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 freeswimming 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 (d-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 b-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 (e-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.

This paper provides the first information on the morphology of different morphotypes of bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans. Ascidians were collected from waters near Helgoland (German Bight, North Sea). The dominant type is represented by extremely high numbers of long, needlelike rods (length 10-30 m, width 0.5 m). The bacteria are motile by means of bipolar monotrichous flagella, generating swift sigmoidal movement. Bacteria are already present during different embryonic stages. It is assumed that they are transferred during sexual propagation from the parental colony to its offspring. As a second morphotype, the tunic harbors screw-like bacteria in low numbers (length 4-10 m, width 0.5 m). Besides these conspicuous morphotypes, occasionally motile rods with spore-like globules at one end and additional coccoid forms in large quantities of unknown meaning (possibly spores) were found. The taxonomic status and ecological functions of these differently shaped bacterial groups are unclear.

Serial sections of non-ovulated and ovulated eggs of Diplosoma migrans (Tunicata, Ascidiacea, Didemnidae) were compared in order to study the process of ovulation on the level of light microscopy. Fully grown eggs are surrounded by an outer follicular epithelium consisting of cubic cells, and an inner follicular epithelium consisting of flat cells. Egg release is initiated by a fusion of the epidermis with the outer follicular epithelium at the distal pole of the egg. A contraction of the outer follicular epithelium leads to an opening of both epidermis and outer follicular epithelium, and their removal from the egg. The envelope of ovulated eggs corresponds to the former inner follicular epithelium. Mainly due to a contraction of the epidermis, the outer follicular epithelium is shifted into the abdomen and separates from the epidermis. It transforms into a vestigial body (corpus luteum) and finally atrophies. The epidermis flattens and resumes its typical appearance. On the basis of these observations, the results of studies on the closely related Diplosoma listerianum are discussed and questioned.

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