научная статья по теме DIVERSITY OF DIAZOTROPHIC GUT INHABITANTS OF PIKAS(OCHOTONIDAE) REVEALED BY PCR-DGGE ANALYSIS Биология

Текст научной статьи на тему «DIVERSITY OF DIAZOTROPHIC GUT INHABITANTS OF PIKAS(OCHOTONIDAE) REVEALED BY PCR-DGGE ANALYSIS»

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

УДК 579.266.1

DIVERSITY OF DIAZOTROPHIC GUT INHABITANTS OF PIKAS(Ochotonidae) REVEALED BY PCR-DGGE ANALYSIS

© 2014 A. K. Kizilova1 and I. K. Kravchenko

Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia

Received March 18, 2013

Abstract—Diazotrophic gut symbionts are considered to act as nitrogen providers for their hosts, as was shown for various termite species. Although the diet of lagomorphs, like pikas or rabbits, is very poor in nitrogen and energy, their fecal matter contains 30—40% of protein. Since our hypothesis was that pikas maintained a diazotrophic consortium in their gastrointestinal tract, we conducted the first investigation of microbial diversity in pika guts. We obtained gut samples from animals of several Ochotona species, O. hyperborea (Northern pika), O. mantchurica (Manchurian pika), and O. dauurica (Daurian pika), in order to retrieve and compare the nitrogen-fixing communities of different pika species. The age and gender of the animals were taken into consideration. We amplified 320-bp long fragments of the nifH gene using the DNA extracted directly from the colon and cecum samples of pika's gut, resolved them by DGGE, and performed phylogenetic reconstruction of 51 sequences obtained from excised bands. No significant difference was detected between the nitrogen-fixing gut inhabitants of different pika species. NifH sequences fell into two clusters. The first cluster contained the sequences affiliated with NifH Cluster I (Zehr et al., 2003) with similarity to Sphin-gomonas sp., Bradyrhizobium sp., and various uncultured bacteria from soil and rhizosphere. Sequences from the second group were related to Treponema sp., Fibrobacter succinogenes, and uncultured clones from the guts of various termites and belonged to NifH Cluster III. We suggest that diazotrophic organisms from the second cluster are genuine endosymbionts of pikas and provide nitrogen for further synthesis processes thus allowing these animals not to be short of protein.

Keywords: nifH, nitrogen fixation, pikas, Ochotona, gut microflora.

DOI: 10.7868/S0026365614010066

Animals have various food preferences, and many of them are herbivores, obtaining energy and nutrients from plant material. Two aspects of herbivore diet have always been of interest: cellulose digestion, since herbivores do not have enzymes for cellulose breakdown [1], and protein synthesis, since plants are low in proteins. Ruminants, such as cattle, rely on symbiotic microflora for cellulose digestion and utilize microbial protein synthesized in a special part of their four-chambered stomach called rumen [2]. Protein synthesis in the rumen is a complex process which involves organisms belonging to Bacteria, Protozoa and Fungi [3]. During this process, dietary protein and non-protein nitrogen are converted to ammonia and amino acids by microbes, which use these compounds to synthesize bacterial protein, partly transformed by ruminants into the body tissue protein and partly excreted with faeces [4].

Termites feed on various substrates, including wood, which is as much as 50 per cent cellulose [5], soil [6] and plant litter, as well as fungi cultivated in fungal gardens for further consumption. Cellulose

1 Corresponding author. E-mail: alegrria@gmail.com

consumed by the termites is usually broken by either cellulolytic protozoa, or by endogenous cellulases [7]. Symbiotic microflora of the termites provides them with nitrogen by means of nitrogen fixation, since termites' diet is usually very poor in this essential nutrient [8]. Diazotrophic symbionts of the termites are quite diverse and mostly belong to Bacteria [9] and metha-nogenic Archaea [10].

Mammalian herbivores face several challenges when consuming plant material as a primary food source. An order of mammals, known as Lagomorpha and comprising hares, rabbits, and pikas, combines the feeding mechanisms of ruminants and termites. Lagomorphs feed on grass and tree branches, but they have no rumen for fermentation purposes. Representatives of all families of Lagomorpha were shown to get protein into diet by means of caecotrophy, a form of co-prophagy [11]. Lagomorphs have two types of faeces — solid faeces containing undigested food and soft faeces, rich in bacterial protein [12]. These mammals consume soft faeces, thus supplying their bodies with protein. The dry substance of soft faeces of hares was shown to contain up to 39% of protein [13]; for rabbits this value was lower — up to 32%. The estimated pro-

Table 1. Sequences of primers used in this study

Primer Primer sequence (5'-3') Target microbes Reference

F1 TAYGGIAARGGIGGIATIGGIAARTC N-fix Bacteria and Archea [37]

R6 GCCATCATYTCICCIG N-fix Bacteria and Archea [37]

nifHfor TAYGGNAARGGNGGHATYGGYATC N-fix Bacteria [38]

nifHrev ATRTTRTTNGCNGCRTAVABBGCCATCAT N-fix Bacteria [38]

nifH-f GGHAARGGHGGHATHGGNAARTC N-fix Bacteria [39]

nifH-r GGCATNGCRAANCCVCCRCANAC N-fix Bacteria [39]

PolF TGCGAYCCSAARGCBGACTC N-fix Bacteria [17]

PolR ATSGCCATCATYTCRCCGGA N-fix Bacteria [17]

PolFI TGCGAICCSAAIGCIGACTC N-fix Bacteria [18]

AGER- GC30 CGCCCGCCGCGCCCCGCGCCCGGCCCG CCCGACGATGTAGATYTCCTG N-fix Bacteria [18]

nifH-F AAAGGYGGWATCGGYAARTCC ACC AC N-fix Bacteria [40]

nifH-R TTGTTSGCSGCRTACATSGCCATCAT N-fix Bacteria [40]

Modied bases: I = Inosine, Y = CT, S = CG, R = AG, B = GCT, W = AT; N = ACGT; H = ACT.

tein content in the food of these animals is between 6 and 13%. Pikas appear to be much more efficient digesters of low-nutrient and low-protein food. They eat legumes and tree branches, which are 2—14 times less rich in protein than their soft faeces that contained 41-48% of protein [13].

Since the staple food of pikas is extremely poor in nitrogen, these animals probably obtain additional nitrogen by ways similar to those of the termites, i.e. via the symbiotic nitrogen fixation. Although the idea of symbionts involved in nitrogen utilization by herbivorous mammals has appeared earlier in the literature [14], nothing is currently known about the organisms inhabiting pika's gut and fixing nitrogen for further utilization of this essential nutrient for protein synthesis. In this study, we concentrated on the diversity of the nifH gene encoding nitrogenase reductase found in the intestines of several species of Ochotonidae family — Ochotona hyperborea, O. mantchurica, and O. dauuri-ca. NifH is the marker gene which encodes a component of the evolutionary conserved key enzyme, responsible for the process of atmospheric nitrogen fixation. It is widely used for assessing the diversity of nitrogen-fixing communities in various habitats [15].

MATERIALS AND METHODS

Sample preparation. All gut samples were obtained at the Faculty of Biology of the Lomonosov Moscow State University. Two individuals of northern pikas Ochotona hyperborea, young male and female were collected during field work of 2009 and 2010 in Za-baykalsky Krai, Transbaikalia, Russia. One Manchuri-an pika Ochotona mantchurica scorodumovi [16] also originated from Transbaikalia. The gender of this pika was unknown. Two Daurian pikas Ochotona dauurica (a very young male and a sub-adult female) arrived to

MSU after fieldwork of 2010 in the Daursky State Natural Biosphere Reserve Park, Siberia, located between two large lakes, Barun-Torey and Zun-Torey. For each animal, except for Manchurian pika, samples for analysis were taken from back end of the ce-cum, initial portion of the colon immediately distal to the cecum, and the distal colon, and tissue samples were stored in ethanol-containing buffer prior to analysis. For Manchurian pika, only the cecum sample was obtained. Food remains were not removed from the guts before DNA extraction.

DNA extraction and PCR amplification. DNA was

extracted from the samples of pika intestines in 2010 and 2011 by means of Power Soil DNA Extraction Kit (MO BIO, United States) according to the manufacturer's recommendations with an obligate bead-beating step. Extracted DNA was used for PCR amplification of nifH gene. PCR was run in triplicates, and the products were pooled prior to further analysis. We tested several primer systems for nifH gene amplification, running PCR according to the authors' recommendations (Table 1), until obtaining a PCR product of expected size with PolF-PolR primers [17]. We used nested PCR approach, when a product of the first amplification round was used as a template for the second amplification round with primers Pol FI/AQER-GC30 [18]. Bradyrhizobium japonicum was chosen as a positive control strain for nifH amplification for both rounds of PCR. The primer AQER-GC30 had a GC-clamp attached to its 5' end for further separation of amplification products by means of denaturing gradient gel electrophoresis (DGGE) [19]. The PCR mixture for both amplification steps contained 1x PCR buffer (17 mM (NH4)SO4, 67 mM Tris-HCl, pH 8.8, 2 mM MgCl2, Helicon, Russia), 0.5 mM of each dNTP, 25 pmol of the forward and reverse primers,

Table 2. Description of samples resolved on DGGE lanes

Lane ID Organism, gender Age, sampling year Part of gut

om Manchurian pika, unknown Unknown, 2009 Cecum

oh1 Northern pika, male Juvenile, 2009 Cecum

oh2 Northern pika, male Juvenile, 2009 Initial colon

oh3 Northern pika, male Juvenile, 2009 Distal colon

oh4 Northern pika, female Sub-adult, 2010 Cecum

oh5 Northern pika, female Sub-adult, 2010 Initial colon

oh6 Northern pika, female Sub-adult, 2010 Distal colon

od1 Daurian pika, male Juvenile, 2010 Cecum

od2 Daurian pika, male Juvenile, 2010 Initial colon

od3 Daurian pika, male Juvenile, 2010 Distal colon

od4 Daurian pika, female Sub-adult, 2010 Cecum

od5 Daurian pika, female Sub-adult, 2010 Initial colon

od6 Daurian pika, female Sub-adult, 2010 Distal colon

1 ^L of DNA template, 20 mg of bovine serum albumin (Fermentas, Canada), 5% of dimethyl sulfoxide (DMSO) and 1.25 U of Taq DNA polymerase (Helicon, Russia). The temperature

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