S U M M A R YThe vast increase in the population density of the rumen flagellate Neocallimastix frontalis shortly after the host animal has commenced eating is caused by stimulation of a reproductive body on a vegetative phase of the organism to differentiate and liberate the flagellates. The stimulant is a component of the host's diet. The vegetative stage of N . frontalis bears a strong morphological resemblance to that of certain species of aquatic phycomycete fungi, and consists of a reproductive body borne on a single, much branched rhizoid. The flagellates liberated in vivo within 15 to 45 min of feeding lose their motility within I h and develop into the vegetative phase, thus producing a rapid decrease in population density of the flagellates. Conditions for maximum flagellate production are similar to those occurring in the rumen: pH 6-5, 39 "C, absence of 02, presence of C02. Differentiation of the reproductive body is inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The organism was cultured in vitro in an undefined medium in the absence of bacteria or other flagellates.

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

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Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.

S U M M A R YThe rumen flagellate Sphaeromonas cominunis showed a significant increase i n population density I to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The lifehistory of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro:) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore be zoospores, Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 ' C , presence of C 0 2 , absence of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absence of bacteria and other flagellates. I N T R O D U C T I O NRumen flagellates possessing a spherical-to-ovoid cell and a single flagellum were described by Liebetanz (1910) and by Braune (1913). Since then, very few references to this type of organism have been made in the literature, and no mention was made by Hungate ( I 966) or by Jensen & Hammond (1964). It appears that the organisms have been overlooked, perhaps from lack of knowledge of their identity, even though population densities of 105 ml-1 have been recorded for flagellates with this morphology in the sheep at the author's laboratory. Both Liebetanz ( I 9 I 0) and Braune ( I 9 I 3) regarded them as flagellate protozoa, an opinion also held by Das Gupta (1935) and Becker & Talbot (1927). Four genera of these rumen flagellates have been named -Monas, Sphaeromonas, Piromonas and Oikomonasbut they were subsequently thought to be synonymous (Braune, 1913; Wenyon, 1926;Das Gupta, 1935; Levine, 1961). However, no mention was made by Kudo (1954) of representatives of Monas or Oikomonas occurring in the rumen, and the genera Sphaeromonas and Piromorzas were not included in his treatise. Since the flagellates described herein fit most closely the description by Braune (1913) of Sphaeromonas communis Liebetanz, I have used this nomenclature.Organisms resembling phycomycete fungi free-living in the rumen were first reported by Orpin (r975), who found that the rumen flagellate Neocallimastix frontalis was probably a zoospore of a phycomycete fungus and not a flagellate protozoon as previously believed (Braune, I 9 I 3). The vegetative phase morphologically resembled aquatic Phycomyces. These investigations were prompted by the observation (Warner, 1966) that the flagellate stages of N . frontalis underwent considerable fluctuations in population density over the feeding

SUMMARYThe rumen flagellate Piromonas communis is the zoospore of a phycomycete fungus inhabiting the rumen. Zoosporogenesis was stimulated by a dietary component (the inducer), and inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The zoospores showed taxis towards the tissues surrounding the inflorescence of Lolium perenne L. in the rumen, invading principally the stomata and damaged tissues. The zoospores germinated on this substratum and the rhizoids of the developing vegetative stage penetrated the tissue, taking up 14C from labelled plant material, which was incorporated into the fungal cells. The conditions for maximum flagellate production (39 "C, pH 6.0 to 7.0, high concentration of C 0 2 , absence of O& resembled those found in the rumen. The organism was cultured in an undefined medium in yitro in the absence of other flagellates. I N T R O D U C T I O NTwo species of rumen flagellates, Neocallimastix frontalis and Sphaeromonas communis, were shown to be the zoospores of species of phycomycete fungi (Orpin, 1g75,1976a, 19773). The vegetative phases of these organisms live free in the rumen fluid. Neocallimastix frontalis flagellates may also be attracted to and germinate on plant particles, especially inflorescence tissue, whilst the vegetative phase grows on and takes up carbon from this tissue (Orpin, Liebetanz (1910) described a flagellate resembling S. communis, but larger and more elongate, which he named Piromonas communis. Subsequent references in the literature are rare, but Braune (1913) described the species more fully. Both Liebetanz and Braune considered P. communis to be a species of flagellate protozoon, but Kudo (1954) did not mention the genus Piromonas in his treatise on the protozoa. The species is less common than N. frontalis or S. communis in the sheep at the author's laboratory, but shows similar population density fluctuations in response to the ingestion of food by the host animal to those shown by N. frontalis and S. communis (Orpin, 1974(Orpin, , 1976a. The demonstration that the sporangia of P. communis contain chitin (Orpin, 19773) showed that, like N. frontalis and S. communis, it was also a species of Phycomycete. This paper describes the life-history and morphology of the organism, together with its relationship with the ingested food of the host animal. I977 4 - METHODS Animals.The sheep used were Clun Forest wethers, each fitted with a permanent rumen cannula, and fed I kg hay chaff and IOO g rolled oats once daily. Defamation was by a method modified from that of Abou Akkada et al. (r968), using dioctyl sodium

The dominant rumen bacteria in high-arctic Svalbard reindeer were characterized, their population densities were estimated, and ruminal pH was determined in summer, when food quality and availability are good, and in winter, when they are poor. In summer the total cultured viable population density was (2.09 1.26) x 1010 cells ml-1, whereas in winter it was (0.36 ± 0.29) x 1010 cells ml-', representing a decrease to 17% of the summer population density. On culture, Butyrivibriofibrisolvens represented 22% of the bacterial population in summer and 30% in winter. Streptococcus bovis represented 17% of the bacterial population in summer but only 4% in winter. Methanogenic bacteria were present at 104 cells ml-' in summer and 107 cells mlV1 in winter. In summer and winter, respectively, the proportions of the viable population showing the following activities were as follows: starch utilization, 68 and 63%; fiber digestion, 31 and 74%; cellulolysis, 15 and 35%; xylanolysis, 30 and 58%; proteolysis, 51 and 28%; ureolysis, 40 and 54%; and lactate utilization, 13 and 4%. The principal cellulolytic bacterium was B. fibnisolvens, which represented 66 and 52% of the cellulolytic population in summer and winter, respectively. The results indicate that the microflora of the rumen of Svalbard reindeer is highly effective in fiber digestion and nitrogen metabolism, allowing the animals to survive under the austere nutritional conditions typical of their high-arctic habitat.