Release of greenhouse gases from millipedes as related to food, body size, and other factors

Highlights

• CO₂ production from millipedes reflected the metabolic response of the animals.

• Stable and substantial CH₄ emission was restricted to two millipede orders.

• The quality of the food and feeding regime affect CH₄ production.

• Traces of N₂O released species of Glomeridae only, depending on the food N content.

• Feeding of pure cellulose decreased metabolism of tested species of millipedes.

Abstract

The release of greenhouse gases from millipede digestive tracts warrants study because of its potential effect on climate change and also as an indicator of microbial processes that transform organic matter during passage through the gut of these animals. Gas chromatography was used to quantify the release of methane (CH₄), carbon dioxide (CO₂), and nitrous oxide (N₂O) from living millipedes in laboratory conditions. The effect of four food types (leaf litter of alder, oak, and maple, and rotten wood) on the release of CH₄, CO₂, and N₂O by 12 species was also assessed. In addition, two julid species were fed pure cellulose to test the ability of these millipedes to obtain energy from cellulose and to determine the effect of this diet on gas production.

All of the tested millipede species produced CO₂ and some produced CH₄. Stable and substantial CH₄ emission was restricted to the large millipedes in the tropical orders Spirobolida and Spirostreptida. This asymmetrical phylogenetic distribution of CH₄ production may be related to body size and the presence of gut commensals, but these factors may influence each other and depend upon geographic distribution of species. The quality of the food and feeding regime can also affect CH₄ production in that CH₄ release was significantly higher when millipedes were fed alder leaf litter rather than oak or maple leaf litter. CO₂ production from millipedes mainly reflected the metabolic response of the animals. Traces of N₂O were only occasionally emitted by millipedes; this release evidently depends on the N content in the food and seems to be restricted to members of the Glomeridae family. Based on gas production, the tested species of millipedes were unable to obtain their energy needs from a diet of pure cellulose.

Introduction

The release of greenhouse gases (GHG) from the intestinal tracts of animals warrants study not only due to its possible effects on GHG concentration in the Earth's atmosphere and therefore on climate change, but also as an important indicator of intestinal microbial processes. The intestines of millipedes represent natural models of fermenting bioreactors that are colonized by taxonomically rich and functionally diverse microbial communities; as such, these communities are useful for studying microbial interactions and as sources of novel organisms (Brune, 1998). The microbiome of millipede guts is rich in several groups of archaea, bacteria, and fungi responsible for a wide range of biological and biochemical processes. Byzov (2006) reviewed microbiological studies of almost 30 species of millipedes, and most of the studies were based on routine isolation techniques and microscopy. Our knowledge about cultivable microorganisms and their enzymatic potential in the intestines of tropical millipedes was subsequently increased by Ramanathan and Alagesan (2012). Ambarish and Sridhar (2015) documented the changes in the diversity and abundance of cultivable heterotrophic microorganisms in the food (leaf litter), guts, and feces of two giant pill-millipedes. Nardi et al. (2016) described the ultrastructural distribution of microorganisms in the hindgut of the spirostreptid millipede Cambala speobia. Glukhova et al. (2018) assessed the antibiotic activity of actinomycetes isolated from a polydesmid millipede.

The classical isolation methods used in the studies cited in the previous paragraph fail to detect non-cultivable microorganisms. The use of molecular tools to detect non-cultivable microorganisms associated with millipedes began with the work of Oravecz et al. (2002), who assessed the microbial community in millipede feces. Using fingerprinting analysis, Knapp et al. (2009) found that the gut microbial community in the millipede Cylindroiulus fulviceps differed from the community in the food source and was not greatly affected by diet change. In a subsequent study, Knapp et al. (2010) also used molecular methods to determine that the community of gut bacteria in C. fulviceps is similar to communities previously found in the intestinal tracts of termites and beetles, which are known to harbor symbiotic bacteria essential for digestive activity. Šustr et al. (2014) used molecular profiling (DGGE) to compare communities of methanogenic archaea in 35 species and 17 families of tropical and temperate millipedes; the authors detected sequences related to Methanosarcinales, Methanobacteriales, Methanomicrobiales, and some unclassified archaea.

The intestines of millipedes also contains eukaryotic commensals or parasites such as nematodes (Kitagami et al., 2019) and ciliates (Hackstein and Stumm, 1994), which in turn contain endosymbiotic or associated prokaryotes. Ciliates were found mainly in the hindgut of spirostreptid and spirobolid millipedes (Tuzet et al., 1957).

Methane (CH₄), which has the second-largest effect on global warming after carbon dioxide (CO₂), is released into the atmosphere from natural gas and petroleum deposits and is also produced during many industrial operations (IPCC, 2001). Another significant source of CH₄ is a specific group of microorganisms, i.e., the methanogens (Dlugokencky et al., 2011). Methanogenic archaea, which are an ancient group of microorganisms that occupy ecological niches with limited oxygen concentrations including the digestive tracts of animals, are the only known biogenic source of CH₄ (Ehhalt, 1974). CH₄ emission from vertebrate hosts (Crutzen et al., 1986) as well as from invertebrate hosts (Breznak, 1982, Cruden and Markovetz, 1987, Gijzen et al., 1991, Rasmussen and Khalil, 1983) was discovered already many years ago. Among invertebrates, termites are recognized as a globally important source of CH₄ in that they are responsible for between 5 and 19% of the global CH₄ emissions (Jamali et al., 2011). CH₄ production and the presence of symbiotic methanogens have been systematically screened in a wide variety of invertebrate taxa (Bijnen et al., 1996, Hackstein and Stumm, 1994, Rosenberg and Hackstein, 1995, Sprenger et al., 2000, Šustr and Šimek, 2009, Šustr et al., 2014). Based on the taxonomic groups of arthropods studied to date, CH₄ production is restricted to millipedes, cockroaches, beetles (Cetonidae), and termites (Hackstein and Stumm, 1994). The symbiotic associations involving methanogens and CH₄ production are likely to be a characteristic property of the host taxon (Hackstein and Stumm, 1994, Šustr et al., 2014).

Because they apparently include producers as well as non-producers of CH₄, millipedes are useful for determining which factors influence gut methanogenesis. Among millipedes, species that release CH₄ have been mostly found in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular taxonomic distribution of CH₄ production is correlated with the presence of the methanogen-specific mcrA gene (Šustr et al., 2014). The qualitative and quantitative variability in CH₄ production among millipede species and taxonomic units seems to reflect the suppression or activation of methanogenic archaea in the digestive tract rather than a fundamental inability to host methanogens (Šustr et al., 2014).

Methane-producing millipede taxa also appear to differ in the functional groups of methanogens that they host. An important part of the taxonomic variability in the presence and quantity of CH₄ emission from millipedes may be related to differences in body size (Šustr et al., 2014). This variability may also result from the presence in large tropical species of anaerobic ciliate protists that host intracellular methanogenic archaea (Hackstein and Stumm, 1994) and from the unique feeding habits of some taxa (Šustr et al., 2014). Although a number of branches of the millipede phylogenetic tree have been investigated with regard to the CH₄ releasing, several branches remain unexplored.

In addition to great taxonomic variability in CH₄ production by millipedes, methanogenic community structure varies greatly among gut and faecal pellet samples, and CH₄ production varies substantially among individual millipedes of the same species (Šustr et al., 2014). Except for temperature (Šustr and Šimek, 2009), however, the external factors that affect CH₄ emission by millipedes have not been examined.

Millipedes represent an exception to the typical anatomy of methane-releasing arthropods in that their hindgut lacks an enlarged pouch, which is typical of other methane-producing invertebrates (Hackstein and Stumm, 1994). However, the inner surface of the millipede hindgut is strongly developed and is covered with structures of various shapes (Hopkin and Read, 1992) that facilitate microbial colonization (Byzov, 2006). According to Hopkin and Read (1992) and Byzov (2006), there is no evidence that millipedes possess a permanent symbiotic microflora similar to that of termites. It follows that food type and feeding regime might affect the production of CH₄ by millipedes. However, most reports have documented that the millipede intestinal tract contains a stable indigenous microbial community that includes facultative anaerobes that can degrade recalcitrant organic polymers.

Another GHG that could be potentially produced by millipedes is nitrous oxide (N₂O). N₂O is an important intermediate and by-product of a number of microbial nitrogen transformations including denitrification. Denitrification is performed by many facultative anaerobic bacteria in habitats in which organic substrates are readily available but oxygen is not. Those conditions occur in the intestinal tracts of various invertebrates, and N₂O release has been confirmed from living earthworms (Karsten and Drake, 1997), some termites (Brauman et al., 2015), and several aquatic invertebrates (Stief et al., 2009). Data suggest that a significant proportion of soil-derived N₂O emissions might be directly or indirectly related to earthworms (Karsten and Drake, 1997, Matthies et al., 1999). However, the release of N₂O by many other soil invertebrate groups due to microbial communities in their guts remains largely unknown (Drake et al., 2006). Accordingly, there is no information on N₂O emissions from millipedes.

CO₂ is produced by aerobic as well as anaerobic organisms when they metabolize organic compounds to produce energy. In addition to being the most significant GHG in Earth's atmosphere, its rate of production is a useful indicator of the metabolic rate of animals and of general microbial activity. The efflux of CO₂ from the soil, which is also termed soil respiration, is the major pathway by which carbon exits terrestrial ecosystems (Ryan and Law, 2005). CO₂ output from a living animal may be interpreted as a measure of its metabolic rate but is affected by the type of food being metabolised, by internal pH changes, and by the activity of intestinal aerobic as well as anaerobic microorganisms. CO₂ production has often been used to estimate the intensity of invertebrate metabolism (Mitchell, 1973, Reichle, 1977, Šimek and Šustr, 1995), but the portion of the evolved CO₂ produced by their intestinal microorganisms has rarely been considered.

As noted earlier, the only external factor known to affect CH₄ emission by millipedes is temperature (Šustr and Šimek, 2009); the possible effects of other external factors have not been assessed. In addition, there is no information about the relationship between the metabolic release of CO₂ and the release of other gases, about N₂O emissions from millipedes, or about the ability of millipedes to cover their metabolic energy expenses from cellulose digestion. In the current study, we attempted to fill these knowledge gaps and also to determine how gas emissions are affected by food type. In a series of laboratory microcosm experiments, we studied the effects of four food sources (alder, oak, and maple leaf litter, and rotten wood) on 12 species of millipedes. The food types were selected because they represent natural food of the most European forest millipedes and because they differ in nitrogen content. Alder leaf litter contains highest amount of nitrogen, probably due to the symbiosis of alder trees with nitrogen-fixing Frankia (Sellstedt et al., 1986). Previous research demonstrated that millipedes prefer some species of leaf litter and that millipede assimilation efficiency differs among food types (Tajovský, 1992). Finally, we determined the ability of two julid species to obtain energy from pure cellulose diet and the effect of this diet on CH₄, CO₂, and N₂O production. We tested five hypotheses: 1) At least some millipede species can release N₂O; 2) There are inter-species differences in the release of GHG from millipedes; 3) Release of GHG from millipedes is affected by food type (leaf litter species or rotten wood); 4) Release of these gases is prevented by starvation; and 5) The metabolic rate (as indicated by CO₂ production) is higher for millipedes reared on a cellulose diet than for millipedes that are starved, indicating that at least some species of millipedes are able to obtain energy from pure cellulose.