DOI: 10.5061/dryad.kd51c5bb0
title: Data for: The source of microbial transmission influences niche colonization and microbiome development

description: Early life microbial colonizers shape and support the immature vertebrate
 immune system. Microbial colonization relies on the vertical route via
 parental provisioning and the horizontal route via environmental
 contribution. Vertical transmission is mostly a maternal trait making it
 hard to determine the source of microbial colonization in order to gain
 insight in the establishment of the microbial community during crucial
 development stages. The evolution of unique male pregnancy in pipefishes
 and seahorses enables the disentanglement of both horizontal and vertical
 transmission, but also facilitates the differentiation of
 maternal vs. paternal provisioning ranging from egg development,
 to male pregnancy and early juvenile development. Using 16s rRNA amplicon
 sequencing and source-tracker analyses, we revealed how the distinct
 origins of transmission (maternal, paternal & horizontal) shaped
 the juvenile internal and external microbiome establishment in the
 broad-nosed pipefish Syngnathus typhle. Paternal provisioning
 mainly shaped the juvenile external microbiome, whereas maternal microbes
 were the main source of the internal juvenile microbiome, later developing
 into the gut microbiome. This suggests that stability of niche microbiomes
 may vary depending on the route and time point of colonization,
 the strength of environmental influences (i.e., horizontal transmission),
 and potentially the homeostatic function of the niche microbiome.
description: Sample
 collection:  Adult <em>Syngnathus
 typhle</em> were caught in Orth on Fehmarn (54°26'N
 11°02'E) and brought to our aquaria facilities at GEOMAR Kiel, for
 breeding. Fish were kept in a flow-through aquaria system at 18°C with 18h
 day/6h night light regime and fed with live <em>Artemia
 salina</em> and frozen <em>Mysidae</em> spp. twice a
 day. In each aquarium three males and three females were kept together to
 allow mating. After the onset of breeding, fish were randomly sampled,
 regarding their sex and gravity stages, on five days between the end of
 May 2019 and end of June 2019. We sampled 89 mature <em>S.
 typhle</em> (18 females, 16 early pregnant males, 19 mid pregnant
 males, 18 late pregnant males and 19 non-pregnant males). Pregnancy stages
 (early, mid and late pregnancy) were defined according to (56). In order
 to detect microbial transfer from parental gonads and pouch tissue to the
 juveniles, we sampled testes and endometrial inner pouch lining tissue as
 well as fertilized larvae from the three pregnancy stages in male fish. In
 female fish, we sampled unfertilized eggs to assess potentially deposited
 microbes into the eggs or on their surface. The hindgut was sampled
 irrespective of sex. To investigate maternal microbial transfer through
 the cytoplasm, we surface-sterilized half of the unfertilized eggs from
 each female in a bath of 0.5% Polyvinylpyrrolidone-iodine (PVP-I, Solution
 in sterile-filtered Phosphate buffered Saline (PBS)) for 5 min with
 subsequent washing three times with 500µl sterile-filtered PBS (adapted
 from (57)). To sample the cytoplasm of surface-sterilized
 eggs (sterilized eggs) without contamination through the chorion, the egg
 was squished in the collection tube. The same sterilization treatment was
 applied to larvae (sterilized juveniles) of different pregnancy stages to
 discriminate between external and the internal microbiome. Non-sterilized
 eggs (untreated eggs) and larvae (untreated juveniles) were directly
 placed in the collection tubes. We pooled three juvenile and eggs sample
 from each of the pregnancy stages and sterilization treatment. All sampled
 organs were collected into collection microtubes from the DNeasy96 Blood
 and Tissue Kit from Qiagen (Hilden, Germany) and stored immediately at -80
 °C. Juvenile microbiota
 development:         <br>Pregnant male <em>S.
 typhle</em> were caught in Orth on Fehmarn (54°26'N
 11°02'E) in late spring 2019, transferred to the aquaria system, kept
 individually in a flow-through system with 18h day/6h night cycle and fed
 twice a day with live <em>Mysidae</em> spp. After parturition,
 free-swimming juveniles of each male were kept in a distinct aquarium and
 fed <em>ad libitum</em> twice per day with
 live <em>Artemia salina</em>. First sampling took place after
 release from the brood pouch, sampling was then continued in 2-3 day
 intervals. During each sampling three juveniles per family tank were
 collected individually in a collection tube for the analysis of whole-body
 microbiome (whole juveniles) development. To test for development of
 internal vs. external microbiome another three juveniles from the same
 family tank were euthanized by immersion in MS222 and the gut was removed
 in a sterile manner (juvenile gut) and collected individually. At each
 sampling day, controls of water and food samples were
 taken.  RNA extraction, library preparation and amplicon
 sequencing: Both datasets have been treated with the same
 DNA extraction and 16S rRNA sequencing protocols. DNA extraction was done
 with DNeasy Blood & Tissue Kit (QIAGEN, Germany) following the
 manufacturers protocol including a pre-treatment for Gram-positive
 bacteria with ameliorations from (58). See Supplementary Material 1 for
 further details. Library preparation was done by the institute for
 experimental medicine (UKSH, Campus Kiel) with 20µl of sample DNA from
 each sample. Amplicons of the V3-V4 hypervariable region (341f/806r) were
 sequenced using the Illumina MiSeq platform (Illumina, USA) with 2x
 300-bp paired-end read settings at the IKMB, Kiel
 University.