Effect of ingested material on Can. Lumbricincola

The anecic model earthworm L. terrestris ingest a wide range of differential material (e.g., plant material, soil, and associated microorganisms [Section 1.3]). In this regard, the feeding behavior of L. terrestris introduces diverse organic matter to the alimentary canal that potentially causes high fluctuations of organic carbon in the earthworm gut. Previous studies demonstrated the emission of gut fermentation-derived H2 by L. terrestris (Wüst et al., 2009b), and further evaluations, including different earthworm diets, indicated a positive correlation between the emitted H2 and the organic carbon content of ingested material (Feustel, Oppermann, Schmidt, and Drake; data not published). Furthermore, the findings of this study also illustrates a potential impact of the dietary material on abundant families of the gut content microbiota, including Mycoplasmataceae. In this regard, this earthworm-associated bacterial family displayed a positive response, when L. terrestris was maintained on organic carbon rich dietary substrates (Feustel, Oppermann, Schmidt, Drake; data not published).

Interestingly, Mycoplasmataceae-affiliated phylotypes closely related to Can. Lumbricincola displayed essentially no positive response to any supplemental nutrient (e.g., amino acids, saccharides, microbial- and plant-derived lysates, protein, RNA) evaluated in this dissertation.

Although these phylotypes are strongly associated with L. terrestris (Nechitaylo et al., 2009), their metabolism and function in this invertebrate is largely unresolved. These considerations prompted the evaluation of the response of Can. Lumbricincola-affiliated phylotypes in gut contents of earthworms maintained on dietary substrates, containing different amounts of organic carbon (worm bedding, turf, or soil [Section 2.1.1 and Section 2.5.3]).

A total of 99,886 bacterial 16S rRNA sequences were obtained from the different gut content samples, yielding 25 phyla (including candidate phyla), and rarefaction analyses indicated that the most abundant taxa were targeted (Figure 73). Based on the 16S rRNA abundance analyses at family-level, only the family Pseudomonadaceae was significantly associated with one of the three different gut contents. Thus, this family displayed a significantly higher 16S rRNA abundance in gut contents extracted from earthworms maintained on worm bedding than in gut contents of earthworms kept on soil or turf (Figure 74; Statistical analyses using LEfSe revealed a LDA score [log 10] of 5.2 [Section 2.7.4.2]). The Tenericutes-affiliated family Mycoplasmataceae displayed no significant response to any of the three dietary substrates.

However, based on the relative 16S rRNA abundance analyses at phylotype-level, three Can. Lumbricincola-affiliated taxa were abundant and responded differentially to the three earthworm-ingested dietary substrates (Figure 75). Thus, phylotype OC8 (closely related to an uncultured earthworm bacterium and distantly related to Can. Lumbricincola) displayed a significant higher relative 16S rRNA abundance in gut contents extracted from worms maintained on worm bedding (Figure 75; Statistical analyses using LEfSe revealed a LDA score [log 10] of 5.3 [Section 2.7.4.2]). Furthermore, this phylotype was hardly detectable in earthworm gut

RESULTS 171 contents derived from dietary substrates with limited organic carbon contents (Figure 75 A and B). Likewise, phylotype OC6 (closely related to Can. Lumbricincola) displayed a higher 16S rRNA abundance in earthworm gut contents derived from worm bedding than in the gut contents derived from turf and soil (Figure 75). In marked contrast, phylotype OC1 (also closely related to Can.

Lumbricincola) displayed a positive response in gut contents of earthworm that ingested turf and soil (Figure 75), an observation more pronounced in gut contents pooled from approximately 20 worm individuals maintained on turf or soil (Figure 75 B).

Based on the mean values, the numbers of detected phylotypes, the number of expected phylotypes (Chao1), and Shannon indices were lowest in gut contents extracted from earthworms maintained on the worm bedding (Table 63), indicating a lower bacterial diversity in alimentary canals of earthworms that are finding on organic carbon rich dietary substrates. That the gut content diversity decreases when the availability of organic carbon increases is corroborated by previous microcosm experiments (Figure 25, Figure 36, and Figure 66).

Figure 73. Rarefaction analyses of bacterial 16S rRNA sequences obtained from gut contents of earthworms maintained on different dietary substrates. Phylotypes were based on a 97% sequence similarity cut-off. Capital letter assigned to a substrate [e.g., Turf A] indicate the respective individual.

Number assigned to a substrate [e.g., Turf 1] indicate the respective replicate of the three replicate analyses of pooled gut content from approximately 20 individuals per substrate. WB, worm bedding.

Figure 74. Effect of dietary substrates on the relative 16S rRNA sequence abundances of gut-associated families. The most abundant families (i.e., families with ≥ 4% relative abundance in at least one sampling period) are displayed in the color of the respective phylum. Information on all detected taxa is provided in Table A11. Capital letter assigned to a substrate [e.g., Turf A] indicate the respective individual. Number assigned to a substrate [e.g., Turf 1] indicate the respective replicate of the three replicate analyses of pooled gut content from approximately 20 individuals per substrate. Abbreviations: OC, organic carbon content; WB, worm bedding.

Bedding Turf Soil Turf

Soil

48% 4.9% 2.3% 4.9% 2.3%

RESULTS 173

Figure 75. 16S rRNA-based overview of the most abundant and responsive Can. Lumbricincola-affiliated phylotypes in gut contents of earthworms maintained on different dietary substrates (A and B), and phylogenetic tree of these phylotypes (C). Phylotypes (OC) are based on a sequence similarity cut-off of 97% and were considered to be abundant when a phylotype displayed a ≥ 4% relative 16S rRNA abundances in at least one of the different gut contents. Panel A: 16S rRNA analyses of three worm individuals per substrate. Panel B: the 16S rRNA analyses of pooled (P) gut content from approximately 20 individuals per substrate. Panel C: The phylogenetic tree was calculated using the neighbor-joining, maximum parsimony, and maximum likelihood methods. Solid circles, congruent nodes in three trees.

Branch length and bootstrap values (1,000 resamplings) are from the maximum parsimony tree. The bar indicates 0.1 change per nucleotide. T. maritima (AE000512) was used as outgroup. Accession numbers occur at the end of each branch.

Table 63. Alpha diversity of the microbial community in gut contents of earthworms maintained on different dietary substrates.^a

Dietary Substrate

Number of sequences

Observed phylotypes^b (normalized)^c

Chao1 (normalized)^c

Shannon (normalized)^c

Worm Bedding A 10525 249 (60) 381 (75) 2.9 (2.4)

Worm Bedding B 9385 304 (78) 412 (102) 3.4 (2.9)

Worm Bedding C 8180 457 (122) 681 (167) 3.4 (2.8)

Turf A 5065 560 (210) 899 (233) 4.4 (3.9)

Turf B 5757 682 (239) 928 (264) 4.8 (4.1)

Turf C 8572 565 (171) 866 (216) 3.6 (3.2)

Soil A 3493 603 (228) 841 (244) 5.4 (4.6)

Soil B 4696 548 (212) 882 (245) 4.5 (3.9)

Soil C 4484 414 (175) 814 (221) 3.3 (2.9)

Turf 1 3776 433 (200) 711 (240) 3.9 (3.5)

Turf 2 7565 643 (216) 919 (250) 4.2 (3.7)

Turf 3 5187 526 (208) 848 (246) 3.9 (3.5)

Soil 1 8678 757 (229) 1075 (257) 4.3 (3.7)

Soil 2 8394 748 (225) 1001 (254) 4.6 (4.0)

Soil 3 6119 595 (216) 902 (255) 4.1 (3.6)

aAnalyses based on 16S rRNA sequences. Capital letter assigned to a substrate [e.g., Turf A] indicate the respective individual. Number assigned to a substrate [e.g., Turf 1]

indicate the respective replicate of the three replicate analyses of pooled gut content from approximately 20 individuals per substrate.

bPhylotypes were clustered based on a sequence similarity cut-off of 97%.

cThe data sets were normalized to 2,500 sequences for comparison of amplicon libraries of different sizes.

Can. Lumbricincola Lt-A1, FM16558 Can. Lumbricincola Lr-B2, FM165583 OC6, LR589801

Can. Lumbricincola Lr-C2, FM165584 OC1, LR589795

OC8, LR589797

uncultured earthworm bacterium, LT626939

70

100 75

79

100 0.10

C