Data_Sheet_1_Exposure to Yeast Shapes the Intestinal Bacterial Community Assembly in Zebrafish Larvae.pdf

Establishment of the early-life gut microbiota has a large influence on host development and succession of microbial composition in later life stages. The effect of commensal yeasts - which are known to create a conducive environment for beneficial bacteria - on the structure and diversity of fish gut microbiota still remains unexplored. The present study examined the intestinal bacterial community of zebrafish (Danio rerio) larvae exposed to two fish-derived yeasts by sequencing the V4 hypervariable region of bacterial 16S rRNA. The first stage of the experiment (until 7 days post-fertilization) was performed in cell culture flasks under sterile and conventional conditions for germ-free (GF) and conventionally raised (CR) larvae, respectively. The second phase was carried out under standard rearing conditions, for both groups. Exposure of GF and CR zebrafish larvae to one of the yeast species Debaryomyces or Pseudozyma affected the bacterial composition. Exposure to Debaryomyces resulted in a significantly higher abundance of core bacteria. The difference was mainly due to shifts in relative abundance of taxa belonging to the phylum Proteobacteria. In Debaryomyces-exposed CR larvae, the significantly enriched taxa included beneficial bacteria such as Pediococcus and Lactococcus (Firmicutes). Furthermore, most diversity indices of bacterial communities in yeast-exposed CR zebrafish were significantly altered compared to the control group. Such alterations were not evident in GF zebrafish. The water bacterial community was distinct from the intestinal microbiota of zebrafish larvae. Our findings indicate that early exposure to commensal yeast could cause differential bacterial assemblage, including the establishment of potentially beneficial bacteria.</p

Data_Sheet_2_Exposure to Yeast Shapes the Intestinal Bacterial Community Assembly in Zebrafish Larvae.pdf

Establishment of the early-life gut microbiota has a large influence on host development and succession of microbial composition in later life stages. The effect of commensal yeasts - which are known to create a conducive environment for beneficial bacteria - on the structure and diversity of fish gut microbiota still remains unexplored. The present study examined the intestinal bacterial community of zebrafish (Danio rerio) larvae exposed to two fish-derived yeasts by sequencing the V4 hypervariable region of bacterial 16S rRNA. The first stage of the experiment (until 7 days post-fertilization) was performed in cell culture flasks under sterile and conventional conditions for germ-free (GF) and conventionally raised (CR) larvae, respectively. The second phase was carried out under standard rearing conditions, for both groups. Exposure of GF and CR zebrafish larvae to one of the yeast species Debaryomyces or Pseudozyma affected the bacterial composition. Exposure to Debaryomyces resulted in a significantly higher abundance of core bacteria. The difference was mainly due to shifts in relative abundance of taxa belonging to the phylum Proteobacteria. In Debaryomyces-exposed CR larvae, the significantly enriched taxa included beneficial bacteria such as Pediococcus and Lactococcus (Firmicutes). Furthermore, most diversity indices of bacterial communities in yeast-exposed CR zebrafish were significantly altered compared to the control group. Such alterations were not evident in GF zebrafish. The water bacterial community was distinct from the intestinal microbiota of zebrafish larvae. Our findings indicate that early exposure to commensal yeast could cause differential bacterial assemblage, including the establishment of potentially beneficial bacteria.</p

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-2

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p>orms longitudinal lamellae that extend into the central ovarian cavity (). A squamous epithelium lines these lamellae at the luminal surface, below which a group of undifferentiated somatic cells were observed (). The ovarian lamellae are formed by nests of oogonia (), pre-perinucleolar oocytes (), early and late perinucleolar oocytes (), yolk vesicle oocytes () and secondary () and tertiary () yolk vesicle oocytes. Oogonia () and somatic cells () only proliferated during testicular involution. Some atretic follicles were observed in the ovarian of males at the beginning of the third RC (). Scale bar = 100 μm (), 50 μm (), 25 μm () and 10 μm (). OL, ovarian lamellae; OC, ovarian cavity; EPO, early perinucleolar oocytes; LPO, late perinucleolar oocytes; YVO, yolk vesicles oocytes; (arrow heads), proliferative cells; (asterisk), ovarian somatic cells; (white arrows), epithelium lines the ovarian lamellae; (white arrows heads), follicular epithelial cell layer

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-4

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p>were obtained after mixing the same amount of mRNA from 4–5 fish/month. Different letters denote statistically significant differences between the groups according to a Student-Newman-Keuls test. Sg, spermatogenesis; S, spawning; PS, post-spawning; TI, testicular involution

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-0

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p> germ cell type cysts formed the tubules of the testis. At spawning (), the tubules are larger and full of free spermatozoa. At post-spawning (), the main cell types in the tubules are spermatogonia stem cells and primary spermatogonia cysts. Some remaining spermatozoa can also be seen. At testicular involution (), the testis is formed by spermatogonia stem cells and primary spermatogonia cysts that compose a dense tissue with no lumina in the tubules. Two morphological areas can be distinguished: the peripheral testicular area () and the internal testicular () area which is close to the efferent duct and the ovarian area and presents large necrotic areas surrounded by eosinophilic granulated cells. Scale bar = 25 μm (,) and 50μm (). Sg, Spermatogenesis; S, spawning; PS, post-spawning; TI, testicular involution; SG, spermatogonia cysts; PSG, primary spermatogonia cysts; SC, spermatocytes cysts; SZ, spermatozoa; PTI, peripheral testicular area in the involution stage; ITI, internal testicular area in the involution stage; OV, ovarian area; (arrow), spermatogonia stem cell; (arrow heads), eosinophilic cells; (white arrows), efferent duct; (asterisk), necrotic areas

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-7

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p>ermatogonia stem cells and primary spermatogonia and spermatocytes cysts proliferate. At post-spawning () and testicular involution () spermatogonia stem cells and primary spermatogonia cysts were inmunostained. Only at post-spawning () and testicular involution () were apoptotic cells observed. Notice that in the testicular peripheral area () the number of TUNEL positive cells is higher than in the testicular internal area (). Scale bar = 25 μm (). (arrowheads), proliferative cells; (arrows), TUNEL positive cells; Sg, Spermatogenesis; PS, post-spawning; TI, testicular involution; PTI, peripheral testicular area in the involution stage; ITI, internal testicular area in the involution stage

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-3

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p>ocytes throughout the second RC and at the beginning of the third (). Data represent means ± SEM n = 10–18 fish/month (), n = 3 fish/month () and n = 111–269 cell/stage (). Different letters denote statistically significant differences between the groups according to a Waller-Duncan test. Sg, Spermatogenesis; S, spawning; PS, post-spawning; TI, testicular involution; NFD, no further development; G, gametogenesis

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-5

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p>nulocytes (), the percentage of gonad acidophilic granulocytes (), the ultrastructure of testicular macrophage-like cells and lymphocyte-like cells and RT-PCR analysis of M-CSFR, TCR-β and IgM-H genes, as appropriate markers of macrophages and T and B lymphocytes, respectively (). The acidophilic granulocytes appeared in the interstitial tissue of the testis at spermatogenesis (), post-spawning () and testicular involution (). Note that they also appeared between the germ cells at post-spawning () and around the necrotic areas at testicular involution (). Scattered acidophilic granulocytes were also observed in the interstitial tissue of the ovarian area (). Heterogeneous granules fused to each other were observed in the acidophilic granulocytes closed to the testicular necrotic areas (). Testicular cell suspensions (n = 3–5 fish/month) were immunostained with the G7 and then analyzed by flow cytometry (). The macrophage-like cells in the interstitial tissue in the vicinity of Leydig cells at spermatogenesis stage (). Lymphocyte-like cells in the interstitial tissue (). Total mRNA was obtained to mix the same amount of each mRNA from 4–5 fish/sample (). Scale bar = 100 μm (), 25 μm (), 1 μm (), 0.5 μm (), 0.3 μm(). Different letters denote statistically significant differences between the groups according to a Waller-Duncan test. (arrow), G7 positive cells; (white arrows), granules fused to each other; MLC, macrophage-like cell; LLC, lymphocyte-like cell; L, Leydig cell; Sg, Spermatogenesis; S, spawning; PS, post-spawning; TI, testicular involution; NFD, no further development; PTI, peripheral testicular area in the involution stage; ITI, internal testicular area in the involution stage; O, ovary; G, gametogenesis and C-, negative control

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-1

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p>ermatogonia stem cells and primary spermatogonia and spermatocytes cysts proliferate. At post-spawning () and testicular involution () spermatogonia stem cells and primary spermatogonia cysts were inmunostained. Only at post-spawning () and testicular involution () were apoptotic cells observed. Notice that in the testicular peripheral area () the number of TUNEL positive cells is higher than in the testicular internal area (). Scale bar = 25 μm (). (arrowheads), proliferative cells; (arrows), TUNEL positive cells; Sg, Spermatogenesis; PS, post-spawning; TI, testicular involution; PTI, peripheral testicular area in the involution stage; ITI, internal testicular area in the involution stage

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration-6

<p><b>Copyright information:</b></p><p>Taken from "Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration"</p><p>http://www.rbej.com/content/5/1/20</p><p>Reproductive Biology and Endocrinology 2007;5():20-20.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1894798.</p><p></p> germ cell type cysts formed the tubules of the testis. At spawning (), the tubules are larger and full of free spermatozoa. At post-spawning (), the main cell types in the tubules are spermatogonia stem cells and primary spermatogonia cysts. Some remaining spermatozoa can also be seen. At testicular involution (), the testis is formed by spermatogonia stem cells and primary spermatogonia cysts that compose a dense tissue with no lumina in the tubules. Two morphological areas can be distinguished: the peripheral testicular area () and the internal testicular () area which is close to the efferent duct and the ovarian area and presents large necrotic areas surrounded by eosinophilic granulated cells. Scale bar = 25 μm (,) and 50μm (). Sg, Spermatogenesis; S, spawning; PS, post-spawning; TI, testicular involution; SG, spermatogonia cysts; PSG, primary spermatogonia cysts; SC, spermatocytes cysts; SZ, spermatozoa; PTI, peripheral testicular area in the involution stage; ITI, internal testicular area in the involution stage; OV, ovarian area; (arrow), spermatogonia stem cell; (arrow heads), eosinophilic cells; (white arrows), efferent duct; (asterisk), necrotic areas