Specificity of the Symbiosis

A powerful tool for the molecular investigation of a symbiosis is a colonization assay that allows one to evaluate the ability of specific strains to colonize the medicinal leech (Graf 1999, 2002; Indergand and Graf 2000). In order to differentiate the introduced test strain from the native microbiota, we use spontaneous rifampin-resistant mutants as test strains. The strains are added to a blood meal that was preheated to 37°C and is fed to the leech in a parafilm covered disposable centrifuge tube. At specified time intervals animals are surface sterilized and killed by placing them in 70% ethanol before recovering the intraluminal fluid. Serial dilution of the samples and plating aliquots on agar plates containing appropriate antibiotics allows us to evaluate the ability of a specific strain to colonize the leech. The versatility of this colonization assay allows us to feed multiple strains carrying different antibiotic resistance markers in a competition assay or add antibiotics to eliminate the native microbiota.

The native symbiont colonized the crop of H. medicinalis rapidly, doubling approximately every 1.2 h, the symbiotic strain HM21R reached a maximum concentration of about 1x108 CFU/ml (Graf 1999) which was maintained for at least 7 days (Indergand and Graf 2000). The growth curve suggests the presence of a rapid proliferation phase and a persistence phase (Fig 2). During the proliferation phase, the symbiont was able to grow at a similar rate inside the animal as in a rich medium at room temperature. The cessation of growth could be due to the depletion of

Fig. 2. A schematic representation of the growth of A. veronii biovar sobria inside the leech. The symbionts proliferate rapidly inside the leech reaching an apparent stationary phase between 12 and 24 h. The colonization process can be divided into three stages. I. Overcoming antimicrobial compounds from the ingested blood. II. Rapid growth phase. III. Persistence phase

Fig. 2. A schematic representation of the growth of A. veronii biovar sobria inside the leech. The symbionts proliferate rapidly inside the leech reaching an apparent stationary phase between 12 and 24 h. The colonization process can be divided into three stages. I. Overcoming antimicrobial compounds from the ingested blood. II. Rapid growth phase. III. Persistence phase nutrients, the accumulation of toxic byproducts, or host modification of the intraluminal fluid. Alternatively, bacteria could be removed or killed by the release of antimicrobial compounds or by phagocytosis by leech hemocytes. These results indicate that the symbiotic A. veronii biovar sobria is well adapted to the digestive tract environment of the leech.

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