Earthworm egg capsules as vectors for the environmental introduction of biodegradative bacteria

Abstract

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2, 4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10(2) or 10(5) CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10(4) CFU R. eutropha (pJP4) g-1 of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2, 4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.

FIG. 1

Degradation of 2,4-D in nonsterile soil with or without added earthworm cocoons. Cocoons contained R. eutropha JMP222N(pJP4), R. eutropha JMP222N, or no degradative bacteria (nonsterile) and were about 1 week from hatching when added to the degradation microcosms at time zero. Juvenile worms containing microcosms were respiked with 100 ppm of 2,4-D on days 10 and 11. Data points represent the mean of two replicate samples. Error bars indicate the standard deviation, and where no standard bar is presented the value is less than the height of the symbol. Symbols: ▴, no cocoons; □, nonsterile cocoons; ○, JMP222N cocoons; ●, JMP222N(pJP4) cocoons.

FIG. 2

Comparison of degradation of 2,4-D in nonsterile soil with cocoons containing high (∼10⁵ CFU cocoon⁻¹; ●) or low (∼10² CFU cocoon⁻¹; ■) densities of R. eutropha JMP222N(pJP4) or without cocoons (▴). Data points represent the mean of two replicate samples. Error bars indicate the standard deviation, and where no standard bar is presented the value is less than the height of the symbol.

FIG. 3

Toxicity of 20 ppm of 2,4-DCP to cocoons formed in uninoculated or R. eutropha JMP222N(pJP4)-inoculated earthworm mating microcosms. Symbols: ◊, nonsterile cocoons; ⧫, R. eutropha (pJP4) cocoons.