TABLE CONTENTS

Production and Efficacy of in vitro Pasteuria spp Parasitizing Belonolaimus
longicaudatus
Hewlett, T.E., S.T. Griswold, J.P. Waters & K.S. Smith
Pasteuria Bioscience Inc., 12085 Research Drive Suite 185, Alachua Florida, USA 32615
Production of in vitro endospores of Pasteuria spp. that parasitize Belonolaimus
longicaudatus began with collecting primary isolates from field soil. Sting nematodes
encumbered with spores were handpicked, surface sterilized and placed into culture media in
6-well plates. The nematode bodies were immediately crushed to release cells. Cultures were
incubated for 2 days to check for contamination and transferred to shaker flasks with more
media added and allowed to shake for an additional 2-3 days. The cells from the shaker flasks
were transferred to 500 ml fermentation reactors and grown for 3 or more days. When cell
count was between 10 6 -10 7 cells/ml cultures were transferred to a 20 liter fermenter and
allowed to grow to 10 9 cells/ml , at which time sporulation was induced. Efficacy of in vivo
produced spores was tested for attachment and infection rate. The outer exosporium spore
coat was removed by passing the spore suspension through a French Press. The spore
attachment rate was compared to P. penetrans (in vivo spores) on root knot nematodes using
a centrifuge technique. The sting nematode in vitro spore attachment rate (100,000
spores/tube) averaged 89% compared to 100% for in vivo root-knot. In vivo spore
encumbered sting were used to determine the infection rate. Spore encumbered nematodes
were placed in clean moist sand at room temperature 23 C for 48 hours. Nematodes were
retrieved, surface sterilized, and placed in media as described above. Approximately 48 hours
later, wells were observed for presences of Pasteuria spp. cells and sporulating structure.
Spore encumbered sting nematodes had an average of 100% infection. Presently, efficacy of
spore penetration, attachment, and infection in soil is being tested.
Molecular Mechanisms of Symbiosis Establishment in Marine Nematodes
Bulgheresi, S.
Department of Marine Biology, Vienna
Laxus oneistus is a marine nematode whose cuticle is obligatorily coated by sulfur-oxidizing
bacteria. It lives few centimetres below the sea bottom, where it migrates between upper sand
layers and deeper ones. These migrations allow the bacteria to alternatively obtain oxygen
and sulfide. In turn, the symbionts are the main component of their host diet. We are
interested in the molecular mechanisms of symbiont recruitment from the environment by
newly hatched or moulted worms. By homology cloning, we identified Mermaid, a Ca 2+ -
dependent mannose-binding protein. This lectin is secreted onto the worm’s cuticle and
localizes to the symbiont coat. Mermaid mediates both bacteria-bacteria and worm-bacteria
attachment and even shows striking structural and functional similarities to a human dendritic
cell-specific immunoreceptor. To identify other genes involved in symbiosis, we created an
EST collection and screened for abundantly expressed transcripts. 21% of the ESTs
corresponds to a gene encoding for a novel, secreted, extremely hydrophilic protein which we
provisionally called Pesto. Pesto localizes to the exit channels of the sub-cuticular glands
which also secrete Mermaid, but does not localize to the bacterial coat. Preliminary results
show that Pesto blocks Mermaid-mediated bacterial agglutination, possibly by chelating Ca 2+ .
The biological significance of this, as well as the function of other abundantly expressed
transcripts, is currently under investigation.
5 th International Congress of Nematology, 2008 96