triphasic life cycle in vestimentifera

Research project P 13762 Triphasic life cycle in Vestimentifera Monika BRIGHT 28.06.1999 Vestimentifera are a small group of very successful invertebrate tubeworms that occur in high abundance at deep sea hydrothermal vents and cold seeps. They grow extremely fast and reach sizes up to 1.5 meters. Their success is made possible by a tight nutritional relationship with chemoautotrophic, sulfur-oxidizing, endosymbiotic bacteria. In this proposed research project, the vestimentiferan tubeworms are used as a model system for understanding the evolution of this type of symbiosis. Horizontal transmission of the symbionts is suggested to take place in each parental host generation. The uptake of symbionts from the environment and the transformation of both partners into a symbiotic entity can be followed studying the life cycle of the host. Three successive ontogenetic phases are distinguished: 1) larva, 2) early juvenile, and 3) late juvenile and adult. Uptake and transformation are considered two crucial stepping points in understanding the development of the symbiosis and the phylogeny of the host. 16S rRNA-targeted fluorescent in situ hybridization (FISH) and transmission electron microscopical (TEM) serial reconstruction are the appropriate techniques for gaining insights into the exact location and time frame of symbiont uptake and the development from the non-symbiotic settled larva, to the early juvenile where transmission takes place, and finally to the symbiotic late juvenile and adult.

A first order question for all obligate symbiotic associations addresses the mechanism of symbiont transmittal between generations. Uptake of the symbiont from a free-living source and specific processes in co-joining of the two partners are fundamental for environmental transmission as we find in the giant tubeworm (Riftia pachyptila) and its chemoautotrophic endosymbionts living at the deep sea hydrothermal vents of the east pacific. The origin of the trophosome - the organ which harbors the symbionts - is recognized as one of the keys to under-standing the evolution of this symbiosis. The present results of this still ongoing study falsify the current hypothesis of infection through mouth and subsequent development of the trophosome from endodermal tissue. Ap-plying transmission electron microscopy with 3-dimensional reconstructions of total animals from ultrathin serial sections and fluorescence in situ hybridization using newly designed sym-biont-specific oligonucleotide probes targeting 165 rRNA (and various group-specific eubacte-ria and archaea probes) on series of paraffin sections, we could distinguish 3 different phases in the life cycle of vestimentiferan tubeworms: 1) non-symbiotic feeding larvae in metamorpho-sis; 2) symbiotic, feeding early juveniles; 3) symbiotic, non-feeding late juveniles and adults. Uptake of the symbiont was specific as no other eubacterial or archaeal phylotypes were detected. It occurs in early juveniles (300 - 400 m). Not the mouth opening, but the skin is the organ of uptake. This finding stands in dear contrast to the previously proposed mode of uptake and led to the development of a new hypothesis: The free- living symbionts infect the skin, migrate through the epidermis, muscle layers, and mesenchyme until they reach the visceral mesoderm where development of the trophosome occurs. Based on the morphology and ultra-structure of the adult, we identified the tissue transforming into the trophosome as mesodermal. Symbiont-like bacteria were located in the dorsal visceral mesoderm and the mesentery. Growth of these transformed peritoneal cells, now termed bacteriocytes, goes hand in hand with proliferations of the blood vascular system until in late juveniles the typical multi-lobed trophosome is fully developed. The original cell cycle hypothesis was rejected too since no evidence of migration of symbiont from one to another host cell was found. Instead, our modified model proposes that some of the central bacteriocytes act as stem cells for a proliferating tissue produced in the center and ultimately degraded at the periphery of each lobule. Similarly, the rod-shaped symbionts in the center act as stem cells and exhibit a simple cell cycle. Differentiation into cocci takes place in the median and peripheral zone, until it terminates with lysis in the degenerative zone. Our findings indicate a very different mode of symbiont acquisition, one that follows proc-esses normally found in pathogenic infections. Our model of the evolution of an obligate sym-biosis, driven by active infection by the symbionts and facilitated by apoptosis in host tissue, is new and will lead to a re-examination of accepted hypotheses on the evolution of a variety of symbiotic associations.