Methanogenesis in the Digestive Tracts of the Tropical Millipedes Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae)

Abstract

Methanogens represent the final decomposition step in anaerobic degradation of organic matter, occurring in the digestive tracts of various invertebrates. However, factors determining their community structure and activity in distinct gut sections are still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration peaked in the anterior hindgut of E. pulchripes. In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tracts of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so-far-unclassified "Methanomethylophilaceae" (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promote gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates. IMPORTANCE Methane (CH₄) is the second most important atmospheric greenhouse gas after CO₂ and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats, including digestive tracts of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked as the third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane-producing and non-methane-emitting species of millipedes have been observed, but what limits their methanogenic potential is not known. In the present study, we show that physicochemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH₄ emission in millipedes. We also found close similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

Keywords: Methanobrevibacter; Methanomassiliicoccales; digestive tract; methane; methanogenesis; methanogenic community; physicochemical parameters; tropical millipedes.

FIG 1

(A) Methane emission rates of Archispirostreptus gigas (n = 10) and Epibolus pulchripes (n = 10) expressed in nanomoles per gram (fresh weight) of a millipede per hour. (B) Weight-specific methane emission rates of isolated midgut and hindgut sections of selected individuals (n = 5) measured at five time points (after 1, 2, 4, 6, and 24 h of anaerobic incubation). The methane production rates are expressed in nanomoles per gram (fresh weight) of a single gut section. All rates are reported as means ± SE. For original data on fresh weight of millipedes and gut sections, see Table S1.

FIG 2

Typical radial profiles of oxygen concentration in agarose-embedded midgut and hindgut sections of millipedes Archispirostreptus gigas and Epibolus pulchripes. “Depth” refers to the distance between the electrode tip and the surface of the agarose. To account for individual variations in the exact depths of embedding, the solid arrowheads indicate the points at which the tip reached the gut wall; the open arrowheads indicate the point of emergence on the opposite side. Readings were taken every 0.5 mm.

FIG 3

Axial profiles of pH and hydrogen concentration along the intestinal tracts of millipedes. (A) pH in A. gigas and E. pulchripes; (B) hydrogen concentration in E. pulchripes. Readings were taken at cardinal points of the respective gut sections.

FIG 4

Short-chain fatty acids in midgut, hindgut, and hemolymph of Archispirostreptus gigas (A) and Epibolus pulchripes (B). Concentrations are means ± SE for two and five individuals, respectively. Results are expressed in millimoles per liter of hemolymph or gut content volume.

FIG 5

Phylogenetic tree (16S rRNA genes) of the genus Methanobrevibacter, illustrating the relationships of uncultured methanogenic archaea from the gut of Archispirostreptus gigas (green; 34 clones) and Epibolus pulchripes (red; 33 clones) to their closest relatives in public databases. Taxa that contain representatives from arthropod guts are in blue. The maximum-likelihood tree was reconstructed using IQ-TREE. Closed and open bullets indicate highly supported nodes with values of ≥95/99 and ≥75/90, respectively (SH-aLRT/ultrafast bootstrap analysis, 1,000 replicates each). Accession numbers are shown only for one representative per subgroup. For more details, including accession numbers of all sequences and outgroups, see Fig. S3.

FIG 6

Phylogenetic tree (16S rRNA genes) of the order Methanomassiliicoccales, showing the relationships of uncultured methanogenic archaea from the gut of Archispirostreptus gigas (green; 34 clones) and Epibolus pulchripes (red; 33 clones) to their closest relatives in public databases. Taxa that contain representatives from arthropod guts are in blue. The maximum-likelihood tree was reconstructed using IQ-TREE. Closed and open bullets indicate highly supported nodes with values of ≥95/99 and ≥75/90, respectively (SH-aLRT/ultrafast bootstrap analysis, 1,000 replicates each). Accession numbers are shown only for one representative per subgroup. For more details, including accession numbers of all sequences and outgroups, see Fig. S3.