In their Correspondence article, Rae and Sommer raise a number of interesting issues concerning the findings we reported recently in The Journal of Experimental Biology (Abebe et al., 2010). Their main thesis that “the authors have failed to demonstrate the pathogenicity of Caenorhabditis species and the symbiotic nature of this association” is incorrect. We would like to restate that our Caenorhabditis briggsae strain, i.e. C. briggsae KT0001, was isolated from independent Galleria mellonella trap experiments from soils collected from three provinces, and in all cases we found the nematode with the bacteria Serratia sp. SCBI. Our subsequent experiments showed that the worms entered G. mellonella, reproduced successfully and emerged from the cadaver as infective dauer-stage juveniles. Very importantly, C. briggsae KT0001 was not able to kill G. mellonella in 24 hours when grown on Escherichia coli, and our results clearly showed that it was Serratia sp. SCBI and not the nematode that killed the insect, indicating that the Caenorhabditis–Serratia sp. SCBI association was critical in producing insect mortality; G. mellonella mortality when topically exposed to Serratia sp. SCBI was not significantly different from similar exposure to E. coli OP50 or mortality in the absence of any manipulation. These results are similar to those reported in the literature for Heterorhabditis and Steinernema (Gouge and Snyder, 2006). So although we know little about the exact nature of the association or how common it is in nature, we clearly demonstrate the ability of this association to hasten the death of an insect.
The umbrella term ‘symbiosis’ represents a diverse array of biological associations between organisms. At this time we don't have data to support speculation on the kind of association that exists between C. briggsae KT0001 and Serratia sp. SCBI. We fully agree with Rae and Sommer that Caenorhabditis lacks specific morphological adaptations that support symbiosis between some nematodes and bacteria. However, similar to Caenorhabditis, the well-recognized entomopathogenic nematode (EPN), Heterorhabditis also lack any morphological adaptations; instead, the bacterial associates of Heterorhabditis colonize the entire intestine (Ciche and Ensign, 2003). Phylogenetically, Caenorhabditis are closer to Heterorhabditis (Kiontke and Fitch, 2005) than to Steinernema. As a result, it is not surprising that Caenorhabditis lack the adaptation that Steirnernema has to host its bacterial associate. In terms of maintaining the association, we don't have evidence to exclude any mechanism, including invasion of the intestinal cells by the bacteria, similar to what is reported for Heterorhabditis (Ciche and Ensign, 2003). Félix and Braendle reported “intestinal colonization by live bacteria” for C. elegans (Félix and Braendle, 2010). Consequently, the current lack of evidence regarding the mechanism of maintenance of the association between the nematode and bacteria does not prove the absence of such a mechanism. However, as is obvious from the comments of Rae and Sommer, these are very interesting questions that will be crucial to understanding the evolution of such associations.
The entry of the bacteria and nematode into the insect varies among well-characterized EPNs. In Steinernema, infective juveniles enter the insect body through natural opening such as the mouth, spiracles and anus; in Heterorhabditis, infective juveniles enter the hemocoel directly through the insect's integument (Wang and Gaugler, 1998). Félix and Braendle pointed out that C. elegans' associations with insects could be “phoretic, necromenic and possibly commensal or parasitic” (Félix and Braendle, 2010). From this, it is plausible to conclude that Caenorhabditis species may enter the body cavities of invertebrates as a common feature of their life cycles.
Rae and Sommers also state that “all known EPNs can only reproduce on one bacterium.” This generalization may not hold true on closer inspection. Gouge and Snyder provided an extensive list of bacterial species reported from Heterorhabditis and Sterinernema in addition to their well-documented bacterial associates (Gouge and Snyder, 2006). Often those associated bacteria, other than Xenorhabdus spp. and Photorhabdus spp., were isolated from surface-sterilized worms (e.g. Babic et al., 2000). Many gram-negative bacteria were also shown to support reproduction of steinernematids (Boemare et al., 1983; Ehlers et al., 1990). There is extensive evidence that Caenorhabditis indeed establish phoretic or necromenic association with insects (Kiontke and Sudhaus, 2006). Some have argued that such associations might have produced entomopathogenic associations (Sudhaus, 1993).
So although we agree with Rae and Sommer that we have not characterized the nature of the association between this strain of Serratia and Caenorhabditis, we have demonstrated that in some cases they are capable of acting as an entomopathogenic complex. We fully recognize that the Caenorhabditis–Serratia association is not as well characterized as the archetypical EPNs. However, we look forward to the possibility that this finding may shed light on the life history of one of our premiere model organisms in science as, although great strides are being made toward finding and describing natural populations of Caenorhabditis (Barrière and Félix, 2005), we remain in the almost dark about the role of this nematode and its relatives in nature (Félix and Braendle, 2010).
