Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria

Earthworms are saprotrophic invertebrates which contribute to the overall structure, drainage, and aeration of soil ecosystems (11). In addition, earthworms directly and indirectly influence soil microbial communities, mainly through the processes of feeding, burrowing, and fecal (cast) deposition. Several studies have shown that earthworms can transport bacteria into the environment (6, 7, 9, 13, 16, 23, 27, 28, 29). In particular, previous studies have assessed the importance of earthworms for the transport of beneficial bacteria for use in biological control (27). More recently, earthworms have been implicated as vectors and contributors for bacterial gene transfer (6, 7). To date, the majority of these studies have focused on adult earthworms, with little attention to how developing cocoons or juvenile earthworms affect microbial communities. Several studies have shown that microorganisms are associated with earthworm egg capsules (cocoons) (8, 18, 32). Morgan and Burrows (18) reported that the internal fluid of Eisenia fetida cocoons contained bacteria of the genera Nocardia, Pseudomonas, and Alcaligenes. Zachmann and Molina (32) were the first to quantify the bacteria present in egg capsules of E. fetida, with up to 108 CFU g−1 of cocoon. Attempts to identify several of the morphologically diverse cocoon isolates were inconclusive. In addition, Zachmann and Molina (32) determined that cocoons may acquire bacteria from the environment from which they are formed; however, the location and function of the bacteria within the cocoon were not addressed in that study. Recently, Daane et al. (8), using scanning electron microscopy, determined the location of bacteria within the developing earthworm cocoon. Examination of cocoon fragments containing well-developed juvenile earthworms revealed the presence of numerous rod-shaped microorganisms within the developing tissues. The microorganisms present did not appear to be detrimental to the developing earthworm tissue, which suggests a mutualistic association. Earthworm cocoon fluid has been found to elicit an immune response against several bacterial species, which indicates that earthworms may be able to control the cocoon microbiota (30). This is also suggested by Daane et al. (7), who reported consistent recovery of Ralstonia eutropha (formerly Alcaligenes eutrophus) from Aporrectodea trapezoides cocoons formed in inoculated soil. In addition, Zachmann and Molina (32) reported that Escherichia coli could not be recovered from cocoons formed in microcosms inoculated with this bacterium. Although cocoons have been found to contain and acquire microorganisms from the environment, few if any studies have been performed to assess how the earthworm egg capsule microbiota influences juvenile development, terrestrial microbial communities, or the fate of toxic chemicals in the environment. The latter is of particular interest because it would determine whether earthworm egg capsules could be used as biovectors for the introduction of beneficial bacteria, such as biological control, biodegradative, or plant-growth-promoting strains, into the environment. The objectives of the present study were to use the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium, R. eutropha JMP222N (pJP4), and the compost earthworm, E. fetida as a model system to determine whether bacterial strains acquired by earthworm egg capsules can be released from the cocoons into the soil for degradation of 2,4-D. In addition, we assessed the ability of a degradative strain within the cocoon to influence the level of tolerance of the developing earthworm to 2,4-dichlorophenol (2,4-DCP).