DEVELOPMENT, LIFE-CYCLE, ULTRASTRUCTURE AND PHYLOGENETIC POSITION OF PASTEURIA-RAMOSA METCHNIKOFF 1888 - REDISCOVERY OF AN OBLIGATE ENDOPARASITE OF DAPHNIA-MAGNA STRAUS

The development, life cycle, ultrastructure and phylogenetic position of an obligate, spore-forming endoparasite of Daphnia magna Straus is described. The microparasite was found in the body cavity of three Dap hnia species (D. magna, D. pulex and D. longispina) collected in Engla nd and Russia during 1992-1994 and maintained in artificial culture by co-cultivation with D. magna. Transmission of the endoparasite occurr ed horizontally through waterborne spores released from the remains of dead infected hosts. Progeny of infected hosts were never infected, i ndicating that vertical transmission does not occur. Egg production by infected mothers ceased soon after infection and death ensued after 4 6 days (+/-7 standard error) at 20 degrees C. Phase contrast light mic roscopy and transmission electron microscopy of the infection process showed the endoparasite to have a polymorphic life cycle beginning wit h the appearance of branched 'cauliflower-like' rosettes and ended wit h the development of single, oval endospores, nippled at one end and w ith complex internal structure. Endospore formation resembled that fou nd in endospore-forming bacteria. Morphologically the parasite has str ong resemblance to the Pasteuria ramosa that Metchnikoff isolated from D. magna and D. pulex in Ukraine and described in 1888. Identificatio n of this parasite has been an enduring puzzle since Metchnikoff. The previously confused phylogenetic position of P. ramosa (it has been cl assified as bacterium, yeast and protozoa) was resolved by sequencing the 16S rDNA molecule. Fluorescent in situ hybridizations confirmed th at the 16S rDNA sequence obtained from the spores within the D. magna body cavity originated from the endoparasite. Maximum likelihood and m aximum parsimony analysis showed that P. ramosa belongs to the low G+C Gram positive branch of the eubacteria and resides within a clade con taining Bacillus tusciae, Alicyclobacillus cycloheptanicus and A. acid ocaldarius as its nearest neighbours. These results confirm suggestion s that this parasite is a bacterium and refute its previous tentative placement based on its morphological complexity among the Actinomyceta les.