Grinding in the gizzard: how earthworms feed their feeders

It is conceptionalized that a fraction of the ingested microbial cells (especially larger cells like fungal hyphae) are disrupted by the grinding activity in the gizzard [35, 391, 484]. Such a disruption would result in the release of disrupted cell-derived biopolymers (proteins, nucleic acids, lipids, and cellwall carbohydrates like chitin) and their breakdown products (amino acids, nucleotides, glycerol, fatty acids, and sugars) [230]. The earthworm may absorb a fraction of the disrupted cell-derived organics as part of its nutrition [35]. Ingested soil anaerobes that are activiated by the beneficial conditions in the gut (e.g., anoxia, a high water content, mucus-derived sugars and glycoproteins) may also feed on organic compounds mucus-derived from disrupted cells. However, such taxa that potentially contribute to the degradation of microbial cells, which were disrupted by the grinding activity of the gizzard, are not resolved. A lysate derived from french pressed yeast cells was supplemented to gut content microcosms to stimulate taxa that in situ may thrive on disrupted cells.

16S rRNA transcript-based analyses of the microbial community indicated that Clostridium bifermentans (Peptostreptococcaceae) and Aeromonas sp. (Aeromonadaceae) were the dominant taxa that initially responded on the supplemented cell lysate (Figure 42 and Figure 43). Both taxa are able to ferment proteinaceous substrates and carbohydrates, and Aeromonas sp. can hydrolyze DNA and lipids [46, 276], making them excellent utilizer of various organics derived from disrupted cells. Acetate, succinate, formate, H2 and CO2 were the major fermentation products that accumulated during the first 6 h of incubation, and these compounds are among the fermentation products that are characteristic for C. bifermentans and Aeromonas sp. [421, 478]. C. bifermentans-related taxa were also abundant but not labled in [¹³C]glucose-supplemented gut content microcosms of L. terrestris, indicating that these taxa did not assimilate supplemental glucose and may have metabolized endogenous carbon compounds in these experiments [490]. This assumption is reinforced by the high abundance of C. bifermentans in unsupplemented controls in the present experiments (Figure 42). Thus, disrupted cell-derived organic compounds represent only one of several substrates on which C. bifermentans can feed in the gut of earthworms.

With time, the generalistic fermenters C. bifermentans and Aeromonas sp. that rapidly responded to the supplemented cell lysate were more and more replaced by saccharolytic Enterobacteriaceae [33], proteolytic Clostridiaceae [34, 285, 363], and physiologically uncharacterized Lachnospiraceae (Figure 42). The Enterobacteriaceae taxa that were stimulated by cell lysates (Figure 43) were highly similar to those observed in gut content

microcosms supplemented with glucose (a constituent of the mucus of L. terrestris) [488, 490].

Thus members of the Enterobacteriaceae may profit from the release of sugars derived from disrupted cells (e.g., ribose and deoxyribose from RNA and DNA) or the mucus in the gut of earthworms. In contrast to the Enterobacteriaceae sp. that were similar in glucose and cell lysate supplemented microcosms, the proteolytic Clostridiaceae observed in the treatments with cell lysate were phylogenetically distinct from those that utilized glucose in the aforementioned study [490]. Therefore, physiologically and phylogenetically distinct fermenters of the Clostridiaceae are active in the degradation of carbohydrates and proteins in the gut of earthworms. OTU_Y6 was the only abundant OTU that was stimulated by supplemental cell lysate but was not closely related to any cultured species (Figure 43). This OTU could be affiliated to the Lachnospiraceae, a family that comprises cellulolytic, pectinolytic, xylanolytic, and other saccharolytic fermenters as well as acetogens, and syntrophic species [344]. No proteolytic species are known and therefore OTU_Y6 may have been stimulated by carbohydrates and not by cell lysate-derived proteins. However, this needs to be verified.

Based on the earlier findings that formate and H2 were produced but not consumed in long term incubated gut section homogenates supplemented with glucose [192] and that acetogens were not detected in glucose-supplemented gut content microcosms [490], it was assumed that acetogenesis is not an important process in the gut of earthworms. However, formate consumption started after 6 h in cell lysate treatments and in parallel the production of acetate and CO2 increased (Figure 40), both observations being indicative for formate-dependent acetogenesis. Furthermore, taxa closely related to C. glycolicum (Peptostreptococcaceae) and C. magnum (Clostridiaceae), two species that comprise formate utilizing acetogenic strains [28, 175, 222], increasesd in 16S rRNA transcript abundances (Figure 43). After 20 h, H2 and CO2 accumulation slowed down whereas acetate still accumulated at high rates in cell lysate treatments, suggesting that acetogens may have utilized H2-CO2 in addition to formate (Figure 40). Ethanol and lactate are also potential substrates for acetogens (Table 4) and accumulated in glucose-supplemented microcosms [490]. Both compounds did not accumulate in cell lysate treatments and therefore may have been scavenged by acetognes.

Some strains of C. glycolicum can decarboxylate succinate to propionate [45], an activity that was potentially a source for the propionate formed in cell lysate treatments. C. glycolicum and C. magnum can convert various sugars [28, 175, 222] and might compete with saccharolytic fermenters for the same substrates.

A hypothetical model showing the proposed trophic interactions between the earthworm and the ingested soil microbes is given in Figure 50. The earthworm takes up microbial cells attached to plant litter and soil. Larger cells like that from fungal hyphae, protists, and also vegetative bacterial cells are partially disrupted probably by the grinding activity in the gizzard

[110, 391]. The organic compounds that are released during cell disruption are potent substrates for ingested soil anaerobes. The initial stimulation of fermenters with a broad susbtrate range (C. bifermentans and Aeromonas sp.) and the subsequent replacement of these taxa by more specialiced fermenters (e.g., proteolytic Clostridiaceae and saccharolytic Enterobacteriaceae) indicated, that different functional clades of fermenters are active during gut passage from the foregut to the hindgut. These fermenters produce organic acids that can be absorbed by the earthworm as part of its nutrition, although this has not been tested yet.

However, the concentrations of organic acids decreased from the midgut to the hindgut in living earthworms [488] and the capacity to absorp these compounds in the digestive tract is a general trait of animals [21, 106, 164, 347, 439]. Thus, it is very likely that earthworms indeed absorbs organic acids derived from fermentation processes in the gut. Acetogens can also contribute to the formation of organic acids and therefore may also play a role in the nutrition of earthworms. In summary, earthworms feed ingested soil anaerobes (i.e., fermenters and acetogens) with disrupted microbial cells, and the anaerobes in turn produce organic acids (acetate, succinate, and propionate) on which the earthworm can feed (Hypothesis 4, 1.5).

Figure 50 Hypothetical model of anerobic processes and associated taxa that are stimulated by disrupted microbial cells in the gut of the earthworm L. terrestris.

Dashed lines, proposed absorption of organic acids by the earthworm; dotted lines, potential anaerobic processes that might occur in addition to fermentation.

4.3. Peatlands and earthworm guts: anoxic ecosystems