Considering the known physiology of the leech and the mutant phenotypes that we have characterized so far, possible mechanisms can be deduced that may interfere with microbial growth and thus contribute to the specificity of this symbiosis. These mechanisms are likely to play important roles at different times, immediately after feeding, during the rapid growth period and during the persistence phase (Fig. 2). The first powerful layer of defense is the antimicrobial properties of the ingested blood. It appears that these properties remain active for some time inside the animal. Our evidence suggests that the complement system is potent even inside the leech and prevents sensitive bacteria from proliferating. Other properties such as iron sequestration by transferrin are likely to be active as well.
During feeding, the leech secrets numerous compounds into the host animal and these would enter the leech during the feeding process with the blood meal. The leech has been shown to remove most of the water from the ingested blood within 48 h. It is possible that the reduced volume restricts bacterial growth. At the same time as the water is removed from the crop, salts are absorbed until the intraluminal fluid is isoosmotic with the leech hemolymph. The osmolarity of the leech hemolymph is approximately two-thirds of that of vertebrate blood. It is not clear if this level of decrease would affect bacterial growth, especially when considering the fact that the bacteria can survive in freshwater that has an even lower osmolarity. These factors or changes have been shown to be present but it is not clear at this point whether any of them contribute to the specificity. Considering that these modifications take over 48 h to occur, it would seem reasonable to assume that they will not play a dominant role during the first 12 h after feeding.
It may be speculated that other factors prevent bacteria from growing within the host. Invertebrate animals have a powerful innate immune response that has many similarities to that of vertebrates, the large exception being the components of the adaptive immunity (Mallo et al. 2002; Tzou et al. 2002). Other factors could include the release of antimicrobial peptides or enzymes into the crop by either the host or symbiont. Some compounds could be release into the ingested blood during feeding for example from the salivary gland. One would assume that these compounds would be active immediately and have an early effect on bacterial growth or survival. In addition, host hemocytes could be present and phagocytose bacteria or generate oxidative stress. Most likely, the different antimicrobial factors act at different times after feeding. The colonization process can be divided into three distinct stages. The first stage consists of overcoming the antimicrobial properties of the ingested blood. The second stage is the rapid proliferation and the third stage the long-term persistence.
The key question is how to identify the mechanisms that are active inside the leech. We are currently undertaking a random transposon mutant screen to identify bacteria that have lost the ability to colonize the leech. One can view each mutant as a sensor of the microenvironment that the bacteria encounter. The characterization of these mutants will reveal conditions that interfere with the proliferation or survival of bacteria, and hence will yield testable hypotheses to elucidate the role of the host or the native symbionts. We have already shown that the analysis of the mutants allowed us to draw conclusions about the complement system. The characterization of the mutants with a colonization defect should provide us with novel and exciting information on the mechanisms underlying the association of bacteria with their leech host.
Acknowledgements. I would like to thank the Swiss National Science Foundation (31-63775), the U.S. National Science Foundation (MCB 0334627) and the Research Foundation of the University of Connecticut for their financial support.
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