不同月份养殖草鱼幼鱼消化道微生物群落动态变化研究

为了评估草鱼消化道微生物群落结构在草鱼食性转化后的变动情况,实验对食性转换完成后的草鱼消化道微生物群落结构进行了PCR-DGGE分析。结果显示,不同月份采集草鱼消化道微生物群落结构存在明显差异(Monte Carlo检验,A:F=3.41,P=0.002;B:F=3.58,P=0.002),而被广泛认为影响宿主消化道微生物群落结构的宿主大小、丰满度等因素则在短期内并没有发现会对草鱼消化道微生物群落结构产生影响。实验表明,短期内环境因素可能是造成草鱼消化道微生物群落结构变动的主要因素。本实验结果将为深入阐述草鱼消化道微生物群落结构的变化规律、消化道益生菌的作用机理、消化道微生物群落结构变动与草鱼疾病的关系等问题积累基础材料。     

Two biomass preparation methods provide insights into studying microbial communities of intestinal mucosa in grass carp (Ctenopharyngodon idellus)

Animal digestive tract is habitat for a large number of autochthonous microbiota, which play central roles in multiple biological and physiological processes of the host. In this study, two different micro‐biomass preparation methods were employed to evaluate the diversity of intestinal mucosa‐associated microbiota in grass carp (Ctenopharyngodon idellus). Genomic DNAs were isolated either directly from intestinal mucosal samples (group A), or from micro‐biomass after microbial dissociation (group B). Community richness, diversity and evenness indices were all higher in group B, but differences were not statistically significant (P = 0.97, P = 0.33, P = 0.34 respectively). Furthermore, group B samples exhibited an increased ratio of bacterial DNA in comparison with group A samples, but the difference was also not statistically significant (P = 0.74). In addition, there were no statistically significant differences between the two groups (P > 0.05) at the taxonomic level. Our results support previous findings that there exists a great abundance of the intestinal mucosa‐adherent microbiota in the grass carp; among these, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Spirochaetes and Fusobacteria were the most common phyla. Within these microbiota, Paenibacillus, Bacteroides, Bacillus and Cetobacterium genera comprise the majority of the community, implicating their functional importance (e.g. as probiotics) to their host. Our results contribute towards a better understanding of the intestinal microbial profile of grass carp. Both micro‐biomass preparation techniques proved to be feasible for studying mucosa‐adherent microbiota of grass carp; however, the second method (group B) provides a protocol that is somewhat more effective than the first method (group A).     

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.     

Diversity of autochthonous bacterial communities in the intestinal mucosa of grass carp (Ctenopharyngodon idellus) (Valenciennes) determined by culture‐dependent and culture‐independent techniques

Traditional culture‐based technique and 16S rDNA sequencing method were used to investigate the mucosa‐associated autochthonous microbiota of grass carp (Ctenopharyngodon idellus). Twenty‐one phylotypes were detected from culturable microbiota, with Aeromonas, Shewanella, Lactococcus, Serratia, Brevibacillus, Delftia, Pseudomonas, Pantoea, Enterobacter, Buttiauxella and Yersinia as their closest relatives. Genomic DNA was directly extracted from the gut mucosa of C. idellus originating from six different geographical regions, and used to generate 609 random bacterial clones from six clone libraries and 99 archaeal clones from one library, which were grouped into 67 bacterial and four archaeal phylotypes. Sequence analysis revealed that the intestinal mucosa harboured a diversified bacterial microbiota, where Proteobacteria, Firmicutes and Bacteroidetes were dominant, followed by Actinobacteria, Verrucomicrobia and Deinococcus‐Thermus. The autochthonous bacterial communities in the gut mucosa of fish from different aquatic environments were not similar (C_s < 0.80), but γ‐Proteobacteria was a common bacterial class. In comparison to bacterial communities, the archaeal community obtained from one library consisted of Crenarchaeota and Euryarchaeota. These results demonstrate that molecular methods facilitate culture‐independent studies, and that fish gut mucosa harbours a larger bacterial diversity than previously recognized. The grass carp intestinal habitat selects for specific bacterial taxa despite pronounced differences in host environments.     

Application of dietary supplements (synbiotics and probiotics in combination with plant products and β‐glucans) in aquaculture

This review addresses the dietary supplements, synbiotics and probiotics in combination with plant products or yeast/β‐glucans in aquaculture. The potential applications of synbiotics have a relatively short history in aquaculture, but have generated interest because of numerous benefits reported in endothermic animals. Since the first study was published by (Aquaculture Science in 2009) the concept has been used in aquaculture to reveal effects on growth performance, gut microbiota, gut histology, immune parameters, haematological and biochemical parameters as well as increased disease resistance. However, a limited number of probiotic bacteria (mainly Bacillus and Enterococcus) and prebiotics (mannan oligosaccharides, fructooligosaccharides, short‐chain fructooligosaccharides, galactooligosaccharides, arabinoxylan–oligosaccharides, isomaltooligosaccharide, chitosan oligosaccharide and inulin) have been used. Additionally, some studies have used plant products or yeast/β‐glucans in combination with probiotics, and these studies suggested that these dietary supplements promote growth performance and boost some immune parameters. The present review identifies evaluations of gut microbiota and gut morphology, and mucosal immune response as significant gaps in existing knowledge and suggests issues that merit further investigations to conclude the potential of dietary supplements in aquaculture.     

Modulation of nutrient digestibility and digestive enzyme activities in aquatic animals: The functional feed additives scenario

Considering the costs of feed costs (nearly 60% of production cost), nutrition, feeding and feed utilization are among the most important factors in commercial aquaculture. During the last decade, administration of functional feed additives has been practiced for enhancing nutrient digestibility and digestive enzyme activities of cultured fish and shellfish. Traditionally, antibiotics were used for boosting growth performance and nutrient digestibility in commercial aquaculture. However, emergence of resistance pathogens and possible risk to human health resulted in limitation or prohibition of prophylactic administration of antibiotics. Recently, there was increasing attentions towards dietary administration of functional feed additives that include probiotics, prebiotics and synbiotics for elevation of digestive enzyme activity and nutrient digestibility. The results of those studies revealed contradictory effects of different pro‐, pre‐ or synbiotics on various fish species. It seems that the effects are species specific and related to modulation of the intestinal microbiota. In view of this issue, the present review provides a comprehensive sight on the effects of different pro‐, pre‐ and synbiotics on digestive enzyme activity and nutrient digestibility in different species with special focus on the mode of action. In addition, the present review highlighted the gaps of existing knowledge as well as suggesting the subjects which needs additional studies.     

Microbial Ecology of the Gastrointestinal Tract of Fish and the Potential Application of Prebiotics and Probiotics in Finfish Aquaculture

Aquaculture is one of the fastest growing industries in the world. The need for enhanced disease resistance, feed efficiency, and growth performance of cultured organisms is substantial for various sectors of this industry. If growth performance and feed efficiency are increased in commercial aquaculture, then the costs of production are likely to be reduced. Also if more fish are able to resist disease and survive until they are of marketable size, the subsequent cost of medication and overall production costs would be reduced drastically. It has been documented in a number of food animals that gastrointestinal microbiota play important roles in affecting the nutrition and health of the host organism. Thus, various means of altering the intestinal microbiota to achieve favorable effects such as enhancing growth, digestion, immunity, and disease resistance of the host organism have been investigated in various terrestrial livestock as well as in humans. Dietary supplementation of prebiotcs, which are classified as non‐digestible food ingredients that beneficially affect the host by stimulating growth and/or activity of a limited number of health‐promoting bacteria such as Lactobacillus and Bifidobacter spp. in the intestine, while limiting potentially pathogenic bacteria such as Salmonella, Listeria and Escherichia coli, have been reported to favorably affect various terrestrial species; however, such information is extremely limited to date for aquatic organisms. Effects of probiotics, defined as live microbial feed supplements, on gastrointestinal microbiota have been studied in some fishes, but the primary application of microbial manipulations in aquaculture has been to alter the composition of the aquatic medium. In general, the gastrointestinal microbiota of fishes including those produced in aquaculture has been poorly characterized, especially the anaerobic microbiota. Therefore, more detailed studies of the microbial community of cultured fish are needed to potentially enhance the effectiveness of prebiotic and probiotic supplementation. This review summarizes and evaluates current knowledge of intestinal microbial ecology of fishes, the various functions of this intestinal microbial community, and the potential for further application of prebiotics and probiotics in aquaculture.     

Effect of dietary components on the gut microbiota of aquatic animals. A never‐ending story?

It is well known that healthy gut microbiota is essential to promote host health and well‐being. The intestinal microbiota of endothermic animals as well as fish are classified as autochthonous or indigenous, when they are able to colonize the host's epithelial surface or are associated with the microvilli, or as allochthonous or transient (associated with digesta or are present in the lumen). Furthermore, the gut microbiota of aquatic animals is more fluidic than that of terrestrial vertebrates and is highly sensitive to dietary changes. In fish, it is demonstrated that [a] dietary form (live feeds or pelleted diets), [b] dietary lipid (lipid levels, lipid sources and polyunsaturated fatty acids), [c] protein sources (soybean meal, krill meal and other meal products), [d] functional glycomic ingredients (chitin and cellulose), [e] nutraceuticals (probiotics, prebiotics, synbiotics and immunostimulants), [f] antibiotics, [g] dietary iron and [h] chromic oxide affect the gut microbiota. Furthermore, some information is available on bacterial colonization of the gut enterocyte surface as a result of dietary manipulation which indicates that changes in indigenous microbial populations may have repercussion on secondary host–microbe interactions. The effect of dietary components on the gut microbiota is important to investigate, as the gastrointestinal tract has been suggested as one of the major routes of infection in fish. Possible interactions between dietary components and the protective microbiota colonizing the digestive tract are discussed.     

池塘和工厂化养殖牙鲆肠道菌群结构的比较分析

为研究池塘和工厂化养殖条件下牙鲆肠道菌群结构差异及其与饵料、水环境、底质等的关系,采用MiSeq高通量测序技术和生物信息学分析手段,构建了牙鲆(Paralichthys olivaceus)肠道、养殖水体、饵料和池塘底泥等7个样品的16s rRNA基因测序文库,分析了不同样品中菌群组成和生物多样性。结果表明：池塘养殖牙鲆肠道（B1）中以厚壁菌门（30.49%）、变形菌门（19.16%）和梭杆菌门（11.11%）为主,其中芽孢杆菌属（27.66%）占绝对优势,弧菌属（0.16%）丰度最小;工厂化养殖牙鲆肠道（B5）中以变形菌门（44.31%）、厚壁菌门（11.57%）和放线菌门（4.79%）为主,其中不动杆菌属（10.37%）丰度最大,弧菌属（4.05%）相对B1中丰度较高。牙鲆肠道优势菌群主要与营养代谢调节相关,同时有益微生物和有害微生物也是肠道菌群的重要组成部分。差异性和系统进化分析表明两种养殖条件下牙鲆肠道菌群结构与饵料中菌群关系密切,此外受养殖水环境中菌群影响较大。研究结果将为今后牙鲆养殖专用高效微生态制剂的研制和养殖环境微生态调控技术构建提供理论依据。     

微生态制剂对草鱼生产性能和肠道菌群的影响

在水温25~30℃下,将体质量为(110.23±0.43)g的草鱼饲养在3.0 m×2.0 m×1.2 m的加盖网箱中,分别投喂添加0%(对照组)、0.5%和2%的由芽孢杆菌、乳酸菌以及酵母菌复配且以麸皮为载体制成的微生态制剂(8.0×10~9 cfu/g)的膨化饲料饲养60 d,探究微生态制剂对草鱼生产性能和肠结构、菌群及酶活性的影响。试验结果显示,饲料中添加2%微生态制剂显著提高草鱼质量增加率、特定生长率(P<0.05),显著降低饲料系数、脏体比(P<0.05);饲料中添加2%微生态制显著提高肠伸展率、中肠肌层厚度和绒毛高度(P<0.05),提高中肠淀粉酶和脂肪酶活性(P<0.05)。饲料中添加微生态制剂增加草鱼肠道菌群α多样性、丰富度;改变草鱼肠道微生物组成,门水平上,对照组的草鱼肠道微生物中梭杆菌门和厚壁菌门含量最高(63.56%、32.52%)。0.5%添加组的草鱼肠道微生物中梭杆菌门和厚壁菌门含量最高(61.82%、20.27%)。2%添加组的草鱼肠道微生物中厚壁菌门含量最高(64.20%)。属水平上,2%添加组草鱼肠道优势菌属直接发生改变,Paeniclostridium和Erysipelatoclostridium丰度大幅上升。随着微生态制剂添加量的增加,肠道微生物的代谢功能增强,组成中与无机离子转运和代谢、碳水化合物转运与代谢、氨基酸转运与代谢等功能相关的菌群丰度升高。综上可知,饲料中添加芽孢杆菌、乳酸菌以及酵母菌等组成的微生物制剂可作为生产草鱼绿色饲料的重要措施。     

Identification of the adherent microbiota on the gills and skin of poly‐cultured gibel carp (Carassius auratus gibelio) and bluntnose black bream (Megalobrama amblycephala Yih)

PCR‐denaturing gradient gel electrophoresis (DGGE) was applied to analyse the microbial community attached to the gills and skin of poly‐cultured gibel carp (Carassius auratus gibelio) and bluntnose black bream (Megalobrama amblycephala Yih) and compare these results with those detected in the rearing water. The microbiota discussed included bacteria, fungi and a specific bacterial taxa of actinomycetes was also analysed. Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria, Ascomycota, Basidiomycota and some unclassified microbiota were identified. Based on our results, we concluded that: (1) the adherent bacterial/fungal communities on the gills and skin were different from those in the rearing water, (2) the bacterial/fungal diversities on fish gills were lower than that on fish skin, (3) the adherent bacterial/fungal communities on gill and skin of gibel carp were different from that of bluntnose black bream and (4) the adherent actinomycetal community showed certain similarity between the skin of different hosts. Based on our conclusions, we suggested that the topic investigated in the present study merits further investigations.     

Microbial communities associated with early stages of intensively reared orange‐spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation‐based and cultivation‐independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.     

Characterization of the bacterial community associated with early‐developmental stages of grass carp (Ctenopharyngodon idella)

Bacterial communities in eggs and larvae of grass carp were analysed. During the early‐developmental stages, grass carp harboured five bacterial phyla, i.e. Proteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes and Firmicutes. However, the composition of bacterial communities varied among the different developmental stages. In eggs, the bacterial communities were dominated by Proteobacteria, while in larvae the dominant bacterial community was Bacterioidetes. With the exception of a large proportion (>50%) of uncultured bacteria, Sphingobacterium (14.75%) and Acinetobacter (13.11%) were the most abundant groups in eggs at the fertilization stage (FS). However, Aermonas was the most abundant group, ranging from 40.54% to 61.76% in eggs at the cleavage (CS), blastula (BS), organ differentiation (ODS) and hatching stages (HS). In larvae after first ingestion (OW), Chitinophagaceae (79.41%) formed the predominant bacterial community. Changes in the bacterial community were further confirmed by statistical analysis, which demonstrated significant differences in the bacterial communities of eggs at FS, eggs from CS to HS and OW. However, no significant difference was found in bacterial communities of eggs from CS to HS. Furthermore, the present study revealed that bacteria related to Chitinophagaceae persisted from CS to OW, suggesting that these bacteria form part of the autochthonous microbiota of the fish.     

Microbial communities in sea cucumber (Apostichopus japonicus) culture pond and the effects of environmental factors

This study investigated microbial community composition as well as their correlation with environmental factors of Apostichopus japonicus culture ponds in northern China by 16S rRNA gene amplicon sequencing. The results showed that microbiota richness varied consistently with diversity in the pond ecosystem. Microbiota richness and diversity were highest in sediment, followed by gut of A. japonicus and water. The dominant bacterial phylum in the pond ecosystem is Proteobacteria. Gammaproteobacteriaeria and Flavobacteria are two dominant bacterial classes in the ecosystem. There is significant difference (p < 0.05) between dominant bacterial communities at the levels of order, family and genus. There is also remarkable regional difference (p < 0.05) between microbial community composition in the pond ecosystems. Specifically, microbial community composition in Changhai and Yingkou show a high similarity, so do those of Laoting and Rushan. According to the redundancy analysis of the microbial community composition and pond environmental factors, chemical oxygen demand is the dominant environmental factor determining microbial community composition in pond water; sulphide has the greatest influence on the microbial community composition in pond sediment; the rest of environmental factors have varied influence on microbial community composition in pond ecosystems.     

Impacts of diet on hindgut microbiota and short‐chain fatty acids in grass carp (Ctenopharyngodon idellus)

Diet is known to influence intestinal microbiota in fish, but the specifics of these impacts are still poorly understood. Different protein/fibre ratio diets may result in differing structures and activities of gut microbiota. We examined the hindgut microbiome of grass carp (Ctenopharyngodon idellus) fed three different diets: fish meal (FM, high protein – low fibre), Sudan grass (SG, high fibre – low protein) and compound feed (CF, intermediate). Microbial profiles of fish fed on FM were significantly different from profiles of fish fed CF and SG (F = 18.85, p < .01). Cetobacterium, known to be positively associated with protein digestion, was the dominant microbial group in FM samples (approximately 75.7%), while Lachnospiraceae and Erysipelotrichaceae, thought to be involved in fermentation of plant polysaccharides, were dominant in CF and SG samples (46.8% and 42.9% respectively). Network analyses indicated that the abundance of Lachnospiraceae and Erysipelotrichaceae was in a significantly positive correlation (r = .895, p = .001). Short‐chain fatty acid (SCFA) levels may indicate that the digestibility of diet by microbiota in the grass carp gut decreased from FM to SG (FM>CF>SG). Overall low SCFA levels indicate that hindgut fermentation probably provides a low proportion of energy requirements in grass carp.     

Intestinal Microbiota in Large Yellow Croaker,Larimichthys crocea,at Different Ages

The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S ribosomal RNA gene was used to investigate bacterial communities in the intestines of large yellow croaker at six different ages (12 d, 18 d, 26 d, 40 d, 3 mo, and 1 yr old) as well as within the corresponding feed and culture water. In addition, Illumina Miseq sequencing was utilized to compare intestinal microbiota between 12‐d‐old and 1‐yr‐old individuals. PCR‐DGGE results revealed that the culture water had the highest bacterial diversity, followed by the feed, while the intestines had the lowest diversity. The intestinal microbiota at six ages changed severely; however, the change did not follow any trend. The large yellow croaker intestines harbored specific bacterial communities that differed from those in both feed and water. Illumina Miseq sequencing results revealed that the diversity of intestinal bacteria in 12‐d‐old fish was higher than that in 1‐yr‐old fish, and the bacterial composition differed significantly between them. γ‐Proteobacteria and Pseudoalteromonas supplied the most abundant phylum and genus in the 12‐d‐old fish intestine. However, in the 1‐yr‐old fish intestine, Firmicutes and Clostridium were the most dominant, respectively. The study may contribute to a better understanding of gut microbiota and dynamics of the large yellow croaker and the relationship with their surrounding environment.     

池塘养殖牙鲆肠道和环境菌群结构对益生菌制剂的响应

为研究益生菌制剂对池塘养殖牙鲆(Paralichthys olivaceus)肠道及环境菌群结构的调控效果,采用高通量测序技术和生物信息学分析手段构建牙鲆肠道、养殖水体、饵料和池塘底泥的16S rDNA基因测序文库,分析不同样品中菌群组成和多样性在益生菌制剂调控过程中的变化趋势.结果显示,添加益生菌制剂后,池塘底泥和牙...     

Enzyme producing bacterial flora isolated from fish digestive tracts

Isolationand enumeration of aerobic bacterial flora in the gastrointestinal tract of nineculturable freshwater teleosts, namely catla, rohu, mrigal, silver carp, grasscarp, common carp, tilapia, walking catfish and murrel have been carried out.Amylolytic, cellulolytic, lipolytic and proteolytic microflora were identifiedfrom the culture plate using selective media. The isolates were qualitativelyscreened on the basis of their extracellular enzyme producing ability. Theselected strains were further quantitatively assayed for amylase, cellulase,lipase and protease activities. Protease activity was exhibited by almost allthe bacterial isolates, while strains isolated from tilapia, grass carp andcommon carp showed considerable amylolytic and cellulolytic activities. Maximumactivity of lipase was exhibited by a strain isolated from silver carp. Thestudy indicates that there is a distinct microbial source of the digestiveenzymes – amylase, cellulase, lipase and protease, apart from endogenoussources in fish gut. The information generated from the present investigationmight contribute towards better feed formulations for carp at low cost,incorporating the enzyme producing bacterial isolates as probiotics.     

Biolog-ECO方法探究饲喂益生菌对凡纳滨对虾肠道微生物代谢及有效作用时间的影响

以霍氏肠杆菌(E3)和乳酸菌(R3)2株益生菌对凡纳滨对虾进行为期4周的养殖饲喂实验,饲喂后期利用Biolog-ECO方法对实验组及空白组的凡纳滨对虾肠道微生物菌群多样性的差异进行比较分析,以评价益生菌对凡纳滨对虾肠道微生物菌群代谢功能的影响。结果显示,添加霍氏肠杆菌(E3)或乳酸菌(R3)的实验组,与空白组相比较,平均每孔颜色变化率显著上升,表明益生菌增强了肠道微生物活性;凡纳滨对虾肠道微生物利用各类碳源的整体能力显著增强,表明益生菌可以促进水产动物的代谢功能;肠道微生物多样性指数(包括Shannon、Simpson和McIntosh指数)有明显差异,表明饲喂2株益生菌可以提高凡纳滨对虾肠道菌群的丰富度。其中,停喂霍氏肠杆菌后第1天和第5天取样结果表明,Shannon指数显著降低,Simpson和McIntosh指数显著升高;停喂乳酸菌后的第1天和第5天取样结果表明,Shannon指数无显著差异,Simpson和McIntosh指数显著升高;二者在第10天取样的结果中均无显著差异,表明饲料中添加益生菌可以改变凡纳滨对虾肠道内原有菌群的数量和结构,促进对虾肠道内微生物群落间复杂的相互作用,进而在维持或者促进对虾健康方面发挥着重要的作用,同时也表明此两株益生菌在凡纳滨对虾肠道中停留时间最少为5 d。