Characterization of N2O emission and associated ...

Characterization of N2O emission and associated bacterial communities from the gut of wood-feeding termite Nasutitermes voeltzkowi Muhammad Zeeshan Majeed, Edouard Miambi, Muhammad Asam Riaz & Alain Brauman Folia Microbiologica Official Journal of the Institute of Microbiology, Academy of Sciences of the Czech Republic and Czechoslavak Society for Microbiology ISSN 0015-5632 Folia Microbiol DOI 10.1007/s12223-015-0379-x

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Author's personal copy Folia Microbiol DOI 10.1007/s12223-015-0379-x

Characterization of N2O emission and associated bacterial communities from the gut of wood-feeding termite Nasutitermes voeltzkowi Muhammad Zeeshan Majeed & Edouard Miambi & Muhammad Asam Riaz & Alain Brauman

Received: 9 June 2014 / Accepted: 6 February 2015 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015

Abstract Xylophagous termites rely on nitrogen deficient foodstuff with a low C/N ratio. Most research work has focused on nitrogen fixation in termites highlighting important inflow and assimilation of atmospheric nitrogen into their bodies fundamentally geared up by their intestinal microbial symbionts. Most of termite body nitrogen is of atmospheric origin, and microbially aided nitrification is the principal source of this nitrogen acquisition, but contrarily, the information regarding potent denitrification process is very scarce and poorly known, although the termite gut is considered to carry all favorable criteria necessary for microbial denitrification. Therefore, in this study, it is hypothesized that whether nitrification and denitrification processes coexist in intestinal milieu of xylophagous termites or not, and if yes, then is there any link between the denitrification product, i.e., N2O and nitrogen content of the food substrate, and moreover where these bacterial communities are found along the length of termite gut. To answer these questions, we measured in vivo N2O emission by Nasutitermes voeltzkowi (Nasutitermitinae) maintained on different substrates with varying C/N ratio, and also, molecular techniques were applied to study the diversity (DGGE) and density (qPCR) of bacterial communities in anterior and posterior gut portions. Rersults revealed that M. Z. Majeed (*) : M. A. Riaz Department of Agri. Entomology, University College of Agriculture, University of Sargodha, 40100 Sargodha, Pakistan e-mail: [email protected] M. Z. Majeed e-mail: [email protected] E. Miambi UMR BioEMCo (IBIOS), Université Paris Est, 94000 Créteil, France A. Brauman Institut de Recherche pour le Développement (IRD), UMR Eco&Sols, 34000 Montpellier, France

xylophagous termites emit feeble amount of N2O and molecular studies confirmed this finding by illustrating the presence of an ample density of N2O-reductase (nosZ) gene in the intestinal tract of these termites. Furthermore, intestinal bacterial communities of these termites were found more dense and diverse in posterior than anterior portion of the gut.

Introduction With about 3000 described species (Eggleton 2011), termites are important eusocial macrofauna found in tropical and subtropical ecosystems of the world, particularly concentrated in forests, savannas, and grasslands with a density factor of 100 kg/m2 (Sanderson 1996). They are often called as the ecological engineers for the reason that they directly or indirectly modulate the availability of resources to other species, by causing physical state changes in biotic or abiotic materials (Jones et al. 1994). These invertebrates nourish on diverse food sources ranging from wood, plant debris, leaf litter, roots to highly humidified soil components. They accommodate a highly dense (108/mL bacteria in gut lumen) and diversified (~300 different phylotypes) microbial communities in their guts (Breznak 2000; Warnecke et al. 2007; Mrázek et al. 2008; Brune 2014). This intestinal assemblage of digestive microbiota of termites reflects an excellent example of symbiotic association between microbial communities and food diversification (Brauman et al. 2001; Miyata et al. 2007). Wood-feeding xylophagous termites (Nasutitermitinae) are capable of subsisting on food aliments essentially deficient in nitrogen contents (Tayasu et al. 1994). A number of studies have shown that wood-feeding termites, consuming wood at different stages of decomposition, normally fix atmospheric

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nitrogen with the help of facultative anaerobic bacteria inhabiting in their guts (Benemann 1973; Breznak et al. 1973). Xylophagous termites emit various trace gases during their metabolic activities with the help of symbiotic microbes of their digestive tracts (Khalil et al. 1990; EPA 2010). Many of these gases such as CH4, CO2, and N2O cause global warming, hence contribute considerably to global budget of these greenhouse gases (Sanderson 1996; Bignell and Eggleton 2000). Carbon dioxide is the product of wood digestion by the termites resulting from aerobic metabolism while an anaerobic metabolism is characterized by the emission of methane which is a typical product of intestinal fermentation process (Brauman et al. 1992; Brune 2014). The estimates of termites’ contribution of CH4 and CO2 to global budget vary, respectively, from negligible to 15 % and 0.5 to 2.25 % (Khalil et al. 1990; Brauman et al. 1992; Sanderson 1996; EPA 2010). However, few studies have been carried out on the emissions of nitrous oxide (N2O), another important greenhouse gas, from termites and most of previous work regarding N2O has focused on soil-feeding termites (Brümmer et al. 2009; Ngugi and Brune 2012). For wood-feeding termites, some studies have characterized nitrifying bacterial communities (Ohkuma et al. 1999; Yamada et al. 2007; Warnecke et al. 2007), but contrarily, there is a lack of work concerning the study of functional anaerobic communities like denitrifying bacteria involved in the termite metabolic activities. N2O, a very important intermediate of denitrification process, is the third major greenhouse gas after CH4 and CO2. Its annual global warming potential is 310 times higher than CO2, and it represents about 9 % of all greenhouse gas emissions (IPCC 2007). Among natural N2O emission sources, primarily, it comes from tropical soils (Breuer et al. 2000; Mosier et al. 2004; EPA 2010). Termites are the major soil macrofauna in these tropical soils (Eggleton et al. 1994; Bignell and Eggleton 2000). In fact, nitrification and denitrification in soils are the major mechanisms involved in the emissions of N2O (Webster and Hopkins 1996), and the process of denitrification is essentially fundamental for the production of N2O via anaerobic reduction of nitrates to N2O and N2 (Braker and Conrad 2011). Nevertheless, the proportion of N2O liberated by denitrification depends on degree of anaerobiosis and on the nitrogen contents and pH of the milieu (Skiba and Smith 2000). Termite gut exhibits certain criteria, such as labile C products, ammonia, and anoxia, favorable for nitrifying and denitrifying microbial activities and, hence, for N2O production (Brune 1998; Schmitt-Wagner and Brune 1999; Brune 2014). In this study, we postulated that the processes of nitrification and denitrification coexist in the gut of xylophagous termites because as demonstrated by microelectrode techniques, the termite gut lumen has been demonstrated an anoxic environment surrounded by a microoxic periphery with a steep gradient of O2 and H2 (Brune et al. 1995; Brune 1998;

Schmitt-Wagner and Brune 1999). The second hypothesis of work was that if there exist the denitrification process inside termite’s gut, the production of N2O would be dependent on the value of nitrogen contents of substrates and also that these microbial processes would occur in different gut compartments. In order to testify these hypotheses, in vivo N2O emission was investigated from xylophagous termite species, Nasutitermes voeltzkowi and the effect of C/N ratio of different food substrates on termites N2O emission dynamics and their gut microbial community was also determined. Furthermore, the structure and density of microbial communities responsible for the last step of denitrification, i.e., N2O-reduction (nosZ), were also determined in different termite gut compartments.

Materials and methods Wood-feeding species, N. voeltzkowi Wasmann (Termitidae; Nasutitermitinae; Isoptera) was selected as model specimen. Colony of this termite species along with its intact mound was collected from a wet forest of Mauritius Island and was maintained for several years in the rearing laboratory (UMR BIOEMCO-IBIOS) of Institute of Research for the Development (IRD), Bondy, France, on damp wood logs under seminatural conditions (27±2 °C and 80 % relative humidity). Only healthy active worker termites were used for experiments. Experiment I: impact of food substrate on termite N2O emission Two experiments were performed in laboratory using microcosms. First was done in order to determine the effect of different substrates with differential C/N ratio on termite N2O emissions. Hundred termite workers of N. voeltzkowi, with an average weight of 0.534 g, were maintained in 40-mL sterilized serum vials on four different substrates viz. dry wood (Pinus spp.), dry acacia leaves, shredded filter paper (Whatman no. 2), and dry grass (Dactylis spp.). Five replications for each treatment were performed. Five-gram sterilized sand and 1.0 mL distilled water were added in each sterilized flask in order to maintain suitable microcosm condition for termites. Quantities of substrates used were 1.0, 0.30, 0.75, and 0.48 g for dry wood, grass, acacia, and filter paper, respectively. Flasks were sealed hermetically with rubber septa and aluminum seals just after the release of termites on substrates. Every 24 h, flasks were opened for about 5 min to ensure the fresh oxygen supply to termites without removing dead termite individuals. Samples for N2O gas analysis were taken out on 9th day of experiment assuming that termites and their digestive symbionts were well adapted on the substrates.

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Experiment II: dynamics of termite N2O emission on wood

DNA extraction

In the second feeding experiment, 100 worker termites of N. voeltzkowi, weighing about 0.534 g, were maintained on approximately 0.22 g of dry wood chips along with two different types of controls, only termites and only wood. There were five replications for each treatment. However, this time, 0.5 mL of distilled water and 3 g of sterilized sand were put in the microcosms, respectively, keeping in view the observations of previous feeding experiment in which high humidity accelerated termite mortality because of mite attack especially in the last couple of days. Moreover, normalized percentage of substrate consumption and termite mortality were calculated at the end of both experiments to find out consumption efficiency and termite survival on different substrates.

Total genomic DNA was extracted from termite guts by utilizing FastDNA® SPIN Kit (MP Biomedical, CA, USA) according to manufacturer’s protocol. Extractions were performed in triplicate for each treatment. After extraction, DNA was verified by electrophoresis using 1.5 % agarose gel and further, quantified using PicoGreen® (Molecular Probes, Inc., Eugene, Oregon, USA). The extracted DNA was stored at −20 °C for downstream analysis.

N2O measurement At the end of 9th day incubation in case of first feeding experiment and on the 2nd, 4th, and 7th day of second experiment, microcosm headspace gas samples (of 6 mL each) were drawn out with the help of a hypodermic luer-lock syringe and were transferred into 5.9-mL prevacuumed sterilized Exetainer® vials (Labco Ltd, High Wycombe, England). Gas samples were analyzed for N2O in the laboratory of Centre d’Ecologie Fonctionnelle et Evolutive (CEFE, Montpellier, France) with the help of Chromatograph CP-38000 VARIAN® STAR. This chromatograph actually contained 90 % argon and 10 % methane as vector gases and works on the principle of gas electronic affinity using 63Ni (15 mCi) as electron capturing detector (ECD). Measurement was carried out after calibration of chromatograph with air and standard N2O gas (N48, Air Liquide, France) with 59.2-ppm molar concentration. A volume of 0.2 mL was injected into chromatograph by 1.0-mL hypodermic luer-lock syringe. STAR™ Workstation Version 6.0 was employed for monitoring and processing of the chromatographic curves. Microbial community studies Dissection of termite gut Twenty termite guts per replication were dissected out under a binocular microscope with fine-tipped sterilized forceps. First, whole gut was dragged out of termite’s abdomen from posterior side on a sterilized glass slide, and then, the gut was carefully separated into anterior and posterior portions by cutting it at the junction of malpighian tubules with the tip of sterilized forceps, and both portions were separately preserved in 95 % alcohol at −20 °C until downstream molecular analysis.

PCR assay for total bacterial community of termite guts by targeting 16S rDNA The amplification of genomic DNA extracted out from different parts of wood-feeding termite guts was performed by polymerase chain reaction (PCR). During this reaction, V3 region (a hypervariable nonspecific gene region; Chakravorty et al. 2007) of 16S ribosomal DNA was amplified utilizing the primers GC338 F and 518 R (Table 1). The ready-made BIllustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) were used in 25 μL of reaction mixture containing 1.25 μL of each 0.00001 mol/L primer. The dNTPs, Taq polymerase, MgCl2, and buffer were in the form of a single lyophilized bead in a small tube in which 23 μL of primer solutions and filtered sterilized MiliQ water and after that 2 μL of sample DNA (10 ng/μL) were added. The amplification was done in thermo-cycler (Mastercycler Epgradient; Eppendorf, Germany) using thermal protocol as detailed in Table 2.

Quantitative PCR assay for nosZ and 16S rDNA bacterial community The primers nosZ 1840 F (2 F) and nosZ 2090R (2R) were used for quantifying nosZ gene encoding nitrous oxide reductase, while primers 341 F and 515 R were utilized to quantify 16S rDNA gene (Table 1). For qPCR reaction, 10 μL of final assay mixture used for this real-time PCR contained 5-μL SYBR green PCR Master Mix (QuantiTect™ SYBR green PCR kit; QIAGEN, France), 1 μL of each 0.00001 mol/L nosZ or 16S rDNA primer, 2 μL of filter-sterilized MiliQ water, and 1 μL of template DNA (10 ng/μL). Standard samples were prepared from linearized plasmid serial dilutions containing between 107 and 102 nosZ copies per μL for nosZ qPCR while the standard dilution range was 107 to 103 for 16S rDNA qPCR. Thermocycler CFX96® Real-Time PCR Sequence Detection System (BioRad, Hercules, California) was used for thermal cycling, fluorescent data collection, and data analysis according to the thermal protocol used by Henry et al. 2006 (Table 2).

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List of different primer sequences used in PCR assays

Sr. Primer name no.

Sequence (written 5′ to 3′)

Target gene

Reference

1.

518R

ATT-ACC-GCG-GCT-GCT-GG

16S ribosomal DNA

Muyzer et al. 1993

2.

GC338F

16S ribosomal DNA

Muyzer et al. 1993

3.

amoA-1 F-GC

4.

amoA-2R

CGC-CCG-CCG-CGC-GCG-GCG-GGC-GGG-GCG-GGG-GCACGG-GGG-GAC-TCC-TAC-GGG-AGG-CAG-CAG CGC-CGC-GCG-GCG-GGC-GGG-GCG-GGG-GCG-GGG-TTTCTA-CTG-GTG-GT CCC-CTC-KGS-AAA-GCC-TTC-TTC

ammonium monooxygenase Avrahami et al. 2002 ammonium monooxygenase Avrahami et al. 2002

5.

nosZ 1840 F (2 F) CGC-(A,G)AC-GGC-AA(C,G)-AAG-GT(C,G)-(A,C) (C,G)(C,G)-GT nitrous oxide reductase

6.

nosZ 2090R (2R)

CA(G,T)-(A,G)TG-CA(G,T)-(C,G)GC-(A,G)TG-GCA-GAA

nitrous oxide reductase

Henry et al. 2006

7.

nosZ-F

CG(C,T)-TGT-TC(A,C)-TCG-ACA-GCC-AG


Throback et al. 2004

8.

nosZ 1622R

CGC-(G,A)A(C,G)-GGC-AA(G,C)-AAG-GT(G,C)-CG



9.

nirK F1aCu

ATC-ATG-GT(C,G)-CTG-CCG-CG

Nitrite reductase


GGC-GGC-GCG-CCG-CCC-GCC-CCG-CCC-CCG-TCG-CCCGCC-TCG-ATC-AGR-TTG-TGG-TT GT(C,G)-AAC-GT(C,G)-AAG-GA(A,G)-AC(C,G)-GG

Nitrite reductase

This study

Nitrite reductase


Nitrite reductase

This study

13. 341 F

GGC-GGC-GCG-CCG-CCC-GCC-CCG-CCC-CCG-TCG-CCCGAS-TTC-GGR-TGS-GTC-TTG-A CCT-ACG-GGA-GGC-AGC-AG

16S ribosomal DNA

Henry et al. 2006

14. 515R

ATT-ACC-GCG-GCT6GCT-GGC-A

16S ribosomal DNA

Henry et al. 2006

10. nirK R3cu-GC 11. nirS cd 3aF 12. nirS R 3 cd-GC

Henry et al. 2006

Gel electrophoresis

Data analysis

The verification and quantification of PCR products of DNA amplification were performed by migration on 1.5 % agarose gel using the gel electrophoresis technique. For each sample, 5 μL of PCR-amplified DNA product was mixed with 2 μL of loading blue dye and 6 μL of BenchTop 100 base pair DNA Ladder (Promega, USA). Migration was carried out at 90 V and 400 mA for 40 min in a gel electrophoresis filled with TAE 1X. Afterward, the revelation of bands was determined by staining the gel with ethidium bromide for 15 min. Gel photography was carried out under UV using software Bio-Capt™ Version 12.6 after rinsing the gel with distilled water.

Statistical software XLSTAT-Pro 5.2 (Addinsoft, France 2007) was utilized for processing data related to N2O. Data analysis for quantitative PCR (nosZ) was performed with CFX96™ Manager Software (BioRad, Hercules, California). Regarding N2O analysis, apart from graphical presentation, Tukey’s honestly significant difference (HSD) test was applied in conjunction with analysis of variance (ANOVA) using 95 % confidence interval to assess the comparative significance of different substrate treatments on N2O emission trends. Matrices of DGGE profiles were built using BrayCurtis algorithm on each gel after a normalization of the band intensities, and the dendrograms were built using unweighted pair group method on arithmetic averages (UPGMA) on Total Lab (TL-120; Nonlinear Dynamics Ltd) software.

Denaturing gradient gel electrophoresis (DGGE) To assess the genetic diversity of different microbial communities in termite gut, DGGE for 16S rDNA PCR products was used using 8 % acrylamide gel. This gel had a linear gradient, varying from 40 to 70 % of denaturing chemicals, i.e., urea and formamide. Separation of 16S rDNA PCR products was performed on gel while immerged in a tampon of TAE 1X maintained at 60 °C, 100 V, 50 W, and 50 mA in an electrophoresis basin (Ingeny phorU, Netherlands) for about 17 h. After staining with Cyber-Gold dye for about 20 min along with gentle agitation over a vortex, different bands of DNA fragments established on the gel were visualized by UV illumination using Bio-Capt™ image acquisition computer program.

Results Termite survival and substrate consumption First termite feeding bioassay was conducted to investigate the impact of substrate quality (mainly C/N ratio) on termite gut microbial community and host metabolism. Substrate C/N ratio appeared to influence the termite survival and food consumption (Table 3). Indeed, at day 9 of the experiment, 79.24 and 55.74 % mortality was observed in termites fed on grass and acacia leaves, respectively. The C/N ratios of these two substrates were quite lower than those of wood and filter

Author's personal copy Folia Microbiol Table 2 Protocols used for PCR assays of gut bacterial communities of N. voeltzkowi PCR step

Number of cycles

PCR of 16S (total bacterial community) Denaturation 1 Denaturation Hybridization 20

94 °C 94 °C 65 °C a

Synthesis Denaturation Hybridization 10 Synthesis End of synthesis 1 qPCR of nosZ (N2O-reductase community) Denaturation 1 Denaturation Hybridization 6 Synthesis Denaturation Denaturation Hybridization 40 Synthesis Denaturation Dissociation stage qPCR of 16S (total bacterial community) Denaturation 1 Denaturation Hybridization Synthesis Denaturation Dissociation stage

Temperature

72 94 55 72 72

°C °C °C °C °C

2 min 30 s 30 s 1 min 30 s 30 s 1 min 10 min

95 °C 95 °C 65 °C a 72 °C 80 °C 95 °C 60 °C 72 °C 80 °C 80 to 95 °C

15 min 15 s 30 s 30 s 30 s 15 s 30 s 30 s 30 s

95 °C

15 min

95 °C 60 °C 72 °C 80 °C 80 to 95 °C

35

Time

15 30 30 30

s s s s

Touch down −0.5 °C/cycle from 65 to 55 °C for 16S PCR and −1.0 °C/ cycle from 65 to 60 °C for nosZ qPCR

a

paper. Dry wood and to a less extent filter paper substrates showed the lowest mortality rate (10.4 and 33 %, respectively). Termites were found very active on these two substrates till the end of experiment. However, no strict correlation was found between food consumption and mortality rate, for example, 33.21 % termites were dead in microcosms containing filter paper for which the consumption rate (58.33 %) was the

highest within all the substrates tested. However, wood and grass were equally consumed (31.54 and 30.17 %) while acacia showed the least consumption efficiency, i.e., 16.22 %. Emission of N2O by termites according to C/N ratio of substrates Analysis of N2O emission (Table 4) revealed that termites fed on grass gave the least emission on per microcosm basis, followed by filter paper and acacia (difference within these two substrates were not statistically significant). The low recorded values for grass originated from high termite mortality. On individual termite basis, production of N2O was twice higher for grass and acacia than for filter paper and wood. Similar emission trend has been observed for the N2O production per g termite basis. There was a strong correlation (r2 = 0.97) between the values of C/N ratio and mean N2O emission by termites (Fig. 1). Bacterial community structure of total 16S rDNA in guts of termites fed on different substrates To assess whether the substrate C/N ratio influence the structure of termite gut bacterial community, PCR-DGGE analysis was performed for 16S rDNA gene fragments from termite gut microbiota using a higly hypervariate V3 region. Since the structure of termite gut bacterial community depends partly on gut localization, we opted to determine this bacterial community structure in two major gut compartments: the anterior part, which provides shelter for the ingested microbes and the posterior part, which harbors gut symbiotic bacterial community. A first visual analysis of the gel showed clearly a weak impact of substrate C/N ratio on gut bacterial community structure, whatever the gut compartment was. However, there was a significant difference between the bacterial community structure of anterior and posterior portions of the termite gut (Fig. 2). Bands were more intense and diverse in the posterior part of the digestive tract than in the anterior one. A cluster analysis of DGGE profiles (Fig. 2) was carried out to determine the genetic similarity between bacterial Table 4 Impact of substrate on mean N2O emission by the termite, N. voeltzkowi

Table 3 Termite mortality and substrate consumption (9th day observation) Substrate

Grass Acacia leaves Wood Filter paper

Substrate C/N ratio

% of food consumption

% of mortality

20:1 60:1 500:1 733:1

30.17 16.22 31.54 58.33

79.24 55.74 10.39 33.21

Substrate

N2O emission (nmol per microcosm)

N2O (nmol per termite)

N2O (nmol per g termite)

Acacia Grass Paper Wood

0.503±0.037 b 0.242±0.041 c 0.430±0.082 b 0.678±0.034 a

0.0113±0.002 ab 0.0118±0.004 a 0.0064±0.001 b 0.0072±0.001 b

2.109±0.309 ab 2.207±0.804 a 1.203±0.160 b 1.345±0.105 b

Different letters show significant difference of N2O emission between substrates (Tukey HSD test (P