Methane Production and Methanogenic Archaea in the Digestive Tracts of Millipedes

13 Pages·2014·2.73 MB·English

Methane Production and Methanogenic Archaea in the
Digestive Tracts of Millipedes (Diplopoda)
Vladimı´r Sˇustr1*, Alica Chronˇa´kova´1, Stanislava Semanova´1,2, Karel Tajovsky´1, Miloslav Sˇimek1,2
1InstituteofSoilBiology,BiologyCentreASCR,v.v.i.,Cˇeske´ Budeˇjovice,CzechRepublic,2FacultyofScience,UniversityofSouthBohemia,Cˇeske´ Budeˇjovice,Czech
Republic
Abstract
Methane production by intestinal methanogenic Archaea and their community structure were compared among
phylogenetic lineages of millipedes. Tropical and temperate millipedes of 35 species and 17 families were investigated.
Species that emitted methane were mostly in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular
phylogeneticdistributionofmethaneproductioncorrelatedwiththepresenceofthemethanogen-specificmcrAgene.The
study brings the first detailed survey of methanogens’ diversity in the digestive tract of millipedes. Sequences related to
Methanosarcinales,Methanobacteriales,MethanomicrobialesandsomeunclassifiedArchaeaweredetectedusingmolecular
profiling(DGGE).ThedifferencesinsubstratepreferencesofthemainlineagesofmethanogenicArchaeafoundindifferent
millipede orders indicate that the composition of methanogen communities may reflect the differences in available
substratesformethanogenesisorthepresenceofsymbioticprotozoainthedigestivetract.Weconcludethatdifferencesin
methaneproductioninthemillipedegutreflectdifferencesintheactivityandproliferationofintestinalmethanogensrather
than an absolute inability of some millipede taxa to host methanogens. This inference was supported by the general
presenceofmethanogenicactivityinmillipedefaecalpelletsandthepresenceofthe16SrRNAgeneofmethanogensinall
tested taxainthe two main groupsofmillipedes, the Helminthophoraand the Pentazonia.
Citation:SˇustrV,Chronˇa´kova´A,Semanova´S,Tajovsky´K,SˇimekM(2014)MethaneProductionandMethanogenicArchaeaintheDigestiveTractsofMillipedes
(Diplopoda).PLoSONE9(7):e102659.doi:10.1371/journal.pone.0102659
Editor:PedroL.Oliveira,UniversidadeFederaldoRiodeJaneiro,Brazil
ReceivedMarch20,2014;AcceptedJune23,2014;PublishedJuly16,2014
Copyright: (cid:2) 2014 Sˇustr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Mostofrelevantdataarewithinthepaperand
itsSupportingInformationfiles.AllsequencesareavailablefromtheGenBankdatabase(accessionnumber(s)KF574048–KF574078andKF739300–KF739307).
Funding:ThisresearchwassupportedbytheCzechScienceFoundation(Spotsofmethaneproductionandconsumptioninterrestrialecosystems:keyfor
understandingmethaneturmover,No526/09/1570).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationof
themanuscript.WebsiteoftheCzechScienceFoundation:http://www.gacr.cz/.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected]
Introduction taxa [19,20,21,22,23]; the results indicate that symbiotic associ-
ations involving methanogens and methane production are likely
About 600 Tg of the greenhouse gas methane is produced on
tobeacharacteristicpropertyofthehosttaxonandthatmethane
Earth each year [1]. Although methane, which has the second-
production is restricted to millipedes, cockroaches, beetles
largestimpactonglobalwarmingafterCO [2],isreleasedtothe
2 (Cetonidae), and termites [19].
atmospherefromhumanactivities[3]includingindustry,agricul-
Usingmicroscopyandspecificautofluorescence,Hacksteinand
ture,andwastemanagement[4],naturalsourcesaccountfor37%
Stumm [19] found free-living as well as endosymbiotic methan-
of global methane emissions. All methane, other than that from
ogens(methanogenslivinginthecellsofProtozoa)inthehindgut
industry, is produced by the only known biogenic source -
of arthropod hosts. Methanogenic Archaea have been detected
methanogenic Archaea. Methanogenic Archaea are an ancient
and their phylogeny analysed in the digestive tracts of methane-
group of microorganisms that occupy ecological niches with
producing arthropods other than millipedes by 16S rRNA-based
limited oxygen concentrations such as wetlands, rice fields,
surveys [24,25,26,27,28]. Moreover, phylogenetic analysis in
swamps, marshes, freshwater and marine sediments, agricultural
combinationwithenrichmentcultureshassuggestedtheexistence
soils, and the digestive tracts of humans and other animals
of a seventh order of methanogens (the ‘‘Methanoplasmatales’’)
[5,6,7,8,9].
associated with intestinal tracts of both invertebrates and
Many authors have estimated methane production from
vertebrates [29]. A detailed assessment of methanogenic diversity
vertebrates [10,11,12,13], and other authors have discussed the
is still lacking in millipedes, however, because microbial commu-
globalimportanceofmethaneoriginatingfrominvertebratehosts
nities in millipede digestive tracts have mainly been studied only
and its potential contribution to the increase in atmospheric
by classical cultivation methods and microscopic observation
methane [14,15,16,17]. Termites have been recognized as a
[19,30].
globallyimportantsourceofmethaneinthattheyareestimatedto
Unlike the digestive tract of most methane-producing arthro-
contribute between 5 and 19% of the global methane emissions
pods, millipedes have a relatively simple digestive tract that lacks
[18]. Methane production and the presence of methanogens has
an enlarged pouch in the hindgut [19]. The inner surface of the
alsobeensystematicallyscreenedinawidevarietyofinvertebrate
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MethaneandMethanogenicArchaeainMillipedes
millipede hindgut, however, is strongly developed and may 2.1/jam.). The research in the Bohemian Karst PLA was
facilitate microbialcolonization[30]. permitted by the Administration of Cˇesky´ kras PLA, Karlsˇtejn,
AccordingtoHopkinandRead[31],thereisnoevidencethat CzechRepublic;intheBlansky´ lesPLAbytheAdministrationof
millipedeshaveapermanentsymbioticmicrobiotasimilartothat Blansky´ lesPLA,Vysˇny´,Cˇesky´ Krumlov,CzechRepublic;inthe
of termites, although microorganisms are important for millipede Moravian Karst PLA by the Administration of Moravsky´ kras
digestion. But the review of Byzov [30] demonstrated that the PLA, Blansko, Czech Republic; and in the Pa´lava PLA by the
millipede intestinal tract harbours a stable, indigenous microbial Administration of Pa´lava PLA, Mikulov, Czech Republic. No
community that includes facultative anaerobes that are able to specific permission was required to collect millipedes in the
degrade recalcitrant organic polymers. Only a few studies have following five localities in the Czech Republic: the Cˇeske´
used molecular techniques to study the microbial community of Budeˇjovice Basin (49u1290.7930N, 14u24925.6280E), the Chelcˇice
millipede faeces[32] ormillipedeintestines [29,33]. (49u06914.0260N,14u07956.4430E),theCˇeskomoravska´ vrchovina
Specificautofluorescence microscopyshowedthat thedigestive Highlands (49u12956.8730N, 15u54955.6120E), the Lanzˇhot
tract of some millipedes contains free methanogenic Archaea or (48u4396.0030N,16u58911.2730E),andtheZˇda´nicky´lesHighlands
Nyctotherus-typeciliatesthathostmethanogensasendosymbionts (49u04941.9460N, 16u56910.5990E). None of the species collected
[19]. van Hoek [34] detected one archaeal sequence obtained intheCzechRepublicareendangeredorprotectedintheCzech
fromasymbioticciliateofanundeterminedmillipede,whilePaul Republic. The species collected in Romania are also not
and colleagues [29] detected methanogens related to the endangered or protected. No specific permission was required
‘‘Methanoplasmatales’’ in the gut of the tropical millipede for the sampling in the locality Mehedin¸ti Mts. (45u04914.900S
Anadenobolus sp.; no information is available, however, about 22u45948.350E). The species collected in Thessaloniki
thedifferencesinintestinalarchaealcommunitiesamongdifferent (40u36956.370N, 23u02908.960E) is not endangered or protected
millipede lineages. in Greece, and no specific permission was required for this
Methane is known to be produced by almost all tropical location.
millipedes but is generally thought not to be produced by Specimens of the millipede Archispirostreptus gigas, which is
millipedes and cockroaches from temperate climates [19]. Sˇustr naturallydistributedinTanzania,wereobtainedfromapetshop
andSˇimek[23],however,detectedmethaneproductioninseveral (M. Krocˇek, Horn´ı Sucha´, Czech Republic). Two species of
species of European millipedes. Their results indicated that some millipedes wereobtained froma laboratory colonymaintainedat
groups of European millipedes (mainly Julida) seem to produce MPI for Terrestrial Microbiology Marburg (Germany); these
methane,whileothergroupsdonot(PolydesmidaandGlomerida). species were Glomeris marginata, which occurs naturally in
Because millipedes apparently include producers as well as non- Germany, and Orthomorpha coarctata, which occurs naturally in
producers ofmethane, theyrepresent an interestingmodel group Southeast Asia. Specimens of Epibolus pulchripes, which occurs
for thedeterminationof factorsinfluencing gutmethanogenesis. naturally in Tanzania, were obtained from a breeding colony
Inthecurrentstudy,wecomparedmethaneproductionandthe maintained bytheMikulovgrammar school(Czech Republic).
community structure of methanogens among different phyloge- AllotherlocalitiesmentionedintheTableS2arecitedassites
neticlineagesofmillipedes.Indoingso,weattempted toidentify of originof species usedby HacksteinandStumm [19].
factors that explain the phylogenetic differences. To accomplish Themillipedeswereidentifiedtospeciesbasedontheavailable
theseobjectives,weusedgaschromatographyinordertomeasure literature; before they were assessed for methane production, the
methane production for a large number of taxonomically diverse millipedeswerekeptinthelaboratoryat10uCforseveraldaysto
millipede species. We verified the presence of methanogens by several weeks in plastic boxes containing top soil and litter from
usingPCRtodetectthespecificgeneticmarker(mcrAgene),and the collection site. Appropriate moisture was maintained by
we used molecular profiling (DGGE) to compare the community regularlymoisteningthesubstratewithwater,andcuttlefishbone
structure of methanogens in the gut and in faecal pellets of powderwasaddedtothesubstrateasasourceofcalcium.Because
different species of millipedes. Finally, we discuss how methane the diet of most European species of millipedes consists of
production relates to millipede phylogeny and millipede charac- decomposed leaf litter, the litter in the boxes served as a food
teristics. source.
Asnotedearlier,thetropicalspeciesE.pulchripesandA.gigas
Materials and Methods were obtained from a pet shop and from individual breeders.
SpeciesdeterminationofA.gigaswasverifiedbasedongonopod
Animals morphology after Mwabvu et al. [35]. The tropical species were
In the current study, most data used to evaluate methane kept at about 25uC on horticultural substrate plus hardwood leaf
productionrelativetomillipedetaxonomicpositionwereobtained litter. E. pulchripes accepted partly decomposed litter from
by gas chromatography. These data were supplemented by the hardwood trees (a mixture of hazel, maple, and oak leaves) as
data published in the recent literature [19,23]. The European the only source of food. A. gigas consumed large amounts of
millipedes used for our measurements were collected in different organicwastessuchaspiecesofpotatoes,cucumbers,andcabbage
habitats in the Czech Republic, Slovakia, Romania, and Greece. heads in addition toleaflitter andgarden substrate. Coprophagy
The localities of origin of the species included in this study are of millipede faecal pellets was not prevented for either of the
listed inTable S2. tropicalspecies.Intotal,35speciesofmillipedesweremaintained
ThepermissionfortheresearchinTatraMountainswasissued and assessed for methane production. Only individuals taken
by the Regional Office of Environment Protection, Presˇov, directlyfromthesubstrateandwithfulldigestivetractswereused
Slovakia (permission No.1/2007/01066-005/KM-R). Millipedes forgut dissectionandsubsequent microbial analyses.
fromtheLatoricaProtectedLandscapeArea(PLA)werecollected
with the permission of the Regional Office of Environment Methane production measurement
Protection, Kosˇice, Slovakia (permission No.2008/00322), and Methane production from intact, living millipedes was mea-
the research in the Slovak Karst was licensed by the Ministry of sured by comparing methane concentrations in closed vessels
theEnvironmentoftheSlovakRepublic(licenceNo.3102/2009- (250 ml,5 ml,or1 mldependingonmillipedesize)thatcontained
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MethaneandMethanogenicArchaeainMillipedes
ordidnotcontainmillipedes–controlvessels.Thelargestspecies, species was testedby logitregression inStatistica v6.0(Advanced
which were A. gigas, E. pulchripes, and the cockroach Blaptica Linear/Nonlinear Models: Nonlinear Estimation Analysis).
dubia (B. dubia was used as a known methane producer), were Thefaecalpelletsofmillipedeswereanaerobicallyincubatedat
measured in 250-ml glass vessels. Intermediate-sized species such 20uC for 7 days to measure their methane production. Fresh
as Julus scandinavius were measured in 5-ml glass vessels. The pelletswereplacedin5-mlglassvesselsandmoistenedwith100ml
250-and5-mlvesselsweresealedwitharubberstopperandkept of distilled water. The atmosphere in the vessels was changed to
at20uCfor3–4h(inthecaseof250-mlvessels)orfor5–13h(in argon,andtheinternalgaswassampledandanalysedafter24,96,
thecaseof5-mlvessels).Apieceoffilterpaper(264cmfor250-ml and168hours byGC asdescribed previously.
vesselsor161cmfor5 mlvessels)moistenedwithdistilledwater
was placed in each glass vessel to maintain air humidity during Molecular evaluation of the presence and diversity of
measurement. The control glass vessels without animals were methanogenic microorganisms in millipedes
assembledandincubatedinthesameway.A0.5-mlsampleofthe Dissected digestive tract contents or freshly released faecal
internalgaswascollectedatthestartandattheendofincubation pellets were frozen and stored at 218uC until DNA extraction.
using gas-tight syringes, and the samples were injected into a gas Two methods of DNA extraction were tested. A phenol/
chromatograph (GC) column. chloroform extraction method based on bead-beating homogeni-
Thesmallestspecieswereincubatedina700-mlvolumein1-ml zation of frozen samples [39] successfully extracted DNA mainly
plastic syringes [36]. A circle of moist filter paper (5 mm in fromthefaecalpelletsortheintestinaltractsoflargeranimals.For
diameter) was placed in the syringe to maintain air humidity. the intestinal tracts of smaller animals (mostly temperate,
Freshoutdoorairwasdrawn intothesyringeimmediately before European millipedes), the NucleoSpin Tissue XS kit (Macherey-
placement of animals. The syringes were incubated at 20uC,and Nagel,Germany),basedonenzymaticandchemicallyses,enabled
the incubation period ranged from 4h for the largest species to the extraction of concentrated DNA from small amounts of
24h for the smallest species in syringes. As controls, the same starting material (0.025–10mg). The quantity and quality of
syringes without animals were assembled, and the mean final extracted DNA was determined with a NanoDrop ND-2000
concentrationofmethaneinthecontrolswassubtractedfromthe spectrophotometer (Thermo Scientific,Wilmington, DE,USA).
final concentration in samples. A 500-ml volume of the internal For detection (expressed as presence or absence) of methano-
atmosphereinthesyringewasinjecteddirectlyintoaGCafterthe gensinthesamples,weusedPCRamplificationofthemcrAgene,
incubation period. The amount of methane was quantified using whichencodesthemethylcoenzymeMreductasea-subunit.The
an HP 5890Series II gas chromatograph (Hewlett Packard, Palo mcrAgenewasamplifiedwithtwoPCRassays,theresultsofwhich
Alto,CA,USA)equippedwitha2 mPorapakNcolumnat75uC, werecombinedfordetectionofpositivesignals(mcrA-1andmcrA-
andaflameionizationdetectorusingnitrogenasthecarriergas.A 2, Table S1). Both reactions were performed in 25-ml reaction
standard mixture of 100ml m23 methane in N was used for volumes containing 1ml of diluted DNA as template (usually
2
calibration purposes [23]. around5–10ng),QiagenTaqDNApolymerase(1.25U),dNTPs
In most cases, one animal was placed in one glass vessel or (0.2mM each), BSA (0.25mg, Thermo Scientific, USA), and
plastic vessel (syringe) to detect the individual variability in primers (5 pmols and 8 pmols, respectively) in the presence of
methane production. For some of the smallest species, for which MgCl2 (1.5 and 2.5mM, respectively) in the PCR buffer
incubation of individuals resulted in non-detectable methane recommended by the manufacturer (1x PCR buffer, Qiagen,
production, several individuals were incubated in one syringe to CA,USA).A5-mlquantityofthePCRproductswasanalyzedina
1%agarosegel(TopVisionagarose,Fermentas)thatwasstained
verifythenegativeresult(forT.costata,forexample,threegroups
with ethidiumbromide (1 mgL–1)for30min[40, 41].
ofthreeanimalsweremeasured).Animalswereweighedattheend
Methanogenic community structure was evaluated by DGGE
oftheincubation,andtheresultswereexpressedasnlofmethane
using a nested PCR approach that targeted the 16S rRNA gene.
producedperindividualperhorasthelive-massspecificmethane
In thefirstround, astretch ofthe16SrRNAgenewasamplified
production (nl ofmethaneper g of livemassper h).
usingprimersAr109FandAr915R(bothat25pmol[42]).Inthe
The ability to detect methane production depends on the
secondround,dilutedPCRproduct(1:100)servedastemplatefor
sensitivity of the detection method and on the precision of
amplification of methanogen-specific 16S rRNA using primers
measurement expressed in the variability of blank controls. If a
MG0357F-gc and MG0691R (both at 25 pmol [43]). Both
smallamountofmethaneisgenerated,thedetectionlimitmustbe
reactions were carried out with Taq DNA polymerase (Qiagen,
determinedtodistinguishbetweennegativeandpositivemeasure-
CA, USA) and reaction buffer supplemented with 4 mg of BSA
ments. We considered a value to be positive if it exceeded the
(Thermo Scientific, USA). The cycling conditions for each assay
value obtained in five control vessels (glass or plastic vessels
are listed in Table S1. DGGE was performed with an Ingeny
without animals) by three standard deviations of controls. In
PhorU system (Ingeny, Leiden, The Netherlands) as follows:
addition, we designated species as non-producers (0% positive
200ng of PCR product was loaded on an 8% (w/v) polyacryl-
samples), accidental producers (at least one but ,50% positive
amide gel with a denaturing gradient range of 30–60% (100%
samples), facultative producers (from 50% to 99% positive
denaturant is equivalent to 7 M urea and 40% deionized
samples), andobligatory producers(100%positive samples).
formamide). Electrophoresis was run in 0.56TAE buffer for
The distribution of methane-positive species among different
16h at 60uC and 100V. The DGGE gels were stained with
taxonomic groups was summarized in contingency tables, and a
SYBR Green I nucleic acid stain (Lonza Rockland, ME, USA),
nonparametric X2 test (Statistica v6.0 Nonparametric Statistics,
andmolecularprofileswereanalyzedwithGelComparIIsoftware
262tables,StatSoft,Inc.,USA)wasusedtotestthehypothesisof
(Applied Maths, Ghent, Belgium). Bands were excised from gels
random distribution of methane production across the millipede usingasterilizeddissector,elutedin20 mlofMilliQwaterwitha
phylogenetic tree. We used the previously published taxonomic freeze-thaw cycle, and re-amplified with primers MG0357F–
classification of millipedes [37] along with the phylogenetic tree MG0691R. The resulting PCR products were purified using a
basedonmoleculardata[38].Theeffectofthemeanspeciesbody MinElutePCRPurificationkit(Qiagen,CA,USA)andsequenced
mass on the frequency of methane-positive individuals in the using an ABI Prism 3100-Avant Genetic Analyser (Applied
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MethaneandMethanogenicArchaeainMillipedes
Figure 1. The distribution of methane-positive species among millipede orders. The red row of columns (OFP) is based on all tested
speciesclassifiedaseitherobligatoryorfacultativeproducers,andtheyellowrow(OFAP)includesobligatory,facultative,andaccidentalproducers.
doi:10.1371/journal.pone.0102659.g001
Biosystems, CA, USA). The sequences were edited with Bioedit producers (OP). Except for Glomeris tetrasticha, which was an
7.0.4.1software[44]andassembledinGeneiousversionPro5.5.6 accidentalproducer,allspeciesintheorderGlomeridawerenon-
createdbyBiomatters(NewZealand,availablefromhttp://www. producers according to GCanalysis (Table S2). Millipedes inthe
geneious.com/). Sequences were tested against the GenBank ordersPolyzoniidaandChordeumatida(TableS2)werealsonon-
database(www.ncbi.nml.nih.gov)usingtheBlastnalgorithm.The producers. The order Callipodida was represented by only one
sequences are available in GenBank under accession numbers individual of Callipodella fasciata, which did not produce
KF574048–KF574078 andKF739300–KF739307. methane. The order Julida included all categories of methane
The relationship between the results of molecular detection of producers, from non-producers to obligate producers. Of the 18
methanogensandGCdetectionofmethaneproductionwastested species tested in the family Julidae, only three were obligatory
by nonparametric statistical methods (Statistica v6.0 Nonpara- producers; non-producers, accidental producers, and facultative
metric Statistics, 262 tables). Differences in the complexity of producers were each represented by five species in the Julidae.
methanogenic communities (numbers of bands detected by Two species were tested in the family Blaniulidae, and neither
DGGE) among different groups of millipedes were tested with a produced measurable methane. The spirobolid species E.
nonparametricKruskal-WallistestorWilcoxonMatchedPairstest pulchripes was designated an obligatory producer, and the same
(Statistica v6.0, NonparametricStatistics). designation probably applies to the other members of the order
Spirostreptida. All A. gigas tested in this study produced large
Results amountsofmethane.Amongthemillipedesbelongingtotheorder
Polydesmidathatweretestedinthisstudy,onlythetropicalspecies
Phylogenetic overview of methane production Orthomorpha coarctata was classified as an obligatory producer
Table S2 summarizes the data on the presence or absence of (onlyoneindividual was tested).
methanogenesis in living and intact millipedes belonging to 46 European members of the order Polydesmida belonging tothe
speciesandeightorders.Datafor35specieswereobtainedinthe families Paradoxosomatidae, Polydesmidae, and Trichopolydes-
current study, and data for the other 11 species were obtained midaedidnotproducedetectablelevelsofmethaneexceptforone
from the literature [19,23]. With respect to methane production, ofthefourtestedindividualsofStrongylosomastigmatosum(Table
the species are designated as non-producers (NP), accidental S2).Thedataavailableforthisorderarebiased,however,because
producers (AP), facultative producers (FP), and obligatory mostpolydesmidmethaneproducersaretropicalandlargespecies
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MethaneandMethanogenicArchaeainMillipedes
(O. coarctata and Pycnotropis acuticollis [19]) but most of the relatedtoaveragelivebodymass(W)byspeciesasindicatedbya
tested polydesmids were Europeanandsmallspecies. doublelogarithmicplot(Fig.3).ThelinearregressionoflogMon
The current data and previously published data [19,23] were log W corresponded to the equation M=32.494?W1.569
used to describe the distribution of methane-positive species (r2=0.899,p=0.0001).Regressionofbodymass-specificmethane
among the main millipede orders (Fig. 1 and 2). The methane- production (M/W) on millipede mass (W) was also positive and
positivespecies(obligatoryandfacultativemethaneproducers)are significant (r2=0.522, p=0.002). It follows that, in addition to
distributed almost exclusively in the juliform groups (the orders taxonomicposition,bodysizemayexplaindifferencesinmethane
Julida, Spirobolida, and Spirostreptida). The order Julida, which production among millipedespecies.
was the most investigated group, had a higher percentage of We also analyzed changes in the percentages of methane-
accidentalproducersandnon-producersthantheSpirobolidaand positive and methane-negative individuals with decreasing mean
Spirostreptida.Inacomparisonofthefrequenciesofallmethane- body mass in an attempt to detect a general lower limit in body
producingspecies(AP,FP,andOPtogether)inthejuliformorders massformethaneproductionbymillipedes.Allfivespecieswhose
Julida,Spirobolida,andSpirostreptidavs.inallothergroups,the body mass was .0.4 g were obligatory producers (OP). The
X2 test of the 262 contingency table indicated that taxonomic intermediate group in terms of body mass (from 0.06 to 0.30 g)
position was significantly related to methane production included eight species representing all four categories of methane
(X2(df=1)=13.61,p=0.0002). producers(twospeciesofOP,twospeciesofFP,onespeciesofAP,
andthreespeciesofNP).Allspecieswhosebodymasswas,0.06 g
Quantitative aspects of methane production were non-producers except for Unciger foetidus (the mean body
One adult individual of the millipede A. gigas produced more massofthetestedindividualswas0.059 g)andtheverysmalljulid
methane than the smaller tropical E. pulchripes in the order speciesEnantiulusnanus(itsmeanbodymasswas0.006g);inthe
Spirobolida and several orders of magnitude more than small caseofE.nanus,onlyoneofthe10testedindividualswaspositive.
European species in the order Julida. The mean individual In the order Glomerida, the largest species (0.28g) was classified
methaneproductionfromA.gigasiscomparabletothemethane asNP,andthedatasetisverysmall.Thesedataarealsoscarcefor
production from the large cockroach, Blaptica dubia. Mean theorderPolydesmida,buttheexistenceofsomebodymasslimit
individual methane production (M) by species was positively formethaneproductionmaybeexpectedinthisgroupbecauseall
Figure 2. Summary of the recent information concerning methane production by millipedes. Methane production detected by gas
chromatography (Table S1) relative to millipede phylogeny. Species belonging to framed groups have been tested to date. Red lines mark the
lineagescontainingsomepositivespecies.ThephylogenetictreewasusedaccordingtoRegieretal.[38].
doi:10.1371/journal.pone.0102659.g002
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MethaneandMethanogenicArchaeainMillipedes
Figure 3. The relationship between individual methane production and the mean body mass. Mean methane production (M) in
methane-positivespeciesofmillipedes(accidental,obligatory,andfacultativeproducers)plottedagainstmeanbodymassofspecies(W).Species
abbreviationsareindicatedinTableS1.MethaneproductionbythecockroachBlapticadubia(Bdu)isincludedforcomparison.Redlines=regression
ofMonWwith95%confidenceintervals.
doi:10.1371/journal.pone.0102659.g003
species thatwere ,0.08gproducedno methaneandonespecies contentsoftheonespeciesintheorderPolydesmida(TableS2).A
that was .0.4g produced methane. For the juliform orders Chi-square test calculated from 262 contigency tables indicated
(Julida, Spirostreptida, and Spirobolida), a body mass limit of that the production of methane as detected by GC was
0.12 g separated the methane-positive and methane-negative significantly related to the detection of mcrA in the gut contents
species. ofthesameanimal(X2(df=1)=4.05,p=0.044,n=24).Detection
of the mcrA gene in gut contents supported the classification of
Methane production from faecal pellets of millipedes species based on GC measurement of methane production. All
Excrements of all tested millipede species produced methane threeofthepositivegutsamplesoriginatedfromspeciesthatwere
under anaerobic conditions (Fig. 4). The time course of the designated FPor OP.
productionwassimilarinallspeciesinthattherateofproduction For most species, the detection of mcrA in the gut contents
quicklyincreasedduringthefirstdayofanaerobicincubationand corresponded to mcrA detection in faecal pellets. Discrepancies
thenslowly increased overthenext 6 days(Fig.5). occurredinonlythreecases(twoinwhichmcrAwasdetectedonly
in faecal pellets and one in which mcrA was detected only in the
gut contents). All of the tested millipede faecal pellets began to
Methanogenic community structure
produce methane early during the incubation under anaerobic,
DNA was successfully extracted from the faecal pellets of 16
moist conditions but the mcrA gene was detected in the faecal
millipedespeciesandfromthegutcontentsof13millipedespecies
pelletsofonlyfiveofthe16speciesthatweretested.Thedetection
(TableS2).Theextractionwaslesseffectivefromgutcontentsthan
of mcrAindifferent millipedelineages isillustrated inFigure 6.
from faecal pellets. Specific amplification of the mcrA gene using
DGGE profiles of the methanogenic microbial communities
twodifferentPCRassaysconfirmedthepresenceofmethanogens
wereobtainedfrom18samplesofthedigestivetractsrepresenting
in the gut of three species of millipedes belonging to the orders
10species.Thismethodrevealedthepresenceofmethanogensin
Julida, Spirostreptida, and Spirobolida. Results were similar for
the digestive tract of members of the orders Glomerida, Julida,
bothassaysexceptforCylindroiulusluridusexcrementsandJulus
Spirobolida, and Spirostreptida and in all categories of methane
scandinaviusgutcontents(TableS2),whichcanbeinfluencedby
production except NP. Molecular profiles of the methanogenic
primer sequence bias. The mcrA gene was not found in the gut
microbial communities were also obtained from 30 samples of
contents of two species in the family Glomeridae or in the gut
faecal pellets representing 15 species. In addition, methanogens
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MethaneandMethanogenicArchaeainMillipedes
Figure4.Methaneproductionfrommillipedefaecalpelletsrelativetomillipedephylogeny.Methaneproductionfromanaerobically
incubatedfaecalpelletswasdetectedbygaschromatography(TableS1).Speciesbelongingtoframedgroupsweretestedinthisstudy.Redlines
markthelineagescontainingsomepositivespecies.ThephylogenetictreewasusedaccordingtoRegieretal.[38].
doi:10.1371/journal.pone.0102659.g004
were detected in the faecal pellets of Polydesmida members. The others did not. Overall, more DGGE bands were obtained in
DGGEanalysisrevealedthepresenceofthemethanogen-specific faecal pellets than in gut contents. This trend was confirmed by
16SrRNAgeneinallofthetestedmillipedeorders(Fig.7,Table nonparametric statistical tests comparing all DGGE profiles
S2). DGGE analysis also revealed large variability in the obtained from guts and faecal pellets (Kruskall-Wallis test, H (1,
methanogenic community structure among different gut and n=48)=3.99, P=0.046) and comparing species averages for
faecalpelletsamplesinmillipedesfromtheordersGlomeridaand pelletsandguts(Wilcoxonpairtest,n=9,P=0.038).Thenumber
Julida.Clusteringofmolecularprofiles(Fig.8)showednorelation ofbandsinDGGEprofileswasgreaterforthetropicalmillipedes
of methanogenic community structure to the pre-defined groups (Spirobolida and Spirostreptida), which included the two largest
NP,AP,FP,andOP.Theresultsrevealedahighdiversityofthe species in this study, than for the European millipedes (Kruskall-
methanogenic community among millipede individuals, because Wallis test,H (1,n=48)=6.95,P=0.008) (Fig.8).
only a few of the molecular profiles were similar (at similarity It was not possible to re-amplify and characterize all of the
cutoff=70%). The molecular profiles of methanogens from the excised bands from each profile, perhaps because of PCR
faecal pellets of G. hexasticha (MK), G. connexa (MK), M. inhibition or a low concentration of the DNA fragment in the
unilineatum (TH), and U. foetidus (BL) were similar and eluate.Atotalof32bands,representingspecificsequencesinthe
significantly different from that of G. hexasticha (HT) and G. DGGEprofiles,weresuccessfullysequencedandidentified(Table
tetrasticha (SK). The methanogenic communities of guts and S3),andthesewereassumedtorepresentthespecificordominant
excrementsofthesamespeciesweresimilarinsomecases.Inother components ofthecommunities.
species (J. scandinavius and A. gigas) the gut and faecal pellet Ingeneral,diversemethanogenswereidentifiedinthegutand
communities formed different clusters. The highest dissimilarity faecal pellets. These included Methanosarcina thermophila, M.
was found between samples of J. scandinavius. Methanogenic siciliae, Methanoregula boonei, Methanobrevibacter woesei, M.
communities of the whole gut content were almost identical thaueri, and M. arboriphilus, as well as other species of these
among individuals of G. tetrasticha originating from the same generathatremainedunidentified.MethanogenicDGGEprofiles
locality,butmethanogensinthedifferentgutcompartmentsofA. obtainedfromgutsandfaecalpelletsofmillipedesbelongingtothe
gigas hadonly40%similarity (Fig. 8). order Julida were dominated by sequences related to Methano-
As indicated by the number of bands in the DGGE profile, microbiales and Methanosarcinales. Methanoregula and other
some species harboured rich methanogenic communities while
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MethaneandMethanogenicArchaeainMillipedes
Figure 5. Change in the rate of methane production per mass of millipede faecal pellets over time. The pellets were anaerobically
incubatedat20uC.AbbreviationsofspeciesareindicatedinTableS1.
doi:10.1371/journal.pone.0102659.g005
unclassified Methanomicrobiales or Methanosarcina were often Discussion
identified inthese samples.
The results presented here show that methane production and
In samples representing the millipede family Glomeridae, we
foundMethanomicrobiales,Methanosarcinales,aswellasMetha- the mcrA gene are unequally distributed among phylogenetic
lineagesofmillipedes(seeFig.2andFig.6).Previousresearchalso
nobacteriales(Methanobacteriumsp.orMethanobrevibactersp.).In
revealed considerable variation in the presence of methane
faecal pellets and gut content samples from S. stigmatosum
production among different groups of arthropods, and these
(Polydesmida), E. pulchripes (Spirobolida), and A. gigas (Spiros-
differenceswerethoughttoreflectdifferencesintheabilitytohost
treptida), only Methanobrevibacter spp. and related Methanobac-
methanogenic microorganisms [19].
teriales were among the dominant bands that were succesfully
identified. Although detection of methane production and the mcrA gene
indicate a rather restricted distribution of methanogens in
millipedes, we found the 16S rRNA genetic marker of methan-
ogens in all tested taxa (members of Julida, Glomerida, Poly-
desmida,Spirobolida,andSpirostreptida)andpotentialanaerobic
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MethaneandMethanogenicArchaeainMillipedes
Figure6.DetectionofthearchaealmcrAgeneinmillipedesrelativetomillipedephylogeny.mcrA,themarkergeneofmethanogenic
Archaea,wasdetectedbyPCRamplification(TableS1)ingutandfaecalpelletsofmillipedes.Speciesbelongingtoframedgroupsweretestedinthis
study.Redlinesmarkthelineagescontainingsomepositivespecies.OnlyfaecalpelletswerepositiveinGlomerida.Thephylogenetictreewasused
accordingtoRegieretal.[38].
doi:10.1371/journal.pone.0102659.g006
methaneproductioninallofthetestedfaecalpellets(seeFig.7and genetic markers is not an accurate indicator of activity and may
Fig. 4).Thus, the methanogens were present inboth of themain representonlythepotentialforactivity.Italsosuggeststhatavery
groupsofmillipedes:theHelminthophoraandPentazonia.Inthe smallorinactivemethanogeniccommunitymaybepresentinthe
currentstudy,thepresenceofmethanogensinthePentazoniawas gutofmostmillipedes.Suchcommunitiesmightbeactivatedwhen
indicatedbymethanogenesisinalloftheirtestedfaecalpellets,by conditionsbecomefavourable.Thisideaissupportedbytheability
the detection of methane production from some of living of methanogens to survive unfavourable conditions (drought and
millipedes, and by the detection of methanogen genes. In a aeration) for long periods [46] and to be activated once anoxic
previous study, the presence of methanogens in the Pentazonia conditions return [47,48]. Our results indicate that the striking
wassupportedindirectlybythedetectionofhydrogenproduction differencesinmethaneproductionamongdifferentmillipedetaxa
inthegutofsomespeciesofGlomerida[19].Thecurrentresults reflect differences in the activity of intestinal methanogens rather
indicate that methanogens may be more broadly distributed in than an absoluteinability ofsome taxa tohostmethanogens.
millipedes than previously assumed. Hackstein and Stumm [19] considered possible sources of
We obtained molecular evidence of methanogens by assessing variation in the activity of intestinal methanogens, resulting in
the ribosomal marker (16S rRNA) and mcrA gene. The results differences in methane production among arthropod individuals.
obtainedwith16SrRNAfrequentlydifferedfromthoseobtained The authors discussed body size, environmental temperature or
with mcrA. Methane production was better correlated with climatic zone of the species distribution, number of intestinal
detection of mcrA than with detection of 16S rRNA. The mcrA- protozoa,and unknowngenetically fixedfactors.
basedapproachmaybelesssensitivebecauseitsprimersaremore Amongthemillipedesinvestigatedinourstudy,thenumberof
specificthaninthe16SrRNA-basedapproach.Thelatterfeature methane-producinganimalsincreasedwithincreasingbodysizeof
has been discussed previously [45] and can be explained by a the species. Moreover, the amount of methane produced was
highergenecopynumberofthe16SrRNAgenethanofthemcrA positively correlated with millipede body size in methane-
gene inthegenomes. producing species. This effect of body size may be explained by
The disagreement between detection of methane production thebetteraerationinthegutofsmallvs.largemillipedes(thegut
and the molecular evidence of methanogens (based on the 16S of smaller animals has a higher surface-to-volume ratio); the
rRNA gene) can be attributed to the fact that the presence of increased aeration in the gut of small animals may prevent the
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MethaneandMethanogenicArchaeainMillipedes
Figure7.Detectionofthearchaeal16SrRNAgeneinmillipedesrelativetomillipedephylogeny.The16SrRNAgene,anindicatorof
methanogenicArchaea,wasdetectedbyPCRamplification(TableS1).Speciesbelongingtoframedgroupsweretestedinthisstudy.Redlinesmark
thelineagescontainingsomepositivespecies.InPolydesmida,onlyfaecalpelletsweretested.ThephylogenetictreewasusedaccordingtoRegier
etal.[38].
doi:10.1371/journal.pone.0102659.g007
proliferation of anaerobic methanogenic Archaea [49]. Although Another possible source of the differences in methane produc-
theoxygenconcentrationinthegutofmillipedeshasrarelybeen tionbetweentropicalandtemperatetaxamaylieinthesymbiotic
measured,themeanredoxpotentialinthegutofthepillmillipede, interaction of large tropical millipedes with ciliate protists related
G.marginata(bodymassabout0.2g),was+232mVinthemidgut to the genus Nyctotherus [19]. The ciliates host considerable
and +204 mV in the hindgut, and these values correspond to numbers of methanogens near hydrogen-producing hydrogeno-
oxidative conditions [50]. This is consistent with our failure to somes [56,57], where the methanogens consume hydrogen.
detect methaneproductionin thisspecies. Therefore, the abundance of ciliates enhances the methanogenic
Thus, differences in body size may explain much of the activity and enhances the rate of methane production from
between-species variability in methane production of millipedes. cockroaches [17]. It follows that protists may be responsible for
The absence of methaneproduction in relatively larger members quantitative differences in the methane production between
ofthePolyzoniidaandCallipodidamaybecausedbytheirunique tropical spirostreptid millipedes and European Julidae. This is
feeding habits.Polyzoniidaconsumemoist,soildetritus[51],and supported by our finding that mainly hydrogenotrophic [58]
Callipodida contains species that have predatory feeding habits Methanobacteriales (Methanobrevibacter arboriphilus and other
and speciesthat consumethedead tissues ofanimals [52,53]. Methanobrevibacter-relatedsequences)dominatedthegutoflarge
HacksteinandStumm[19]speculatedthatmethaneproduction members of the Spirostreptida (A. gigas); hydrogenotrophic
wouldbemorecommonamongtropicalthantemperatespeciesof Methanobacteriales were previously found in the cytoplasm of
arthropods.Theeffectofclimaticoriginofspeciesmaybemasked ciliates of the genus Nyctotherus [17,34]. Moreover, we observed
by the effect of body size because, like other terrestrial ciliates in the hindgut and fresh faecal pellets of A. gigas
invertebrates, millipedes tend to be larger in the tropics than in (unpublished data).
otherregions[54].Althoughmethaneproductionfromindividual MostmethanogenicArchaealineagespreviouslyreportedfrom
millipedes decreased with decreasing temperature [23], the the insect gut (Methanosarcinales, Methanobacteriales, and
inference that high environmental temperatures are required for Methanomicrobiales; see [59] for a summary) were identified in
methanogenesis was disproved by the evidence of methane thepresentstudyofmillipedes.Thelistofmethanogeniclineages
production from European species of the family Julidae [23] as identifiedheremaybecompletedonlybythepreviouslypublished
well as bymethaneproduction inpermafrost soils [55]. evidence of the Thermoplasmatales-related methanogens in the
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