Does the natural diet influence the intestine's ability to regulate glucose absorption?

Summary Two fish species (rainbow trout and common carp) that differ in natural diet also exhibit differences in the adaptive flexibility of their intestinal nutrient transport mechanisms in response to changes in dietary nutrient composition. When carp ingested a feed that was 24% glucose by weight, there was an increase in both the intestinal length and rates of nutrient absorption, particularly for glucose, when compared to carp fed an isonitrogenous diet devoid of digestible carbohydrate. As a result, the intestine's uptake capacity (nmol of glucose and proline absorbed per min per g body weight) was higher in carp fed the 24% glucose feed. Due to the greater increase in glucose uptake, the ratio of glucose uptake relative to proline (G/P ratio) was higher in carp fed the 24% glucose. Thus, carp are able to adapt to the quantity, and apparently also to the type, of digestible carbohydrate in the diet. In contrast, glucose uptake by trout was unresponsive to the quantity of dietary carbohydrate. Insted, the elevated glucose paradoxically resulted in a greater uptake capacity for proline and a lower G/P ratio. Hence, the adaptive capabilities of the intestinal nutrient transport processes are matched to the potential variation in the carbohydrate content of the natural diet.Abbreviation PEG polyethylene glycol     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2)of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes.Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp, and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gynogenetic progeny of allotetrapioid hybrids of red crucian carp × common carp had important significances in both biological evolution and production application.     

High glucuronidation activity of environmental estrogens in the carp (Cyprinus carpino) intestine

Many adverse effects on carp reproductive organs have been reported to be caused by exposure to environmental estrogens, such as nonylphenol and bisphenol A, which contaminate the aquatic environment. The glucuronidation activities of xenoestrogens (bisphenol A and diethylstilbestrol) and phytoestrogens (coumestrol, genistein and biochanin A), but not nonylphenol and octylphenol, were observed in microsomes prepared from carp organs. The highest levels of glucuronidation of environmental estrogens, for which the optimum temperature was 25-30 degrees C, were observed in the intestinal microsomes of 2-year-old carp. These activities in carp intestine increased developmentally, and the maximum levels corresponded to 5-10 % of that in rat liver microsomes. However, the glucuronidation of phytoestrogen by carp intestinal microsomes corresponded to that of rat liver microsomes. Only bisphenol A-glucuronide was excreted from the everted intestine, indicating that bisphenol A is metabolized in the carp intestine mainly as glucuronide.These results suggest that glucuronidation by carp intestine plays an important role for the detoxification of xenoestrogens and phytoestrogens, except for nonylphenol and octylphenol.     

Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio).

1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle. 2. On a Sephadex G25 column (3 x 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530. 3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A. 4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A. 5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P. 6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb.     

Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio)

1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle.2. On a Sephadex G25 column (3 × 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530.3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A.4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A.5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P.6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb.     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

转基因鲤鱼与对照鲤肠道微生物群落差异研究

以转“全鱼”生长激素基因鲤(Cyprinus carpio L.)和野生对照鲤为对象, 采用454高通量测序技术对其肠道微生物16S rRNA基因进行测序并分析了3个不同发育阶段微生物群落结构的变化, 进而探讨了转基因鲤与对照鲤肠道微生物群落的差异。基于转基因鲤和对照鲤不同发育时期(6日龄、2月龄、5月龄)肠道微生物群落组成的DCA排序分析显示, 2月龄转基因鲤与对照鲤肠道微生物组成不同。Alpha多样性及均匀度都显示转基因鲤肠道微生物多样性高于对照鲤。从门水平的比较分析发现, 转基因鲤肠道中存在较多的厚壁菌门(Firmicutes)细菌, 而对照鲤中拟杆菌门(Bacteroidetes)细菌较多, 其中2月龄转基因鲤肠道内Bacteroidetes/Firmicutes比值低于对照鲤。研究结果表明, 在所分析的3个发育时期, 转基因鲤的肠道微生物组成与对照鲤相比发生了改变, 且在2月龄时存在差异。该研究为进一步揭示转基因鱼肠道微生物与宿主的相互影响和作用机制提供了很好的参考。     

关于草鱼肠炎微生态调节的研究

对健康草鱼肠道菌、患肺炎病草鱼肠道菌进行菌群分析,发现正常草鱼肠道中有气单胞菌(Aeromonas)、哈夫尼亚菌(Hafnia)、黄杆菌属(Flavobacterium)等多种菌,肠炎病鱼肠道中一般仅有气单胞菌的单一菌群。拮抗试验表明哈夫尼亚菌与气单胞菌之间有一定的拮抗性。回归试验表明哈夫尼亚菌无致病性,3种气单胞菌有致病性。微生态治疗试验表明哈夫尼亚菌对患肠炎病的草鱼肠道有促进肠道微生态平衡作用。     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

不同倍性鱼的血细胞和精子DNA含量比较

我们以前的研究表明, 以红鲫 (2n=100) 为母本及湘江野鲤 (2n=100) 为父本的杂交后代的F1-F2 为二倍体 (2n= 100)。在二倍体 F2 个体中, 存在能分别产生二倍体卵子和二倍体精子的雌、雄个体, 二倍体卵子和二倍体精子结合, 形成了两性可育的四倍体鱼 (F3)。目前四倍体鲫鲤已连续繁殖了 12 代 (F3-F14), 形成了一个遗传性状稳定的四倍体鱼群体 (4n= 200) (Liu et al.,2001; 孙远东等, 2003)。雌性四倍体鲫鲤产生的二倍体卵子经紫外线照射的散鳞镜鲤精子激活后,无须染色体加倍处理, 可发育为全雌性二倍体雌核发育后代 (G1) (2n=10…     

Constitutive expression of stromal derived factor-1 by mucosal epithelia and its role in HIV transmission and propagation

HIV particles that use the chemokine receptor CXCR4 as a coreceptor for entry into cells (X4-HIV) inefficiently transmit infection across mucosal surfaces [1], despite their presence in seminal fluid and mucosal secretions from infected individuals [2] [3] [4]. In addition, although intestinal lymphocytes are susceptible to infection with either X4-HIV particles or particles that use the chemokine receptor CCR5 for viral entry (R5-HIV) during ex vivo culture [5], only systemic inoculation of R5-chimeric simian-HIV (S-HIV) results in a rapid loss of CD4(+) intestinal lymphocytes in macaques [6]. The mechanisms underlying the inefficient capacity of X4-HIV to transmit infection across mucosal surfaces and to infect intestinal lymphocytes in vivo have remained elusive. The CCR5 ligands RANTES, MIP-1alpha and MIP-1beta suppress infection by R5-HIV-1 particles via induction of CCR5 internalization, and individuals whose peripheral blood lymphocytes produce high levels of these chemokines are relatively resistant to infection [7] [8] [9]. Here, we show that the CXCR4 ligand stromal derived factor-1 (SDF-1) is constitutively expressed by mucosal epithelial cells at sites of HIV transmission and propagation. Furthermore, CXCR4 is selectively downmodulated on intestinal lymphocytes within the setting of prominent SDF-1 expression. We postulate that mucosally derived SDF-1 continuously downmodulates CXCR4 on resident HIV target cells, thereby reducing the transmission and propagation of X4-HIV at mucosal sites. Moreover, such a mechanism could contribute to the delayed emergence of X4 isolates, which predominantly occurs during the later stages of the HIV infection.     

Hematophagous insects as vectors for frog trypanosomes

Experimental infections of three hematophagous arthropods (Rhodnius prolixus, Aedes aegypti, and Culex pipiens) with a trypanosome of the Trypanosoma rotatorium complex found in the frogs Hyla crepitans and Leptodactylus insularum revealed that A. aegypti is a good host for the flagellate; the course of development in the intestinal tract of the mosquito is described from 15 minutes to 168 hours. C. pipiens showed only low intestinal infections and R. prolixus did not permit development of the parasite. It is postulated that, in addition to the transmission of T. rotatorium by leeches, batrachophilic mosquitoes may transmit the parasite to frogs of more terrestrial habits by being ingested by these anurans.     

Cell proliferation and gill morphology in anoxic crucian carp

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.     

中草药复方对草鱼鱼种肠道菌群与血液有形成分的影响

目的探讨中草药复方对鱼种肠道菌群和血液有形成分的影响。方法以草鱼鱼种为动物模型,分别采用平板倾注法和ATB半自动化细菌鉴定法,测定投喂中草药复方药饵前、后鱼种肠道菌群数量及其优势菌群种类。同时使用常规计数法和改良沙利法,测定鱼种血液中红细胞、白细胞和血红蛋白的含量。结果对照组鱼种肠道内的细菌总数为6．6×10^8CFU／g,其优势菌群为气单胞菌、假单胞菌和肠杆菌;投喂2．5％、5．0％和7．5％中草药复方药饵10d后,其肠道内细菌总数均有所减少,分别为3．0×10^7CFU／g、5．0×10^6CFU／g和1．5×10^7CFU／g,其优势菌群为吲哚黄杆菌、产碱假单胞菌和野油菜黄单胞菌。实验组与对照组鱼种的红细胞数量差异无显著性,但白细胞与血红蛋白的含量差异有非常显著性（P〈0．01）。结论以黄连、大黄、金银花、连翘、黄芪和甘草组成的中草药复方对鱼种肠道菌群和血液有形成分均有一定影响,表现出该复方具有较好的免疫抗病作用。     

不同草鱼池塘混养系统结构优化的实验研究

研究采用陆基围隔实验法,对草鱼(Ctenopharyngodon idellus)、鲢鱼(Hypophythalmichthys molitrix)、鲤鱼(Cyprinus carpio)和凡纳滨对虾(Litopenaeus vannamei)不同混养系统的养殖产量、投入产出比、氮磷利用率和综合养殖效果进行了研究。实验设置6个处理组,分别为草鱼单养(G)、草鱼和鲢鱼二元混养(GS)、草鱼、鲢鱼和鲤鱼按照不同比例放养的三元混养(GSC1和GSC2)和草鱼、鲢鱼和凡纳滨对虾按照不同比例放养的三元混养(GSL1和GSL2)。研究结果表明,各处理组总产量在770480—11492.50 kg/hm2之间;饲料系数在1.00—1.38之间,以GSC1最低,各处理组之间差异显著(P<0.05)。N、P总相对利用率在2.01—7.37之间,以GSL2最高,G最低;投入产出比在1:1.41—1:2.12之间,以GSL2最高,G最低;综合效果指标在0.59—1.39之间,以GSL2最高,G最小。研究结果表明,混养组能显著提高养殖系统的产量,降低饲料系数,提高N、P利用率,从而有效提高草鱼养殖的经济效益和生态效益。     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

在患CyHV-2病的异育银鲫肠道黏膜中胆固醇、胆汁酸代谢通路基因的差异表达

为了探讨CyHV-2疾病条件下异育银鲫胆汁酸肠肝循环代谢途径主要基因表达活性的变化, 以患CyHV-2病的、正常的异育银鲫肠道黏膜为材料, 提取总RNA, 采用RNA-Seq测序、对单一基因进行注释, 并进行KEGG富集分析、单一基因差异表达分析。结果显示, 肠道黏膜组织有7770个基因显著性差异表达, 其中, 差异表达上调基因数为3335个、差异表达下调的基因数为4435个, 表明CyHV-2病的发生对肠道黏膜组织基因表达产生了重大的影响。病鱼胆固醇、胆汁酸生物合成代谢途径的酶、蛋白质的基因显著性差异表达, 显示肠道黏膜胆固醇、胆汁酸合成代谢受到显著性影响。参与胆固醇、胆汁酸合成代谢调节作用, 以及胆固醇、胆汁酸分泌、吸收、转运等生理过程的蛋白质、酶的基因也是差异表达下调, 胆汁酸肠肝循环有出现代谢障碍的趋势。结果表明, CyHV-2病的发生对肠道黏膜组织基因表达产生了严重影响, 对胆固醇、胆汁酸的合成代谢途径、胆汁酸肠肝循环途径的基因表达产生了严重影响, 将导致病鱼体内胆固醇、胆汁酸量的不足。患CyHV-2病病鱼血清胆汁酸含量下降了99%、胆固醇含量下降了10%, 证实了上述基因差异表达的趋势。     

异源四倍体鲫鲤雌核发育后代的RAPD分析

在优化RAPD(随机扩增多态性DNA)检测条件基础上,从134个随机引物中筛选出53个扩增较好且多态性强的引物,对异源四倍体鲫鲤第1代(G1)、第2代(G2)人工诱导的雌核发育二倍体后代群体的DNA多态性及分子标记进行了分析。结果显示,53个随机引物在G1群体和G2群体中检测到的位点数分别为541、511,其中多态性位点数分别为70、52,多态位点比例分别为12.94%、10.18%。两个群体的平均遗传距离分别为0.0732、0.0464。研究表明,经过连续2代人工雌核发育,G2的遗传多样性明显减少,种质进一步纯化。还从53个随机引物的扩增谱带中找到了2个引物(S50、S223)的特异扩增谱带,可以作为第1、2代雌核发育群体间的分子遗传标记。由计算机软件程序构建的分支系统树清晰地反映了两个雌核发育群体及其个体间的相互关系。       

Effects of three strains of intestinal autochthonous bacteria and their extracellular products on the immune response and disease resistance of common carp,Cyprinus carpio

The study isolated three strains of intestinal autochthonous bacteria Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio). To reveal the effects of these three strains of bacteria on the innate immunity of carp, the lysozyme, complement C3, total serum protein, albumin and globulin levels, respiratory burst activity, phagocytic activity by blood leucocytes and the expression of IL-1b, lysozyme-C, and TNF-α were examined after feeding with seven different diets for up to 28 days. Also the survival of carp against Aeromonas hydrophila was challenged for 14 days. The carp were fed seven different diets: one control, three diets supplemented with 1 × 10⁸ cell g⁻¹ of carp intestinal bacteria BA-1 (Group D-I), BA-2 (Group D-II) and BA-3 (Group D-III), and three diets supplemented with extracellular products FA-1 (Group E-I), FA-2 (Group E-II) and FA-3 (Group E-III) which were corresponding to the strains BA-1, BA-2, and BA-3, respectively, up to 28 days. For groups D-I, D-III, E-I and E-III, the innate immune parameters of carp were significantly increased, the expression of three immune-related genes in blood was significantly up-regulated examined during 7, 14, and 21 days of feeding, and the survival rate was improved. The study indicates that the two isolated intestinal autochthonous bacteria A. veronii BA-1 and F. sasangense BA-3 could positively influence immune response and enhance disease resistance of carp against A. hydrophila infection.