ABSTRACTEarthworms emit denitrification-derived nitrous oxide and fermentation-derived molecular hydrogen. The present study demonstrated that the earthwormEudrilus eugeniae, obtained in Brazil, emitted methane. Other worms displayed a lesser or no capacity to emit methane. Gene and transcript analyses ofmcrA(encoding the alpha subunit of methyl-CoM reductase) in gut contents ofE. eugeniaesuggested thatMethanosarcinaceae,Methanobacteriaceae, andMethanomicrobiaceaemight be associated with this emission.

The anoxic earthworm gut augments the activity of ingested microorganisms capable of anaerobiosis. Small earthworms (Lumbricidae) emit denitrification-derived N(2)O, whereas the large Octochaetus multiporus (Megascolecidae) does not. To examine this paradox, differently sized species of the families Glossoscolecidae (Rhinodrilus, Glossoscolex, Pontoscolex), Megascolecidae (Amynthas, Perionyx), Acanthodrilidae (Dichogaster), and Eudrilidae (Eudrilus) from Brazil were analyzed. Small species and the large Rhinodrilus alatus emitted N(2)O, whereas the large Glossoscolex paulistus did not, even though its gut could denitrify. N(2) and N(2)O were emitted concomitantly, and R. alatus emitted the highest amount of N(2). Denitrifiers and dissimilatory nitrate reducers were analyzed by barcoded amplicon pyrosequencing of narG, nirK, and nosZ. Gene sequences in gut and soil of the large G. paulistus were similar, whereas sequences in gut and soil of the small Amynthas gracilis were different and were also different compared with those of the gut and soil of G. paulistus. However, the denitrifying gut microbiota for both earthworms appeared to be soil-derived and dominated by Rhizobiales. The results demonstrated that (1) the emission of denitrification-derived N(2)O is widespread in different earthworm families, (2) large earthworms can also emit nitrogenous gases, and (3) ingested members of Rhizobiales are associated with this emission.

The earthworm gut is an anoxic nitrous oxide (N 2 O)-emitting microzone in aerated soils. In situ conditions of the gut might stimulate ingested nitrate-reducing soil bacteria linked to this emission. The objective of this study was to determine if dissimilatory nitrate reducers and denitrifiers in the alimentary canal were affected by feeding guilds (epigeic [Lumbricus rubellus], anecic [Lumbricus terrestris], and endogeic [Aporrectodea caliginosa]). Genes and gene transcripts of narG (encodes a subunit of nitrate reductase and targets both dissimilatory nitrate reducers and denitrifiers) and nosZ (encodes a subunit of N 2 O reductase and targets denitrifiers) were detected in guts and soils. Gut-derived sequences were similar to those of cultured and uncultured soil bacteria and to soil-derived sequences obtained in this study. Gut-derived narG sequences and narG terminal restriction fragments (TRFs) were affiliated mainly with Gram-positive organisms (Actinobacteria). The majority of gut-and uppermost-soil-derived narG transcripts were affiliated with Mycobacterium (Actinobacteria). In contrast, narG sequences indicative of Gram-negative organisms (Proteobacteria) were dominant in mineral soil. Most nosZ sequences and nosZ TRFs were affiliated with Bradyrhizobium (Alphaproteobacteria) and uncultured soil bacteria. TRF profiles indicated that nosZ transcripts were more affected by earthworm feeding guilds than were nosZ genes, whereas narG transcripts were less affected by earthworm feeding guilds than were narG genes. narG and nosZ transcripts were different and less diverse in the earthworm gut than in mineral soil. The collective results indicate that dissimilatory nitrate reducers and denitrifiers in the earthworm gut are soil derived and that ingested narG-and nosZ-containing taxa were not uniformly stimulated in the guts of worms from different feeding guilds.