High throughput sequencing of 16S and 18S rRNA genes was utilized to characterize bacterial, archaeal and protist communities in the dairy cattle manure (fresh, aged and larval grazed (hereafter grazed)) and evaluated the effect of house fly larval grazing and age of manure in those communities.
Archaeal community profiles
Archaeal communities comprised a total of 19 operational taxonomic units (OTUs). Ten out of 19 archaeal OTUs were shared among three manure types (fresh, aged, grazed manure) (Fig. 1a). Shared OTUs were represented by the most abundant OTUs in each manure type which consisted of the largest proportion of the total abundance in each manure type: fresh (99.0%), aged (98.4%) and grazed (98.5%). Interestingly, all archaeal communities were classified to a single phylum, Euryarchaeota; therefore, no variation in abundance of Euryarcheaota was expected across manure types. At the family level, manure type significantly affected the abundance of Methanobacteriaceae, Methanocorpusculaceae and Methanomassiliicoccaceae. Multiple comparison of means revealed that the relative abundance of Methanobacteriaceae was significantly greater in fresh (92.5%) compared to aged (27.7%; p < 0.0001) and grazed (14.6%; p < 0.0001) (Fig. S1a) and lower in grazed compared to aged (p = 0.003) manure. Similarly, the relative abundance of Methanocorpusculaceae was significantly greater in both aged (62.9%; p < 0.0001) and grazed (73.9%; p < 0.0001) compared to fresh (4.38%) manure (Fig. S1b). The relative abundance of family Methanomassiliicoccaceae was significantly greater in both aged (7.5%; p < 0.0001) and grazed (7.6%; p < 0.0001) compared to fresh (1.1%; Fig. S1c), however, no difference was observed between aged and grazed (p = 0.99) manure.
The archaeal genera Methanobrevibacter, Methanocorpusculum, Methanomassiliicoccus and Methanosphaera (Fig. 1b, c) were dominant in manure, and manure type significantly affected the abundance of all three genera (p < 0.0001). The relative abundance of Methanobrevibacter was significantly greater in fresh (82.52%) compared to aged (26.19%; p < 0.0001) and grazed (13.81%; p < 0.0001) and lower in grazed compared to aged (p = 0.001) manure (Fig. 1c). The relative abundance of Methanomassiliicoccus was significantly greater in both aged (7.5%; p < 0.0001) and grazed (7.6%; p < 0.0001) compared to fresh (1.5%; Fig. 1c) manure. Similarly, the relative abundance of Methanocorpusculum was significantly higher in grazed (73.9%) compared to both fresh (4.4%; p < 0.0001) and aged (62.9%; p < 0.0001) manure (Fig. 1c). The relative abundance of Methanosphaera was significantly lower in both aged (1.5%, p < 0.0001) and grazed (0.8%; p < 0.0001) compared to fresh (10.0%) manure (Fig. 1c).
Bacterial community profiles
Manure bacterial communities comprised 1474 OTUs, among them only 21.64% of OTUs were shared across the manure type (Fig. 2a). These shared OTUs represented the largest portion of the total abundance within each manure type (fresh: 75.4%, aged: 84.8% and grazed: 87.5%). At the higher taxonomic levels, manure bacterial communities were dominated by the phyla Bacteroidetes, Firmicutes and Proteobacteria. Manure type significantly influenced the abundance of all of those phyla (p < 0.0001). Further, the relative abundance of Bacteroidetes was significantly greater in both grazed (34.0%; p < 0.0001) and aged (30.9%; p < 0.0001) compared to fresh (24.8%; Fig. 2b), and greater in grazed compared to aged (p = 0.0060) manure. The relative abundance of Firmicutes was significantly lower in both grazed (10.7%, p < 0.0001) and aged (11.7%; p < 0.0001) compared to fresh manure (28.7%, Fig. 2b). Similarly, the relative abundance of Proteobacteria was significantly greater in both grazed (43.0%; p < 0.0001) and aged (45.2%; p < 0.0001) compared to fresh manure (34.8%) but there was no difference between grazed and aged manure. At the family level, manure types significantly affected the abundance of selected families (p < 0.0, Fig. S2), where the relative abundances of families Veillonellaceae and Succinivibrionaceae were significantly lower in grazed compared to both fresh (p < 0.0001 and < 0.0001 respectively) and aged (p = 0.0002 and 0.0003 respectively; Fig. S2 e, k). The relative abundances of the most dominant families, Moraxellaceae, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Aeromonadaceae, Clostridiaceae, Erysipelotrichaceae, were significantly lower in both aged (p < 0.0001, 0.0004, 0.0004, 0.0004, < 0.0001, < 0.0001, < 0.0001, respectively) and grazed (p < 0.0001, < 0.0001, < 0.0001, < 0.0001, < 0.0001, 0.0003, 0.0033, respectively) compared to fresh manure (Fig. S2 j, b, c, g, l, a, d). Interestingly, the relative abundances of families Flavobacteriaceae, Porphyromonadaceae, Sphingobacteriaceae, Comamonadaceae, Pseudomonadaceae and Xanthomonadaceae were greater in both aged (p = 0.0004, 0.0003, 0.0004, 0.0003, < 0.0001, < 0.0001, respectively) and grazed (p < 0.0001, < 0.0001, < 0.0001, 0.0001, 0.0004, 0.0004, respectively) compared to fresh manure (Fig. S2 h, i, m, n, o, p).
The abundance of several bacteria at the lowest taxonomic level was significantly affected by manure type (Fig. 2c, S3). Rumen-associated bacterial taxa such as Acinetobacter, Ruminococcaceae unclassified, Succinivibrio, Lachnospiraceae unclassified, Clostridiales unclassified, Clostridium, Phascolarctobacterium, Alistipes dominated the fresh manure whereas environmental bacteria such as Petrimonas, Pseudomonas, Taibaiella, Rhodobacteriaceae unclassified, Flavobacterium, Sphingobacterium, Sphingopyxis dominated the grazed manure (Fig. 2c, S3). The relative abundance of the most dominant genus Acinetobacter was significantly lower in aged (9.4%; p < 0.0001) and grazed (11.6%; p < 0.0001) compared to fresh (22.7%) manure (Fig. 2c, Table S2), but no difference was observed between aged and grazed manure (p = 0.28). The second most dominant genus Pseudomonas had significantly greater relative abundance in aged (8.1%; p < 0.0001) and grazed (6.8%; p < 0.0001) compared to fresh (0.9%) (Fig. 2c) manure, and a significant difference was observed between aged and grazed manure (p = 0.0291). The relative abundance of the genus Flavobacterium was significantly greater in the grazed (5.6%; p < 0.0001) compared to fresh (2.1%; p < 0.0001) and aged (2.1%; p < 0.0001) manure (Fig. 2c, Table S2). Similarly, the relative abundance of Bacteroides was significantly lower in grazed (1.9%; p < 0.0001) compared to both fresh (3.4%; p = 0.0001) and aged (3.1%; p = 0.0027) manure (Fig. 2c, Table S2).
Protist community profiles
Manure protist communities consisted of 275 OTUs and only 15.27% of total OTUs were shared among manure types (Fig. 3a). These shared OTUs represented a high percentage of the total abundance within each manure type (fresh: 87.94%, aged: 88.91% and grazed: 92.83%). At the phylum level, Ciliophora, Apicomplexa, Metamonada, Discoba, Cercozoa, Lobosa, Ochrophyta and Stramenopiles were the most dominant groups (Fig. 3b). Manure type significantly affected the abundance of all groups (p < 0.05). Further, multiple comparison of means among manure types revealed that the relative abundance of Ciliophora was significantly lower in grazed (0.5%) compared to both fresh (35.7%; p = 0.002) and aged (40.0%; p = 0.0005) manure. However, the relative abundance of Metamonada was significantly lower in both grazed (0.9%; p < 0.0001) and aged (0.98%; p < 0.0001) compared to fresh (44.2%). The relative abundance of Discoba was significantly greater in grazed (28.9%) compared to both fresh (0.8%; p < 0.0001) and aged (6.6%; p < 0.0001) manure. Similar results were observed for Lobosa and Ochrophyta, where the relative abundances were greater in grazed (15.3 and 13.3%, respectively) and lower in both fresh (0.5 and 0.2%; p < 0.0001 and < 0.0001, respectively) and aged (13.1 and 3.2%; p = 0.26 and 0.0003, respectively) manure. The relative abundance of Apicomplexa was greater in grazed (19.2%) compared to fresh (15.4%, p = 0.29) and aged (2.9%; p < 0.0001) manure. The relative abundance of Stramenopiles was greater in both grazed (15.0%; p = 0.0001) and aged (17.2%; p < 0.0001) compared to fresh manure (1.4%).
The relative abundance of most dominant families varied among manure types (p < 0.05, Fig. S4). The relative abundance of Hexamitinae-Enteromonadida, Trichostomatia and Trichomonadidae were significantly reduced to very low or undetectable levels in both aged (0.9%, p < 0.0001; 0.0% p < 0.0001; and 0.0%, p < 0.0001 respectively) and larval grazed (0.9%, p < 0.0001; 0.0%, p < 0.0001; and 0.1%, p < 0.0001; respectively) compared to fresh (40.5, 35.5 and 3.7% respectively) manure (Fig. S4 a, b, c). Interestingly, manure aging significantly influenced the abundance of Colpodellidae, where the relative abundance in aged was lower (2.8%,) compared to grazed (19.0%, p < 0.0001) and fresh (15.3%, p < 0.0001) manure (Fig. S4d). Relative abundances of the Euglyphida, Nolandellidae, Thecamoebidae and Oxytrichidae were greater in aged (7.7, 1.7, 0.2 and 29.9%, respectively) compared to both fresh (0.0%, p < 0.0001; 0.1%, p = 0.0003; 0.0%, p < 0.0001; and 0.2%, p = 0.001; respectively) and grazed (0.1%, p = 0.004; 0.0%, p = 0.015; 0.0%, p < 0.0001; and 0.4% p = 0.008, respectively) manure (Fig. S4 e, f, g, h). Also, the abundances of Chrysophyceae, Parabodonid and Vannellidae were greater in grazed (12.5, 28.8, 12.3% respectively) compared to aged (2.5%, p = 0.014; 6.3%, p = 0.014; 3.7%, p = 0.082, respectively) and fresh (0.2%, p < 0.0001; 0.3%, p < 0.0001; 0.4%, p < 0.0001; respectively) manure. Thraustochytriaceae were greater in grazed (14.9%) compared to fresh (0.2%, p < 0.0001) and lower compared to aged (16.8%, p = 0.73) manure.
At the lowest taxonomic levels, manure type significantly affected the abundance of dominant taxa (p < 0.05, Fig. 3c, S5). For instance, the relative abundance of Buxtonella (Ciliophora) and the genera of Metamonada: Enteromonas, Trepomonas, Trimitus and Tetratrichomonas (Fig. 3c) were significantly higher in fresh (35.5, 31.0, 5.0, 2.4 and 3.7%, respectively) compared to aged (0.0%, p < 0.0001; 0.3% p = 0.0001; 0.0%, p < 0.0001; 0.0%, p < 0.0001; and 0.0%, p < 0.0001, respectively) and grazed (0.0%, p < 0.0001; 0.4% p = 0.0004; 0.3%, p = 0.0003; 0.2%, p = 0.0002; 0.1%, p = 0.0002, respectively) manure. Although, the abundance of Ciliophora was higher in both fresh and aged manure, the genus Buxtonella was exclusively found in fresh while Oxytricha was found in aged manure (Fig. 3c). The relative abundance of Oxytricha, Cercomonas, unclassified Nolandellidae, unclassified Euglyphida and unclassified Thraustochytriaceae were greater in aged (29.8, 3.4, 1.7, 7.7 and 16.8%, respectively) compared to fresh (0.1%, p = 0.0005; 0.0%, p < 0.0001; 0.1%, p = 0.0003; 0.0%, p < 0.0001; and 0.2%, p < 0.0001, respectively) and grazed (0.4%, p = 0.014; 0.1%, p = 0.008; 0.1%, p = 0.015; 0.1%, p = 0.004; and 14.9%, p < 0.0001; respectively) manure. Interestingly, abundances of Parabodo, Colpodella, Vannella, unclassified Filosa-Sarcomonadea and unclassified Chrysophyceae were greater in grazed (28.8, 19.0, 12.3, 5.5 and 12.5% respectively) compared to fresh (0.3%, p < 0.0001; 15.3%, p = 0.15; 0.4%, p < 0.0001; 0.1%, p < 0.0001; 0.2%, p < 0.0001, respectively) and aged (6.3%, p = 0.014; 2.8%, p < 0.0001; 3.7%, p = 0.08; 3.2%, p = 0.26; 2.5%, p = 0.014, respectively) manure (Fig. 3c).
Microbial diversity and community composition
Manure type significantly affected the archaeal Shannon (p = 0.018) and Simpson (p < 0.0001) diversity indices but not species richness (p = 0.15). Multiple comparisons of means showed that the archaeal Shannon diversity index of grazed (1.05) was significantly lower than fresh (1.27, p = 0.0180) but non-significantly lower than aged (1.26, p = 0.0530) (Fig. 4b, Table S3). The effect of manure type on bacterial Shannon diversity index (p < 0.0001), Simpson diversity index (p = 0.0007) and species richness (p < 0.0001) were significant. Multiple comparisons of means revealed that the bacterial Shannon diversity index was significantly lower in grazed (4.61) compared to fresh (4.96, p < 0.0001) and aged (4.83, p = 0.005) manure type (Fig. 4a, Table S3). Also, manure type significantly influenced the protist Shannon (p = 0.02) and Simpson (p = 0.03) diversity indices and species richness (p < 0.0001). Pairwise comparisons showed the protists Shannon diversity index was significantly greater in grazed (2.59) than fresh (0.76, p = 0.002) but non-significantly greater than aged (2.33, p = 0.14) manure (Fig. 4c, Table S3).
The overall patterns of bacterial (Fig. 4d, S6a, b), archaeal (Fig. 4e, S6c, d) and protist (Fig. 4f, S6e, f) community compositions among manure types were distinctly separated in the first two axes of the principal coordinates analysis based on Bray-Curtis, Uni-Frac, and Jaccard (binary) dissimilarity indices. Permutational multivariate analysis of variance revealed that those distinct patterns were statistically significant for bacterial (p < 0.0001), archaeal (p < 0.0001), and protist (p < 0.0001) communities (Table S4). Moreover, microbial community compositions also were well separated on canonical correspondence analysis, where manure properties (total carbon (TC), total nitrogen (TN) and carbon to nitrogen ratio (CN)) significantly correlated with the bacterial, archaeal and protist community compositions (Fig. S7, Table S5).
Relationships of manure properties, microbial communities and diversity
The major bacterial phyla (Proteobacteria, Bacteroidetes) and protist phyla (Discoba, Lobosa, Ochrophyta and Stramenopiles) were negatively correlated with TN and TC but positively correlated with CN (Table S6). However, the bacterial phylum Firmicutes, and protist phyla Ciliophora and Metamonada were positively correlated with TN and TC (Table S6). In the lower taxonomic levels, significant positive correlations between TN and/or TC and bacterial taxa: Acenetobacter, Alistipes, Bacteroides, Campylobacter, Clostridium sensu stricto, Clostridiales unclassified, Lachnospiraceae unclassified, Ruminococcaceae unclassified, Succinivibrio, Phascolarctobacterium, Acholeplasma and Tissierella; archaeal genera: Methanosphaera, Methanobacter; and protist taxa: Blastocystis, Buxtonella, Enteromonas, Hexamitinae-Enteromonadida unclassified, Trepomonas, Trimitus and Tetratrichomonas (Table S7) were observed. Other bacterial taxa: Pseudomonas, unclassified Comamonadaceae unclassified, Taibaiella, Bacteroidetes unclassified, Sphingobacteriaceae unclassified, Acidaminococcaceae unclassified; archaeal genera: Methanomassiliicoccus and Methanocorpusculum; and protist taxa Parabodo, Vannella, Lobosa unclassified, Filosa-Sarcomonadea unclassified were negatively correlated with TN and TC (Table S7). The analysis of correlation showed that α-diversity indices for bacteria (Shannon and Species Richness) and archaea (Shannon, Simpson, Pielou’s Evenness) were positively correlated with TN and TC but bacteria (Simpson), and protist (Shannon and Species Richness) were negatively correlated with TN and TC (Table S8).
House fly larval grazing changes the manure properties
In dry manure, TC content ranged from 87.3–127.9 g/kg, TN ranged from 9.2–17.2 g/kg and CN ranged from 6.72–10.22. There was strong correlation between TC, TN and CN, and statistically significant effects of manure types on TC (F(2, 13) = 11.04, p = 0.0015), TN (F(2, 13) = 113.6, p < 0.0001) and CN (F(2, 13) = 64.96, p < 0.0001) were observed. Pairwise comparisons of means showed that TN was significantly lower in grazed compared to both fresh (p < 0.0001) and aged (p = 0.0058) manure (Fig. S8b) whereas TC was significantly lower in grazed compared to fresh manure (p = 0.0039, Fig. S8a), CN was significantly higher in grazed compared to both fresh (p < 0.0001) and aged (p < 0.0002) manure (Fig. S8c).