Declaración de los Derechos del Niño

To compare the species composition of the archaeal community, as determined by DGGE, with another method, we selected one of the sheep (no. 4 in Figure 4.1b) fed winter pasture. Libraries of PCR-amplified 16S rRNA genes were prepared using two primer pairs, and randomly-selected PCR products were sequenced and the sequences placed phylogenetically by tree construction. The two primer sets, although amplifying different regions of the 16S rRNA gene, resulted in very similar library compositions (Table 4.3; P = 0.11 in χ2-test for differences). Three of the four major archaeal groups were the same ones that were also detected using DGGE, viz., the Methanobrevibacter gottschalkii clade, the Methanobrevibacter ruminantium clade, and Methanosphaera

spp.. Both libraries contained sequences from the RCC group, making up 22 and 28% of the libraries. In addition, small numbers of sequences affiliated with other lineages were found (Table 4.3). The amount of sequence variation, measured as uncorrected sequence distances, was greatest (≥ 5.3%) among the sequences affiliated with RCC, and lower (≤ 3.1%) among sequences affiliated with the other major groups (Table 4.3).

147 Figure 4.7. DGGE fingerprints of RCC in sheep, red deer, and cattle fed summer pasture. The similarities of the band patterns are indicated by the dendrogram to the left of the gel image. Individual lanes are not in the same order as they were on the original gel, but all are from the same gel. The gel origin is on the left.

148 Table 4.3. Abundance of different clades of archaea in libraries of PCR-amplified 16S rRNA genes from rumen contents of sheep no. 4 fed winter pasture. The differences between sequences were calculated from percentage identities between pairs of aligned sequences.

---

Primer pair Measure Archaeal group

---

Mbb. gottschalkii Mbb. ruminantium Methanosphaera RCC Other

clade clade spp. clade clades

---

109f + 915r No. of sequences 41 29 9 21 3*

Mean sequence difference (± S.D.) 1.4 ± 0.7% 2.1 ± 1.3% 1.0 ± 0.8% 5.3 ± 3.3% n.a.† Range of sequence differences 0 – 4.7% 0 – 6.7% 0 – 2.5% 0 – 10.5% n.a.

915f + 1386r No. of sequences 40 12 14 27 2‡

Mean sequence difference (± S.D.) 1.8 ± 1.5% 2.2 ± 1.2% 3.1 ± 1.9% 5.8 ± 3.2% n.a. Range of sequence differences 0 – 7.9% 0 – 5.0% 0 – 7.7% 0 – 10.8% n.a. ---

*

Two sequences only basally-affiliated with RCC, and one sequence from a Methanobrevibacter sp. outside the Mbb. gottschalkii and Mbb. ruminantium clades.

†

n.a., not applicable.

‡

148 The abundance of 16S rRNA genes from total bacteria, total archaea, and from members of the RCC clade of archaea in samples taken from different sheep on a range of diets was measured using quantitative real-time PCR. Bacterial 16S rRNA gene numbers ranged from 1.0  1011 to 1.2  1012 (mean = 4.5  1011, S.D. = 3.8  1011) copies per g of dry rumen contents. Archaeal 16S rRNA genes were not as abundant, from 3.5  109 _{to 6.2  10}10 _{(mean = 1.4  10}10_{, S.D. = 1.3  10}10

) copies per g of dry rumen contents, and RCC 16S rRNA genes were less abundant again, from 1.3  109

to 7.2  109 _{(mean = 2.6  10}9_{, S.D. = 1.2  10}9

) copies per g of dry rumen contents. There was no detectable correlation between the number of bacterial and archaeal 16S rRNA genes in the samples (r = 0.06), but RCC numbers were moderately correlated with total archaeal numbers (r = 0.64). On average, archaeal 16S rRNA genes were present at only 5.1% of the abundance of bacterial 16S rRNA genes, and the data suggest that RCC made up an average of 26.5% of the archaeal pool. ANOVA of log-transformed data indicated that there were differences in archaeal (P = 0.002) and bacterial (P <0.001) abundances between the different groups of sheep. Abundances of RCC groups did not appear to be significantly different (P=0.84) among the different groups of sheep.

Quantitative real-time PCR indicated that 16S rRNA genes from RCC made up 35.5% of the archaeal pool in sheep no. 4 fed winter pasture, in agreement with clone library estimates of 22 to 28% (Table 4.2).

149

4.4 Discussion

4.4.1 General archaeal community structure

DGGE is a useful method for examining gross differences between microbial communities in environmental samples. DGGE has previously been used to investigate bacterial diversity in the rumen (Kocherginskaya et al., 2001; Tajima et al., 2001a; Klieve et al., 2007), and there have been some studies conducted on rumen archaea. The first application of a technique similar to DGGE to analyze the populations of rumen archaea was performed by Nicholson et al. (2007). They analyzed sequences of bands from Temporal Temperature Gradient Gel Electrophoresis profiles to provide a picture of the ruminal archaea in grazing cattle and sheep. Later, Ouwerkerk et al. (2008) and Cheng et al. (2009) used DGGE to examine the archaeal community structure in a number of rumens. Ohene-Adjei et al. (2008) used DGGE as a tool to study the effects of plant extracts on the diversity of archaea in lambs. Recently Hook et al. (2009) used this technique to identify the changes in archaeal communities when animals were supplemented with monensin.

In our study we compared the communities of archaea in rumen samples from cattle, sheep and red deer fed with different diets. The DGGE fingerprints of archaea from different species of ruminants (sheep, cattle, and red deer) fed different diets (autumn pasture, winter pasture, summer pasture, silage, a concentrate-based diet, and willow) were all similar, regardless of ruminant species or diet. Time after feeding also had very little impact on the community composition. The overall similarity of most of the profiles suggests that similar dominant archaeal populations were present in these animals, despite being from different individuals and different ruminant species or from animals fed different diets. Similar findings were reported by Ouwerkerk et al. (2008), who observed no major differences among DGGE patterns when they examined the archaeal communities of cattle and sheep fed a variety of forages (hay from different grass types, Leucaena sp.-grass mix, lucerne pellets and fresh grass). Ouwerkerk et al. (2008) did observe greater variation between archaeal communities in individuals fed a barley-based diet, and these patterns were different from those fed forages. The higher grain content (75% [w/w]) in these diets compared with ours (45% [w/w]) may have been the reason for this. High grain contents can result in lower ruminal pH values, which may adversely affect some methanogen species (Lana et al., 1998).

150