overseas for culture (including to China). The genetic structure of three Chinese redclaw crayfish culture lines from three different geographical locations in China (Xiamen in Fujian Province, Guangzhou in Guangdong Province and Chongming in Shanghai) were investigated for their levels and patterns of genetic diversity using microsatellite markers. Twenty‐eight SSR markers were isolated and used to analyse genetic diversity levels in three redclaw crayfish culture lines in China. This study set out to improve the current understanding of the molecular genetic characteristics of imported strains of redclaw crayfish reared in China. Microsatellite analysis revealed moderate allelic and high gene diversity in all three culture lines. Polymorphism information content estimates for polymorphic loci varied between 0.1168 and 0.8040, while pairwise F_ST values among culture lines were moderate (0.0020–0.1244). The highest estimate of divergence was evident between the Xiamen and Guangzhou populations.     

Assessment of the bacterial community diversity associated with the queen conch Strombus gigas (Linnaeus, 1758) from the Caribbean coast of Colombia using denaturing gradient gel electrophoresis and culturing

The biotic diversity of Strombus gigas has not been thoroughly studied despite the status of the queen conch as an important ecological resource. The bacteria associated with the conch influence their host in several ways, including through the metabolism of nutrients, protection against invasive bacteria and the regulation of the physical conditions. In this study, conventional microbiological methods and molecular tools such as denaturing gradient gel electrophoresis (DGGE) were used to assess the composition of the bacterial communities associated with the queen conch (S. gigas) in wild and captive habitats in Colombia. A genetic analysis of the bacterial communities revealed a high level of diversity based on the large number of bands detected using DGGE. In addition, differences in bacterial community structure were found between the conchs in captivity and the wild populations. The dominant phylogenetic affiliations of the bacteria, as determined using 16S rRNA gene sequencing, were grouped into four classes, namely, Betaproteobacteria (16%), Gammaproteobacteria (70%), Firmicutes (7%) and Actinobacteria (2%). These groups are related to host defence processes and the decomposition of organic matter. The 16S rDNA sequence analysis of the cultured bacteria and the resulting DGGE profiles are useful tools for characterizing the diversity of the bacteria associated with the analyzed conchs.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

Site fidelity,diel and seasonal activities of yellow‐phase Japanese eels (Anguilla japonica) in a freshwater habitat as inferred from acoustic telemetry

Japanese eels (Anguilla japonica) perform large‐scale oceanic migrations between their spawning ground and growth habitats in continental waters during life history. However, between these migrations, they spend most of time in growth habitats such as rivers. To investigate the diel and seasonal activities, homing behaviour and home range of yellow‐phase Japanese eels in the lower reach of the Tone River, we tracked them throughout a year using a fine‐scale positioning system (VPS) based on acoustic telemetry. The tracked eels were generally nocturnal, but not exclusively. They were mainly mobile from spring to autumn, with little or no activity observed during winter. A transport‐release experiment showed that most eels returned to their original capture area within 13 days after release. The eels had very small home ranges (mean ± SD = 0.085 ± 0.068 km²), core areas (0.014 ± 0.014 km²) and linear home ranges (744 ± 268 m). They also tended to be distributed on one particular side of the river (right or left bank) and in one particular shore type (revetment or vegetation), rarely moving from one to the other. This study provides evidence for nocturnal, dormancy, homing behaviours, limited habitat use and small home range size in Japanese eels. The eels clearly showed strong fidelity to a “familiar” site, which contrasts with the long distances travelled during upstream and downstream migration phases in the river, and during spawning migrations in the ocean.     

16SrDNA序列分析鉴定一株合浦珠母贝共附生乳酸菌

从合浦珠母贝（Pinctada fucata）粘液中分离纯化得到一株抗菌物质产生菌F9,利用16SrDNA方法鉴定该菌株为棒状乳杆菌（Lactobacillus coryniformis）。通过琼脂扩散法测定了该菌的抑菌谱,发现该菌发酵液上清液在排除有机酸和过氧化氢（H2O2）的影响后仍对金黄色葡萄球菌（Staphyloccocus aureus）等多种食源性致病菌有明显的抑菌作用。对该菌发酵液抑菌物质进行超滤分析及蛋白酶处理,可推断该抗菌物质为分子量在5～10kDa的蛋白类物质。     

Effects of dietary protein sources on growth,survival and digestive capacity of Octopus maya juveniles (Mollusca: Cephalopoda)

We propose two hypotheses to explain the inexistence of adequate prepared diet for Octopus maya at this date: Hypothesis 1 is related to changes in protein structure during protein cooking, which affects the digestibility. Hypothesis 2 is related to changes on nutritional characteristics during ingredient process, which affects the nutritional composition of diet. To test hypothesis 1, experiments one and two were directed to determine if protein cooking reduces digestibility and growth of animals when compared to fresh or lyophilized protein sources. For hypothesis 2, three experiments were conducted, testing seven different dietary protein sources offered in isolation or combined in artificial diets fed to O. maya. Results demonstrated that the diets that promoted growth were the ones based on fresh crab paste, and both lyophilized crab and squid tentacles paste. In consequence hypothesis 1 was accepted. The cooking process also changed nutritional characteristics of protein sources, affecting the growth of O. maya. Results obtained when squid and crab were mixed suggest that nutritional requirements of octopuses were covered with that diet in similar forms compared to when using fresh or lyophilized crab, also confirming hypothesis 2. Based on growth rates obtained, we can conclude that nutritional requirements of O. maya must be between 80% and 86% Protein (P), 5.1–5.6% Lipids and a protein: energy ratio between (P/E) 38.9 and 42.2 g MJ^?1.     

Correlation between gut microbial diversity and flatulence of silver pomfret (Pampus argenteus) during ontogenesis

Gut microorganisms play an important role in host health and have susceptibility to diseases, undergoing a distinct transformation at early stages of development. This study aimed to determine the regulation of gut microorganism development from fertilized eggs to 150 DAH (days after hatching) and the differences between flatulent and healthy silver pomfret (Pampus argenteus). High‐throughput sequencing was used to analyze the diversity and relative abundance of microorganisms. PICRUSt was used to predict functional alterations of dominant microorganisms. The results showed that from all 42 samples, a total of 3,746,895 reads were obtained, representing 6,081 operational taxonomic units (OTUs). Top 3 phyla were Proteobacteria, Firmicutes and Bacteroidetes. From 0 to 32 DAH, the relative abundance of Proteobacteria continuously increased and peaked at 94.99%. After that, the abundance of Proteobacteria decreased, but Bacteroidetes and Firmicutes increased instead. Alpha diversity is the lowest at 32 DAH, the difference is significant compared with others, p < .05. Comparing flatulent fish with healthy fish, the former has more specific OTUs, higher diversity and abundance. There were four genera Ethanoligenens, Methanobacterium, Caproiciproducens and Leptolinea that only obtained from the flatulent samples, but almost none in the healthy samples. All of these four genera are directly or indirectly involved in gas metabolism. This study enriched the basic research of silver pomfret, for the first time, focused on flatulent individuals which would provide a theoretical basis for the subsequent breeding.     

Genetic relatedness and differentiation of hatchery populations of Asian seabass (Lates calcarifer) (Bloch, 1790) broodstock in Thailand inferred from microsatellite genetic markers

After more than 20 years of hatchery production of Asian seabass in Thailand, genetic information is still lacking for effective genetic management and a selective breeding programme. This study aimed to evaluate genetic status of existing hatchery populations and genetic consequences of a selective breeding attempt. We examined genetic relatedness in seven hatchery samples, including a selectively bred population (RACF‐F1), compared with three wild samples using 11 microsatellite loci. Genetic diversity and relatedness values within most hatchery samples, except for RACF‐F1, did not differ from those of wild populations (P > 0.05). RACF‐F1 had the lowest allelic diversity and effective population size (A_r = 6.99; N_e = 7.8) and highest relatedness values (mean r_xy = 0.075–0.204). Pairwise Φ_ST values, principal component analysis and model‐based cluster analyses revealed three genetically distinct hatchery groups: Eastern Thailand (CHN, RACF, NSCF and SKCF), Southern Thailand (NICA) and the Andaman Sea (STCF). Results suggest that exiting domestic populations capture reasonable amount of genetic variation and can be useful for a base population for genetic improvement programmes. In addition, given the rapid increase in relatedness that we observed in one selectively bred population, we recommend using selection methods and hatchery practices that reduce variability in family contribution in the subsequent generations.     

Evaluation of some intestinal cytokines genes expression and serum innate immune parameters in common carp (Cyprinus carpio) fed dietary loquat (Eriobotrya japonica) leaf extract

The aim of this study was to illustrate the effects of dietary loquat leaf extract (LLE) on the expression of some intestinal cytokines as well as serum innate immune parameters in common carp (Cyprinus carpio) fingerlings. The fish were fed with experimental diets supplemented with 0 (control), 0.25% (0.25LLE), 0.5% (0.5LLE) and 1% (1LLE) LLE over a 7‐week period. At the end of the trial, the serum lysozyme (Lys) activity, ACH50, total Ig as well as the expression of IL1B, IL8, TNF‐alpha, LYZ and TGF‐β in intestine were evaluated. The results showed that administration of 0.5% or 1% LLE significantly increased serum total Ig. However, in case of serum Lys activity significant elevation was observed just in fish fed 0.5% LLE. Also, supplementation of diet with LLE significantly increased ACH50 compared to the control group, regardless of inclusion levels. Gene expression studies revealed upregulation of TNF‐alpha, IL1B, IL8 and LYZ in intestine of fish fed LLE. However, the effects varied dependent on LLE levels and the tested immune related gene. Also, in case of TGF‐β significant downregulation was observed just in 1% LLE treatment. In conclusion, dietary LLE supplementation significantly upregulated immune related genes in intestine and improves innate immune responses. Altogether, LLE can be recommended as fish immunostimulant in early stage of carp culture.     

鲳亚目鱼类线粒体16S rRNA基因序列变异及其分子系统进化关系

为探讨鲳亚目鱼类的系统进化关系,通过测定中国沿海8种鲳亚目鱼类的线粒体16SrRNA基因部分序列,并结合GenBank上其他鲳亚目鱼类的同源序列,对其序列变异和分子系统进化树进行分析.结果表明,鲳亚目5科13属32种鱼类的16S rRNA基因序列的碱基组成为T 22.2％、C 24.5％、A 30.0％、G 23.3％;科间遗传距离为0.060～0.120,属间遗传距离为0.009 ～0.125,种间遗传距离为0.000 ～0.163;长鲳科位于系统进化树的基部,鲳科的鲳属处于系统进化树的顶端,无齿鲳科、方尾鲳科、双鳍鲳科与鲳科的低鳍鲳属和真鲳属聚类.结合形态学研究结果,认为:长鲳科是鲳亚目中最先分化的原始单系群;无齿鲳科和方尾鲳科为单系群,它们与非单系群的双鳍鲳科有较近的亲缘关系;鲳科为并系群,内部存在与地理区系相对应的2个分支,提示了该科鱼类早期的分化模式.同时,也对16S rRNA基因在鲳亚目鱼类系统进化研究中的适用性进行了剖析.     

Genetic diversity and population structure of a giant freshwater prawn (Macrobrachium rosenbergii) breeding nucleus in China

The giant freshwater prawn breeding programme in China has been carried out as a closed nucleus from 2006. The programme aimed to improve the harvest body weight and pond survival and has achieved remarkable progress. In this study, the genetic diversity and population structure of the giant freshwater prawn breeding nucleus was analysed. Pedigree completeness, the status of genetic diversity, inbreeding accumulation and effective population size of the breeding nucleus were analysed according to the pedigree record. From 2006 to 2014, a total of 107,941 individuals with 555 males and 967 females in nine generations were included in the analyses. Pedigree completeness index in six generations was at least 0.92. After performing eight selections, the genetic diversity decreased by 3%, 67% of which was caused by random genetic drift. After performing 2 years’ selection, the inbreeding level began to show a marked upward trend. The regression of the average rate of inbreeding and the average rate of co‐ancestry was 0.0041 and 0.0051 from 2006 to 2014. The effective population size calculated from the above two parameters was 122 and 98 respectively. The effective population size is still within the recommended level for a population to continue the genetic improvement programme, but below the level required (500) for retaining the evolutionary potential. To restrain the increasing level of inbreeding and maintain effective population size, measures are required to develop a breeding programme which integrates management of genetic variability and selection.