Folia Microbiol. 49 (2), 179–182 (2004) http://www.biomed.cas.

cz/mbu/folia/

Anaerobic Bacteria in the Gut of Terrestrial Isopod

Crustacean Porcellio scaber
R. KOSTANJŠEKa, A. LAPANJEa, M. RUPNIKa, J. ŠTRUSa, D. DROBNEa, G. AVGUŠTINb*
aUniversity of Ljubljana, Biotechnical Faculty, Department of Biology, 1000 Ljubljana, Slovenia
bUniversity of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair for Microbiology and Microbial Biotechnology,
1230 Domžale, Slovenia
fax +386 1724 1005
e-mail gorazd.avgustin@bfro.uni-lj.si

Received 20 October 2003

Revised version 18 December 2003

ABSTRACT. Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated
using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determi-
ned by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts
of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and
Enterococcus were retrieved, as well as sequences from ‘A1 subcluster’ of the wall-less mollicutes. Bacteria
from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In
contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system
due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the
moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results
indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable,
i.e. predominately oxic, conditions.

Porcellio scaber (Isopoda, Oniscidea) is a common terrestrial isopod living in temperate climate,
which feeds mainly on decayed plant material (Szlavecz and Maiorana 1998). Since the diet of P. scaber is
composed mainly of cellulose and other complex polysaccharides, it is generally believed that digestion pro-
cess of these animals strongly depends on microbial activity. In spite of their assumed importance in diges-
tion, the role of microorganisms in digestive system of terrestrial isopods animals is still not understood.
In contrast to the gut of termites (Breznak and Brune 1994) and cockroaches (Cruden and Marko-
vetz 1987) where the resident anaerobic microbiota is known to contribute to the digestion of lignocellulose
in their host’s diet, the tube-like hindgut of terrestrial isopods seems a rather unsuitable environment for deve-
lopment of similar microbial community (Hassall and Jennings 1975). The later assumption has been com-
monly based on the short retention time of the food in the tube-like gut (Clegg et al. 1994), frequent renewal
of gut cuticle (Štrus and Storch 1990) and unsuccessful attempts to cultivate microbes under anaerobic con-
ditions (Reyes and Tiedje 1976; Ullrich et al. 1991).
The vast majority of the microbiota in the gut of terrestrial isopods appears to consist mainly of
ingested microbiota (Reyes and Tiedje 1976; Ineson and Anderson 1985), however, the existence of resident
microbiota was also indicated repeatedly by authors who have used traditional microbiological techniques
(Ineson and Anderson 1985; Gunnarson and Tunlid 1986; Ullrich et al. 1991). Recently, the presence of resi-
dent bacteria associated with the inner gut surface of P. scaber was indicated also by the molecular approach
employing PCR amplification and analysis of 16S rRNA sequences (Kostanjšek et al. 2002). Since some of
the sequences retrieved were closely related to rRNA sequences from obligately anaerobic bacteria, additi-
onal studies were performed in order to determine whether truly anaerobic bacteria inhabit the gut surface of
P. scaber, and whether we can isolate and grow them under anaerobiosis in laboratory conditions.

MATERIAL AND METHODS

The hindguts of five adult isopods were isolated and washed thoroughly. Total DNA from guts was
extracted, and small subunit (ssu) ribosomal genes were amplified with universally conserved bacterial
primer pairs 27f–1495r (Binaciotto et al. 1996) or fD1–1392r (Lane 1991; Weisburg et al. 1991). The ampli-

*Corresponding author.
180 R. KOSTANJŠEK et al. Vol. 49

cons were cloned into the plasmid vector according to Kostanjšek et al. (2002). Selected clones were
sequenced with fD1, 1392r and M13 sequencing primers at Microsynth (http://www.microsynth.ch/)
on our request, thus providing almost total 16S rRNA gene sequence information. The retrieved sequences
were compared to the sequences from related bacteria deposited in the RDP, GenBank and EMBL (EBI)
nucleotide sequence databases in order to find the most related sequences. Sequences were aligned using
Clustal X (Thompson et al. 1997) and phylogenticaly analyzed with programs included in the PHYLIP (ver-
sion 3.6.) (Felsenstein 2002) and Mega version 2.1 phylogenetic package (Kumar et al. 2001).

Fig. 1. Simplified phylogenetic tree showing the position of Mollicutes related 16S rRNA gene sequences
which were retrieved from the gut wall of P. scaber; Escherichia coli 16S rRNA gene sequence was used
as the outgroup; the scale represents 0.1 substitution per nucleotide position.

The sulfate-reducing bacteria were isolated under anaerobic conditions from the guts of 1-d food-
deprived P. scaber. The procedure was done in an anaerobic chamber (Ruskin Technology, UK). Modified
lactate medium (g/L: sodium lactate 4, yeast extract 1, K2HPO4 1, Na2SO4 0.5, NH4Cl 0.5, MgSO4·7H2O
0.2, CaCl2·2H2O 0.1, FeSO4·7H2O 0.1) with vitamins (VA solution; Ballows et al. 1991) and microelements
(SL6 solution; DSMZ 1993) was used for enrichment and further subcultivation.

RESULTS AND DISCUSSION

Analysis of the directly retrieved 16S rRNA gene sequences showed that a fair portion of them ori-
ginates from organisms closely related to Enterococcus faecium, E. saccharolyticus and members of the
genus Bacteroides (Kostanjšek et al. 2002) which are all known anaerobic inhabitants of vertebrate gastro-
2004 ANAEROBIC BACTERIA IN GUT OF P. scaber 181

intestinal tracts. Subsequently further sequences related to anaerobic bacteria were retrieved from the gut
wall of P. scaber. Five of these were found to share the highest similarity with sequences from members of
the class Mollicutes, although the similarity values never exceeded 80 %. Phylogenetic analysis clustered se-
quences retrieved from P. scaber into a deeply branched lineage within Mollicutes (Fig. 1) which was sup-
ported by 99 % confidence value. Since the nearest known sequences were those clustered in a so-called
unknown cluster A (Daly et al. 2001), in which directly retrieved 16S rRNA sequences from equine, bovine,
pig and human intestines are grouped, the provisional name ‘A1 subcluster’ was suggested for the lineage. It
appears plausible that the mollicutes from A1 subcluster that inhabit the P. scaber gut have anaerobic meta-
bolism since all known taxa within Mollicutes are obligate or facultative anaerobes (Razin and Freundt
1984). However, our attempts to grow these bacteria on various mycoplasma-specific media under anaerobic
or aerobic conditions were unsuccessful.

Fig. 2. Phylogenetic tree showing the relationships of Eig1 sequence retrieved from P. scaber hindgut with 16S rRNA gene
sequences from sulfate-reducing bacteria; Aquifex pyrophylus 16S rRNA gene sequence was used as the outgroup; the scale
represents 0.05 substitution per nucleotide position; the numbers at the nodes indicate bootstrap values based on 1000 samplings
(values below 60 are not shown).

Another 16S rRNA gene sequence (Eig1) was retrieved from organisms that are associated with the
gut wall of P. scaber. The phylogenetic analysis affiliated Eig1 to the 16S rRNA sequences of sulfate-redu-
cing bacteria, more specifically to the members of the genus Desulfotomaculum. The Eig1 sequence shared
182 R. KOSTANJŠEK et al. Vol. 49

the highest similarity with Desulfotomaculum ruminis (99.2 %) 16S rRNA gene sequence which was further
confirmed by the phylogenetic analysis (Fig. 2) and supported by 100 % confidence value. Sulfate-reducing
bacterium related to D. ruminis with 16S rRNA sequence homologous to Eig1 was subsequently success-
fully isolated from inner surface of hindgut wall of P. scaber under anaerobic conditions.
Our results confirmed the presumptions that anaerobic bacteria are present in the gut of terrestrial
isopod P. scaber (Kostanjšek et al. 2002). Although at first glance this seems in incongruity with reports on
peripheral oxic ring of the gut lumen (Zimmer 2000), the presence of anaerobic microniches just above the
gut surface in otherwise oxic environments is analogous to the conditions described in the termite gut (Brune
et al. 2000). Since bacteria on gut surface of terrestrial isopod are closely related to bacteria commonly found
in anaerobic digestive systems of other animals, we consider that these bacteria are resident and indeed
autochthonous in the isopod gut rather than being transient passengers that were ingested with the food.

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