Integrative Zoology 2014; 9: 583–589

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doi: 10.1111/1749-4877.12069

ORIGINAL ARTICLE

Influence of dietary feathers on the fecal microbiota in captive Arctic fox: Do dietary hair or feathers play a role in the evolution of carnivorous mammals? Liang ZHANG,1 Shuhui YANG,1 Yanchun XU1,2 and Thomas D. DAHMER3 1

College of Wildlife Resources, Northeast Forestry University, Harbin, Heilongjiang, China, 2State Forestry Administration Detecting Center of Wildlife, Harbin, China and 3Ecosystems Ltd., Aberdeen, Hong Kong, China

Abstract Hair and feathers are composed of keratin and are indigestible, inalimental and unpalatable for carnivores. However, carnivores often ingest hair and feathers during feeding or when grooming. We hypothesized that ingestion of hair and feathers changes species diversity and relative abundance of bacteria in the gut of carnivores. To test this hypothesis, we added disinfected poultry down feathers to the normal diet of captive Arctic foxes (Alopex lagopus). We then used fluorescently labeled terminal restriction fragments (T-RFs) to examine changes in fecal bacterial diversity and abundance. The results showed that the number of bacterial species increased significantly after feather ingestion, but that total abundance was unchanged. This demonstrated that addition of disinfected feathers to the diet stimulated increased production among less abundant bacteria, resulting in a balancing of relative abundance of different bacterial species, or that some newly-ingested microbial species would colonize the gut because a suitable microhabitat had become available. This implies that the overall production of bacterial metabolites would be made up of a greater range of substances after feather ingestion. On one hand, the host’s immune response would be more diverse, increasing the capacity of the immune system to regulate gut microflora. On the other hand, the animal’s physiological performance would also be affected. For wild animals, such altered physiological traits would be subjected to natural selection, and, hence, persistent geographic differences in the character of ingested feathers or fur would drive speciation. Key words: carnivore, diet, evolution, feather, gut microbiota

INTRODUCTION

Correspondence: Yanchun Xu, College of Wildlife Resources, Northeast Forestry University, Harbin, Heilongjiang 150040, China. Email: [email protected]

Carnivores often ingest hair and feather when feeding or grooming. Hair and feathers are composed of keratins. Keratins contain disulfide bridges formed by the sulfur-containing amino acid cysteine, which confer additional strength and rigidity through permanent, thermally-stable crosslinks (Menefee 1977). This gives hair and feather extraordinary thermal and chemical stabili-

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ty and renders them almost indigestible, inalimental and unpalatable to carnivores (Reynolds & Aebischer 1991; Jordan 2005). This raises the question: does the ingestion of hair or feathers have a physiological role in carnivore digestion, or is it simply a passive event? If ingested hair and feathers have physiological functions or influence physiological performance in the gut, they would be related to fitness of carnivores in the natural environment and involved in evolution. To determine whether this is the case, the physiological functions of hair and feathers in the digestive system of carnivores need to be clarified. To date, however, this topic has been poorly studied. It has been proved that hair and feathers are usually retained longer in the stomach and gut than liquid components and play an important role in regulating gastrointestinal motility (Mayer 1994). With their extended retention in the gut it is possible that hair and feathers help to mix different diet components with digestive enzymes through agitation and further improve the digestion of nutrients. It can also be hypothesized that in the large intestine, where there is a complex microbiota (Ley 2008), hair and feathers influence the structure and abundance of the microbiota through agitation and by providing physical complexity, such as sequestration spaces that will favor some microbe species over others (Demeyer & Van Nevel 1986). The gut microbiota is a multifunctional symbiotic system and plays an essential role in the maintenance of host health, including synthesizing B-group vitamins and vitamin K (Albert et al. 1980; Hill 1997), enhancing gut development (Castillo et al. 2007; Willing 2007), protecting the host from colonization by pathogens (Quigley 2006), regulating host metabolism (Karasov & Carey 2009), stimulating development of immunity and post-developmental immunity (Noverr & Huffnagle 2004; Kelly et al. 2007; Hrncir et al. 2008), influencing behavior and central nervous system function (Neufeld & Foster 2009), and even affecting phenotypic properties (Holmes & Nicholson 2005; Li et al. 2008). If hair and feathers in carnivores’ diet influence the gut microbiota, they might also influence physiology, and, therefore, would be involved in the evolution of carnivores. In the present study, we used captive Arctic foxes [Alopex lagopus (Linnaeus, 1758)] as experimental animals to compare the species diversity and abundance of gut microbiota before and after addition of sterilized feathers to their diet.

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MATERIALS AND METHODS Influence of feathers on growth rate of Escherichia coli in vitro Downy poultry feathers were collected from a slaughterhouse and roughly cut into short fragments. The cut feathers were washed and disinfected using 1:1 mixture of 90% ethanol and ether in a large beaker for 20 min with shaking at 60 rpm. Feathers were further disinfected at 120 °C for 45 min and then dried in an oven at 110 °C for 1 h after being rinsed thoroughly using deionized water. Sterilized liquid LB culture medium (Sambrook & Russell 2001) was transferred into 4 flasks, with 100 mL in each. A total of 500 µg of ampicillin (Sigma, USA) and approximately 105 ATCC 25922 Escherichia coli (Migula 1895) Castellani and Chalmers 1919 carrying pMD18-T vector (Takara, Dalian, China) containing an ampicillin resistant gene was added to each flask. Then, 1.5, 3.0 and 4.5 g of feathers were added to 3 of the flasks separately, and the last flask was a negative control. Flasks were moved into a closed shaking bed to culture bacteria at 37 °C and 120 rpm. Using a DU640 Spectrophotometer (Beckman-Coulter, USA), the OD600 value of the culture medium was measured as an indicator of bacteria density every 30 min until the value reached a plateau. The growth of E. coli was demonstrated using the plotting method and growth rates of the 4 treatments were compared using the plot trends.

Influence of feathers on fecal microbiota in vivo A total of 15 male 140-day-old Arctic foxes were selected from a fur farm in Harbin, Heilongjiang Province, China. All animals were supplied with commercial Arctic fox feed (Hualong Feed, China) twice daily (75 g dry matter at each feeding). Water was supplied ad libitum. Whole fresh feces defecated in the morning were collected on 2 successive days for each fox. A 2.0 g sample of fresh feces was transferred from droppings to a 10 mL sterilized tube, sealed and chilled on dry ice until transported to the laboratory. From the third day, 1.5 g of sterilized downy feathers prepared as above was added to the same diet and supplied to the foxes twice a day for 5 days. Sampling and storage of morning feces continued until the end of the experiment. The use of the animals was approved by the Academic and Ethics Committee of the Northeast Forestry University. A subsample weighing 200 mg was transferred from each fresh fecal sample into a 2 mL centrifuge tube and placed on ice. Total DNA was isolated and purified using a QIAamp DNA Stool Mini Kit (QIAGEN,

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USA) according to the user’s manual. DNA was resolved in 200 μL TE buffer (pH = 8.0). A pair of primers developed by Nagashima et al. (2003) was used to amplify the 16S rRNA gene of fecal bacteria: 516f: 5′FAM-TGCCAGCAGCCGCGGTA-3′; 1510r: 5′-GGTTACCTTGTTACGACTT-3′. Primer 516f was labeled using 6-FAM (Sangon Biotech, Shanghai, China) at the 5′ end. A polymerase chain reaction (PCR) was set up in a 20 μL system containing 1× PCR Buffer (Mg2+ plus), each of 4 dNTPs 0.2 mM (Qiagen, Tokyo, Japan), each of primers 0.2 μM, HotStarTaq DNA polymerase 1.0 U (Qiagen, Tokyo, Japan) and DNA template 1.0 μL. Amplification was performed on a ABI9700 Thermal Cycler (Applied Biosystems, USA) using a cycling program containing a denaturation at 95 °C for 15 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min, followed by an extra extension at 72 °C for 10 min. All reactions were repeated 3 times, with the position of each sample on the thermal cycle changed randomly each time to reduce the influence of unequal amplification efficiency due to random factors. Polymerase chain reaction products of the same sample were incorporated into one centrifuge tube, concentrated to approximately 15 μL using a Savant DNA120 SpeedVac concentrator (Thermo Fisher Scientific, USA), and isolated through electrophoresis on 2% agarose gel. Target bands were excised and recovered using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). In this procedure, the target bands were completely contained in the gel block, although the volume of gel block was minimized. The final volume of dilution was 30 μL. The PCR products were subjected to 2 enzymatic digestions. The first digestion was performed using Rsa I (recognition site 5′-GT|AC-3′) plus Bfa I (5′-C|TAG-3′) in a 20 μL system containing 2.5 U each of Rsa I (New England BioLabs, USA) and Bfa I (New England BioLabs, USA), 1× NEB buffer 4 (New England BioLabs) and 10 μL of the purified PCR product, and executed at 37 °C for 3 h. The second digestion was performed using Bsl I (5′-CCNNNNN|NNGG-3′) only in a 20 μL system containing 2.5 U Bsl I (New England BioLabs, USA), 1× NEB buffer 3 (New England BioLabs) and 10 μL of the purified PCR product, executed at 55 °C for 3 h. The fluorescently labeled terminal restriction fragments (T-RF) were analyzed using capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA) in GeneScan mode. 2.0 μL of each

digestion product was mixed with 0.5 μL of fourfold-diluted GeneScan-2500 size standard (Applied Biosystems, USA) and 12.0 µL of deionized formamide, followed by denaturation at 96 °C for 2 min and immediate chilling on ice. The injection time was 20 s for analysis of T-RFs from the digestion with Rsa I plus Bfa I and 40 s for that of T-RFs from the digestion with Bsl I. The run time was 40 min. The area of signal peaks 20 pix higher than the baseline was measured using GeneMapper V3.2 for each T-RF. Bacterial species in vivo exhibit abundant single nucleotide polymorphisms in the 16S rRNA gene. Digestion by restriction enzymes can generate different profiles for different bacterial species as represented by T-RFs (Nagashima et al. 2003). For this reason we used the number of T-RFs as the indicator of species diversity of fecal bacteria. The numbers of T-RFs occurring in each individual before (Nbef) and after (Naft) feather feeding were counted. The ratio of T-RF numbers after and before feather feeding (RNaft/Nbef) was calculated as an indicator of alteration of species diversity in the fecal microbiota. The total areas of all T-RFs signal peaks before (Abef) and after (Aaft) feather feeding were calculated for each individual. The ratio of total peak area after and before feather feeding (RAaft/Abef) was calculated as an indicator of alteration of species abundance in the fecal microbiota. Significance was tested using a paired t-test with α = 0.05 executed by SPSS 13.0 statistical software (SPSS, Chicago, IL, USA).

RESULTS Influence of feathers on growth rate of Escherichia coli in vitro The E. coli growth in all 4 flasks fit the logistic model, with R2 between 0.891 and 0.959. The growth rates for the flasks containing different doses of feathers were all significantly faster than the negative control (Fig. 1). The growth rate of E. coli was roughly correlated to feather quantity between 0 and 3.0 g of feathers and reached a plateau between feather quantities of 3.0 and 4.5 g.

Influence of feathers on fecal microbiota in vivo Bacterial diversity before and after ingestion of feathers Throughout the experiment, foxes ate all of the feed supplied every day. The addition of feathers to the diet did not impact daily food intake. For combination digestion using Rsa I and Bfa I, Nbef ranged from 5 to 26

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Figure 1 In vitro abundance of Escherichia coli at time intervals after addition of feathers.

in the 15 Arctic foxes, with an average of 13.7 T-RFs. In contrast, Naft ranged from 10 to 62, averaging 24.7 T-RFs. The ratio of T-RF numbers after versus before feather ingestion (RNaft/Nbef) indicated that 92.9% of foxes exhibited elevated bacterial species diversity (RNaft/Nbef > 1.00), 7.1% of foxes showed stable species diversity (RNaft/Nbef ≈ 1.00), and no fox showed reduced species diversity (RNaft/Nbef < 1.00) (Fig. 2a). The elevation of species diversity was significant (t = −3.454, P = 0.004). For digestion using Bsl I, Nbef ranged from 8 to 31, averaging 14.3. Naft ranged from 14 to 63, averaging 26.6. After ingestion of feathers, elevated species diversity was seen in 84.6% of foxes (RNaft/Nbef > 1.00), 15.4% of foxes showed stable species diversity (RNaft/Nbef ≈ 1.00) and no fox showed reduced species diversity (RNaft/Nbef 1.00) occurred in 46.7% of foxes, 33.3% of foxes showed reduced bacteria abundance (Aaft/bef < 1.00) and 20.0% showed stable bacteria abundance (Aaft/bef ≈

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DISCUSSION

Figure 3 Ratio of mean total peak area after and before ingestion of feathers. (a) Terminal restriction fragments (T-RFs) derived from combination digestion of PCR products using Rsa I and Bfa I. (b) T-RFs derived from digestion of PCR products using Bsl I.

1.00) (Fig. 3a). For digestion using Bsl I, the mean A bef ranged from 7070.5 pix 2 to 98 013.0 pix 2, averaging 31 975.6 pix2. The mean Aaft ranged from 9699.2 pix 2 to 9 8848.7 pix2, with an average 32 150.2 pix2. Elevated bacteria abundance (Aaft/bef > 1.00) was shown by 53.9% of foxes, 38.5% of foxes showed reduced abundance (Aaft/bef < 1.00) and 7.7% showed stable abundance (Aaft/ bef ≈ 1.00) (Fig. 3b). The abundance of bacteria after ingesting feathers did not change as determined by either combination digestion of Rsa I and Bfa I (t = 0.152, P = 0.882) or single digestion of Bsl I (t = 0.016, P = 0.987).

Feathers of birds and fur of mammals are made of keratin. Keratin is highly resistant to decomposition. Even after ingestion by carnivores, hair and feathers of prey remain almost completely undigested (Ogara et al. 2010). This suggests that hair and feathers do not play a biochemical role but might simply play a physical or mechanical role when they pass through the digestive system. The experiment in vitro demonstrated the positive correlation between feather quantity and abundance of E. coli. When the weight of feathers exceeded the capacity of the cultural system (4.5 g in this experiment), agitation was restricted due to the limitation of space. A similar situation might occur in the digestive systems of carnivores, in which ingestion of moderate amounts of feathers would stimulate the growth of bacteria. The experiment in vivo proved the implication of the in vitro experiment. We observed elevated species diversity of fecal bacteria after feather ingestion (Fig. 2). The feathers used in this experiment were disinfected at 120 °C for 45 min and then dried at 110 °C for 1 h after being rinsed thoroughly using deionized water. This eliminated the possibility that the feathers themselves were the source of novel non-spore-forming bacterial species in the feces. The explanation for elevated bacterial species diversity after ingesting feathers is the change in relative abundance among intrinsic bacteria species. Some bacteria that occurred at low abundance before ingestion of feathers were not detectable using PCR methods. When feathers entered the gut they presumably altered the structure of the ecosystem with the result that the numbers of (some of) these formerly scarce bacteria were elevated to levels at which they were detectable, or some newly-ingested microbial species would colonize the gut because a suitable microhabitat had become available as a consequence of the ingestion of the feather material. This increase in species diversity might result in greater stability of the gut microbiota (higher resistance to perturbations and quicker return to equilibrium after a perturbation), as shown by DeAngelis (1980). Animal fiber in the diet of carnivores, including hair and feathers, affects the production of metabolites of gut bacteria that influence carnivore physiology (Depauw et al. 2011). The present study showed that such effects are the consequence of alteration of the abundance of bacterial species because of the influence of animal fiber. The overall abundance of fecal microbiota did not

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significantly change in response to ingestion of feathers. This might be because the overall abundance of bacteria before ingestion of feathers has reached the maximum capacity of the gut. Anyway, it suggests that overall production of bacterial metabolites is unaffected by ingestion of feathers. However, the increased diversity of bacteria after feather ingestion, regardless of whether it is due to a change in the balance of abundance or to colonization by newly-ingested microbial species. This implies that the overall suite of bacterial metabolites would be made up of a greater range of substances. On one hand, the host’s immune response would be more diverse, increasing the capacity of the immune system to regulate gut microflora (Duerkop et al. 2009). On the other hand, physiological performance would also be affected. The present study used disinfected feathers in order to remove the influence of bacteria inhabiting the feather. In the wild, bacteria are ingested by carnivores together with the feathers that they inhabit, so the changing pattern of gut microbiota as well as the output of metabolites production would be even more complex. The resulting altered profile of bacterial metabolites would influence the physiological performance of hosts, so that physiological traits are subject to natural selection. This process would promote the localization of animals in those traits, leading toward speciation.

ACKNOWLEDGMENTS This study was supported by the Program for New Century Excellent Talents in the University of China (NCET-10-0280) and the Program of Wildlife Conservation and Breeding of the State Forestry Administration of China (2008). We are very grateful to Mr Xing Yuan Miao and his colleagues of Heilongjiang Hualong Feed, China for their help in providing experimental animals and feed, and aiding in sample collection.

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Influence of dietary feathers on the fecal microbiota in captive Arctic fox: do dietary hair or feathers play a role in the evolution of carnivorous mammals?

Hair and feathers are composed of keratin and are indigestible, inalimental and unpalatable for carnivores. However, carnivores often ingest hair and ...
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