Diversity of intratunical bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans

This paper provides the first information on diversity based on sequence data of the 16S rDNA of intratunical bacteria in the colonial ascidian Diplosoma migrans and its embryonic offspring. Ascidians were collected from waters near Helgoland ( German Bight, North Sea). Sample material comprised tunic tissue, bacteria collected from tunic tissue, eggs with single embryos at different developmental stages, and free-swimming larvae. Bacterial 16S rDNA from D. migrans was directly amplified using PCR. DNA species were separated using denaturing gradient gel electrophoresis (DGGE). DGGE profiles generated ca. ten different distinguishable operational taxonomic units. Eleven bands from different sample materials were successfully re-amplified and sequenced. Sequence data generated five different subgroups of intratunical proteobacteria. The dominant band, detected in all of the samples tested, showed a low degree of relationship ( 84 - 86%) to Ruminococcus flavefaciens (delta-subgroup). A weaker band, located above, which was not detected in all of the samples, was also similarly related to R. flavefaciens. Other bands derived from tunic material and embryonic stages showed closer relationship ( ca. 97 - 99%) to Pseudomonas saccherophilia, a knallgas bacterium, and Ralstonia pickettii, a pathogen bacterium ( both members of the beta-subgroup). A solitary band generated from tunic material was assigned to a typical marine Flavobacterium symbiont ( 95%). Finally, a band from isolated bacteria was related (96%) to pathogen Arcobacter butzleri (epsilon-subgroup). At this state of the investigation, a reliable interpretation of the ecological functions of intratunical bacteria cannot yet be given. This is due to the low degree of relationship of some of the bacteria and the fact that not all of the characteristic bands were successfully sequenced. However, the intratunical bacteria represent a unique bacterial community. Their DGGE profiles do not correspond to the profiles of the planktonic bacteria generated from surface seawater close to the ascidian habitat. The allocation of DNA sequences to the different morphotypes, their isolation and culturing, and the elucidation of the physiological functions of intratunical bacteria are in progress.