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

Abstract

Methanogens represent the final decomposition guild in anaerobic degradation of organic matter, occurring in digestive tract of various invertebrates. However, factors determining their community structure and activity in distinct gut sections is 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 culminated in 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 tract 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 promotes the 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 tract 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 third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane producing and non-emitting species have been observed in millipedes, but what limits their methanogenic potential is not known. In the present study, we show that physico-chemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also find great similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

Metatranscriptomic holobiont analysis of carbohydrate-active enzymes in the millipede Telodeinopus aoutii (Diplopoda, Spirostreptida)

“…The contribution of the eukaryotic microbiota (nematodes, ciliates, and fungi) to the CAZyme production was less significant. The expression of fungal CAZymes was very rare in the intestine of T. aoutii, which may be due to the anaerobic conditions in most intestinal sections (Horváthová et al, 2021). The expression of amylolytic or chitinolytic activities in nematodes and ciliates, living in the hindgut, supported their commensal feeding on the rich bacterial community.…”

“…Within prokaryotes, Bacteria are potentially more important producers of GHs than the Archaea (Geng et al, 2021). In the millipede intestine, Archaea are mainly involved in methanogenesis as a final step of the fermentation processes (Horváthová et al, 2021).…”

“…Bacterial ligninases were expressed in the hindgut of T. aoutii as members of the AA2 family. However, the actual activity of LMEs in the hindgut of millipede is questioned by the fact that low oxygen levels prevail here (Horváthová et al, 2021), as in the caeca of isopods (Bredon et al, 2018) or in the P3 lumen fluid of termites (He et al, 2013). The minor role of aerobic fungi, usually capable of lignin degradation, in the holobiont, corresponds with oxygen limitation.…”

“…The intestinal tract of millipedes is a straight tube without distinct regions specialized for the harboring of intestinal symbionts (Hopkin and Read, 1992). However, anoxia and reducing conditions in the gut contents (Horváthová et al, 2021) and the structured inner surface of the hindgut wall (Fontanetti et al, 2015) promote the proliferation of the fermenting microbial community. Their activity in the gut has been demonstrated by the presence of fermentation end-products (short-chain fatty acids and hydrogen) and methane production in the millipedes Archispirostreptus gigas (Spirostreptida) and Epibolus pulchripes (Spirobolida) (Horváthová et al, 2021).…”

The largest arthropod in Earth history: insights from newly discovered Arthropleura remains (Serpukhovian Stainmore Formation, Northumberland, England)

“…However, a cylinder is not representative of the true form of Arthropleura, which is better envisaged as a hemi-ellipsoid with a flat underside and raised topside tapering towards the lateral, anterior and posterior edges. In addition, the density of water (997 kg m −3 ) is not representative of the densities of modern giant millipedes, which are typically 350-550 kg m −3 (Bercovitz and Warburg 1985;Mwabvu et al 2010;Horváthová et al 2021).…”