Editor: Christoph Tebbe
Inter- and intraspecific comparison of the bacterial assemblages in the hindgut of humivorous scarab beetle larvae (Pachnoda spp.)
Version of Record online: 9 JUL 2010
© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Ecology
Volume 74, Issue 2, pages 439–449, November 2010
How to Cite
Andert, J., Marten, A., Brandl, R. and Brune, A. (2010), Inter- and intraspecific comparison of the bacterial assemblages in the hindgut of humivorous scarab beetle larvae (Pachnoda spp.). FEMS Microbiology Ecology, 74: 439–449. doi: 10.1111/j.1574-6941.2010.00950.x
Present address: Janet Andert, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany
- Issue online: 9 JUL 2010
- Version of Record online: 9 JUL 2010
- Received 25 November 2009; revised 29 June 2010; accepted 30 June 2010.Final version published online 3 August 2010.
- gut microbiota;
- microbial diversity;
- host specificity
The larvae of scarab beetles are model organisms for studying the role of physicochemical gut conditions and intestinal microbiota in symbiotic digestion, particularly of humus. Here, we address the question of whether the enlarged hindgut paunch of Pachnoda ephippiata and Pachnoda marginata, two closely related, but allopatric species, harbors a specific bacterial microbiota. Terminal restriction length fragment polymorphism (T-RFLP) analysis revealed that in both species, the bacterial hindgut community differs strongly from that in the midgut, food soil, and fecal pellets. High intra- and interspecific similarities between the T-RFLP profiles of different larvae indicate the presence of a hindgut-specific microbiota. Nevertheless, we found a clear separation of the two species. A 16S rRNA gene clone library from the hindgut of P. ephippiata identified the major phylogenetic groups as members of the Clostridia, Betaproteobacteria, and Bacteroidetes, followed by Bacillales and Deltaproteobacteria. A comparison with a previously obtained clone library of the same species corroborates both the similarities and the intraspecific variance of the hindgut microbiota.
The intestinal microbiota of insects is an important component in the symbiotic digestion of lignocellulose and its degradation products (reviewed by Brune, 2006; Brune, 2009). In the case of termites, evidence is accumulating that the gut microbiota is characteristic of its particular host lineages. The hindgut of termites harbors a large proportion of termite-specific microbial lineages, and a close relatedness of the bacterial phylotypes within one termite genus has prompted the hypothesis that many lineages of the intestinal microbiota are coevolving with their host (Schmitt-Wagner et al., 2003b; Hongoh et al., 2005; Shinzato et al., 2005; Yang et al., 2005). Furthermore, it is possible to differentiate individuals of different termite colonies on the basis of the structure of their bacterial gut communities (Minkley et al., 2006), suggesting that species- and genus-specific similarities are superposed by significant intra-specific differences at the colony level that may be caused by differences in the environment or the diet.
Information on the intestinal microbial communities in insects of other orders is rather scarce. An important example is the larva of rose chafers (Pachnoda spp.; Coleoptera: Scarabaeidae), which has served as a model organism to study the role of physicochemical gut conditions and intestinal microbiota, particularly in the symbiotic digestion of humus (e.g. Egert et al., 2003; Lemke et al., 2003; Li & Brune, 2007; Andert et al., 2008). It was recognized early that the cellulolytic and hemicellulolytic activities in the hindgut of these scarab beetle larvae are of microbial origin (Werner, 1926b; Wiedemann, 1930; Rössler, 1961). Later, microorganisms were shown to be responsible for the formation of fermentation products (Cazemier et al., 1997; Lemke et al., 2003; Egert et al., 2005), which are presumably resorbed and used as carbon and energy sources by the host (Brune, 2009). So far, only a single study has addressed the bacterial diversity in the rose chafer hindgut (Egert et al., 2003) and revealed a diverse microbiota consisting of numerous novel lineages apparently typical for intestinal tracts. However, only a few clones were sampled, and host specificity remains to be addressed. Moreover, it remains to be clarified whether similarities of the gut microbiota of related species are caused by their similar lifestyle (e.g. diet) or whether the host species maintain their specific gut microbiota owing to the spatial separation of the respective microbial communities.
In this study, we analyzed the larval gut microbiota of two closely related scarab beetles. Pachnoda ephippiata and Pachnoda marginata are allopatric species occurring in East Africa and in West Africa, respectively. Their larvae share the same lifestyle, feeding on soil organic matter in all stages of decay, and harbor a very abundant gut microbiota (Cazemier et al., 1997; Egert et al., 2003). Using terminal restriction length fragment polymorphism (T-RFLP) analysis, we compared the bacterial community structure in the midgut and hindgut of individuals of the two species and tested the influence of different batches of humus on the composition of the microbiota. The major components of the hindgut community were characterized by 16S rRNA gene cloning and phylogenetic analysis.
Materials and methods
Larvae and soil
Third instar larvae of P. marginata and P. ephippiata were purchased from a commercial breeder (b.t.b.e. Insektenzucht, Schnürpflingen, Germany). A second batch of P. ephippiata larvae was obtained from the zoological teaching collection at the University of Konstanz (Germany).
Larvae were kept on a peat-based and clay-enriched potting soil (Compo, Münster, Germany) at 25 °C for at least 2 weeks before sampling of the gut microbiota (for details, see Lemke et al., 2003). The influence of feeding on different food soils on the composition of the bacterial gut community was examined with P. ephippiata and P. marginata larvae fed with the above-described soil (soil I), with a mixture of forest soil (Schnürpflingen; soil II) and peat, and with clay-enriched potting soil from a different supplier (Oekohum, Herbertingen, Germany; soil III). For sampling, larvae were dissected, and the intestinal tract was divided into midgut and hindgut compartments as described previously (Lemke et al., 2003). Fecal pellets were collected 24 h after the larvae were placed on fresh food soil.
Nucleic acid extraction and 16S rRNA gene amplification
DNA was extracted from gut segments, soil, and fecal pellets using the bead-beating protocol of Henckel et al. (1999), with the modifications introduced by Egert et al. (2004). Bacterial 16S rRNA genes were amplified using 25 PCR cycles with a combination of the 27f primer [5′-GAG-TTT-G(AC)T-CCT-GGC-TCA-G-3′; Lane, 1991] and 907r [5′-CCC-GTC-AAT-TC(AC)-TTT-GAG-TTT-3′; Lane, 1991; MWG, Ebersberg, Germany] according to Egert et al. (2003). For T-RFLP, primer 27f was labeled with IRD 700 (pentamethine carbocyanin; MWG). To prevent inhibition by humic substances, 0.4 mg mL−1 bovine serum albumin was added to each amplification reaction; if necessary, extracted DNA was diluted 10-fold.
16S rRNA gene clone library
The PCR product was cloned in Escherichia coli JM109 using the pGEM-T easy Vector system (Promega, Mannheim, Germany) following the manufacturer's instructions. The correct size of the inserts was confirmed after PCR amplification with vector-based primers and subsequent gel electrophoresis. The inserts of 90 randomly selected clones were sequenced in their full length by ADIS (Cologne, Germany). The sequences were imported into the Silva database (Pruesse et al., 2007) using the arb software package (Ludwig et al., 2004). Sequences were automatically aligned, and the alignments were refined manually. A 50%-consensus filter was used to exclude highly variable positions. Phylogenetic trees were calculated using fastdnaml (Olsen et al., 1994), a maximum-likelihood method implemented in arb. The stability of the branching pattern was tested using the maximum-parsimony method included in the phylip package (dnapars; Felsenstein, 1989) implemented in arb. Shorter sequences from other studies documenting neighboring clusters of interest were added with the arb parsimony tool, which does not change global tree topologies. The clones were checked for chimera using mallard (Ashelford et al., 2006; http://www.cardiff.ac.uk/biosi/research/biosoft). Suspected chimerae were validated in arb by fractional treeing (Ludwig et al., 1997). Three sequences identified as chimerae and one clone clustering with 16S rRNA genes of chloroplasts were excluded from further analysis. The remaining sequences were submitted to GenBank (accession numbers FJ374174-FJ374260; the numbering follows the order of appearance in the phylogenetic trees).
Bias-corrected Chao1 richness estimates and rarefaction analysis were performed with dotur (Schloss & Handelsmann, 2005), using sequence similarity cut-offs at 97% for the species and 95% for the genus level.
The PCR products were purified using the MinElute PCR Purification Kit (Qiagen, Hilden, Germany). The DNA concentration in the purified PCR products was determined spectrophotometrically (Eppendorf). Seven different restriction enzymes (MboI, MspI, PauI, RsaI, SspI, HhaI, HaeIII; Promega) were tested to identify the one resulting in the highest possible resolution. All restrictions were carried out according to Egert et al. (2004), except that 80 ng DNA was digested.
The T-RFLP profiles were generated on a LI-COR sequencer as described in Pester et al. (2004) with IRDye (50–700 bp, LI-COR) as a size marker and analyzed using gel-pro 4.5 (Media Cybernetics). Before statistical analysis, the T-RFLP profiles were background-subtracted and normalized by peak height according to Dunbar et al. (2001); T-RFs with a relative height of <1% of the total peak height were rejected. Great care was taken to ensure that independent aliquots of the same sample resulted in virtually identical profiles. As the base pair length of identical T-RFs can vary within a range of 1–2 bp between gels and/or lanes of the same gel, T-RFs differing by only one or two base pairs (early or late peaks, respectively) were summarized as operational taxonomic units (OTU), as described by Egert et al. (2004). In the case of the hindgut sample subject to cloning analysis, clones were restricted in silico using the trf-cut software (Ricke et al., 2005) and assigned to OTUs based on T-RF length.
Indices of diversity and similarity
The species richness was defined for our analysis as the number of OTUs recognized in a profile (peak height>0). To also consider the abundance of OTUs, we calculated a diversity index (Krebs, 1994) using the relative peak size of T-RFs as a proxy of relative abundance (Blackwood et al., 2007). We selected the Simpson index because this index has a low SD (Lande, 1996).
Community similarity was estimated using the Morisita index as modified by Horn, which takes into account both the presence and the relative abundance of a species (Krebs, 1989). This index varies between 0 (no species shared) and 1 (communities with an identical structure). The statistical evaluation of community similarities and distance followed Anderson (2001). This method, referred to as ‘permutational manova’ or ‘nonparametric manova’, partitions the sums of squares of a multivariate data set analogous to manova using a multivariate analogue of Fischer's F-ratio. This ratio is calculated directly from any symmetric distance matrix (in our case, the 1-Morisita index). We used the function adonis in the package vegan, a package implementing ecological analyses in r (Oksanen et al., 2009). P-values were obtained with 9999 permutations. For ordination of different assemblages and therefore visual inspection of similarity patterns, we used nonmetric multidimensional scaling using metamds in vegan, which attempts to find a stable solution using several random starts. Again, we used the modified Morisita index as calculated by vegdist in vegan.
Comparative analysis of the communities in different gut compartments
A comparison of the T-RFLP profiles showed that the bacterial communities of the food soil and of the feces differed from that in the midgut and hindgut content in both P. marginata and P. ephippiata (Fig. 1), indicating that the community structure changes considerably both after ingestion of the soil and after excretion of the residual gut content. The strong differences between the midgut and hindgut samples underline that a fundamental shift in the bacterial populations also occurs between these gut compartments.
In the larvae of both species, the richness of microbiota in the midgut was considerably lower than that in the hindgut (two-way anova with species and gut compartment as factors; gut compartment P<0.001). The anova also revealed differences between species (P<0.001), with an interaction between species and gut compartment (P=0.03; Table 1). The difference between the two gut compartments was much more pronounced in P. ephippiata than that in P. marginata (Table 1). An analysis of the diversity of the microbiota using the Simpson diversity index produced almost the same results, although the interaction was not significant (anova, factor species P<0.07; factor gut P<0.001; and interaction P=0.19).
|Species richness||Simpson diversity|
|P. ephippiata||11 ± 2.7||25 ± 4.4||0.79 ± 0.11||0.93 ± 0.021|
|P. marginata||11 ± 3.5||17 ± 4.6||0.82 ± 0.12||0.87 ± 0.045|
The multivariate analysis of the dissimilarity matrix (calculated as the 1-Morisita index) among microbiota showed significant differences between host larvae (P=0.0002) and gut compartments (P=0.0001), with a significant interaction between the two factors (P=0.0012; 9999 permutations). The ordination of the similarity matrix using nonparametric multidimensional scaling shows the clear differences in the composition of the microbiota between the two species as well as between the midgut and the hindgut. Note that the variation between individuals is much more pronounced in P. marginata than that in P. ephippiata (Fig. 2a; Table 2). Most major T-RFs of hindgut communities were found for both species, albeit at a different relative abundance. For instance, the T-RF of 92 bp length was present in almost all hindgut profiles obtained with both species, but its relative abundance ranged from 1% to 27% (details not shown).
|Morisita index (mean ± SD)|
|P. ephippiata : P. ephippiata||0.26 ± 0.24||0.62 ± 0.10|
|P. marginata : P. marginata||0.11 ± 0.18||0.35 ± 0.21|
|P. ephippiata : P. marginata||0.14 ± 0.15||0.35 ± 0.18|
We had sufficient hindgut samples to test for differences in the composition of the microbiota in larvae living in different soils (Fig. 2b). Other than the already shown differences between species, the ordination revealed no clear-cut differences in the microbiota (nonparametric manova: species P=0.0001, soil P>0.3, interaction P>0.3; 9999 permutations).
The bacterial community of the P. ephippiata hindgut
To identify the individual elements of the hindgut community of P. ephippiata, we compared the T-RFs of the respective profiles with the sequences obtained from a clone library prepared from the 16S rRNA gene fragments amplified from the hindgut content. Phylogenetic analysis showed that the residual clones represent four different phyla of bacteria.
The clone library covered 83% and 98% of the diversity predicted by bias-corrected Chao1 richness estimates at the species and genus levels. This is in agreement with the incomplete sampling at the species level indicated by rarefaction analysis (details not shown). However, the slope of the rarefaction curve at the end of sampling indicated that new genera should also have been discovered if sampling had been continued (four novel OTUs per 10 samples).
The majority of the clones belonged to the Firmicutes (33%), Betaproteobacteria (22%), and Bacteroidetes (22%), followed by Bacillales (5%) and Deltaproteobacteria (5%). Only a single clone was affiliated with the Planctomycetes.
Detailed comparison with the existing sequences in public databases revealed that the clones related to the Bacteroidetes formed five separate clusters among the Bacteroidales (Fig. 3). Several clones (Cluster 1) were affiliated with Bacteroides eggerthii as the closest relative (89–90% sequence similarity). Another group (Cluster 2) was most closely related to Tannerella forsythensis (87–88% sequence similarity) and also comprised several clones obtained previously from a larva of the same species. Other Bacteroidetes clones clustered with Dysgonomonas gadei (92–93% sequence similarity), including a bacterium isolated from the hindgut of the termite Reticulitermes speratus (Cluster 3), were loosely affiliated with Alistipes massiliensis (Cluster 4), or formed distinct lineages with no cultured representatives (Cluster 5). Also, most of the remaining clones were closely related to the clones obtained from termite guts.
The Firmicutes clones mostly fell into the Clostridiales, forming novel clusters containing only clones from P. ephippiata (Fig. 4). Often, clones from the intestinal tracts of other insects were closely related. The largest clone group (Cluster 1) formed a distinct lineage among the Clostridiales that contained no cultivated representatives. Also, numerous other clones (Clusters 2 and 3) were only distantly related to Clostridium sporosphaeroides and Ruminococcus species; their closest relatives were clones from termite guts. Two clones clustered with Clostridium oroticum (93–94% sequence similarity) and other isolates from mammalian intestinal tracts; two other clones fell into the genus Sporomusa.
Most of the clones belonging to the Proteobacteria are affiliated with the Betaproteobacteria (Fig. 5). The largest clone groups (Clusters 1 and 2) were most closely related to Bordetella petrii (97–99% sequence similarity) and Azoarcus buckelii (90–94% sequence similarity), respectively. Several other lineages were loosely affiliated with Hydrogenophaga flava and other members of the Comamonadaceae. Also, the remaining clones either represented new lineages or had clones obtained from termite guts as close relatives. Almost all of the clones affiliated with the Deltaproteobacteria formed a distinct lineage (Cluster 3) of sequences with clones obtained from the larval gut of the scarab beetle Melolontha melolontha and from termite guts. They were only distantly related to their next cultivated neighbor, Desulfovibrio cuneatus (82–84% sequence similarity).
Most major T-RFs in the hindgut profiles of P. ephippiata could be assigned to clones in the clone library obtained from this sample; all phylogenetic groups were represented (Fig. 6). The T-RFs with 76 and 85 bp lengths shared by almost all larvae corresponded to Clostridiales and Betaproteobacteria clones, whereas the large cluster with (T-RF 89–96) corresponded exclusively to Bacteroidetes clones. Another T-RF cluster was assigned to Clostridiales (T-RFs 282–299), also including Deltaproteobacteria (288) and Planctomycetes (296). Most of the longer T-RFs were assigned to Betaproteobacteria. However, a considerable number of clones (33%) had predicted T-RFs that were not represented in the T-RFLP profile. Because there were no undigested products, these clones are most likely rare phylotypes that are below the detection limit of the T-RFLP analysis. This is in agreement with the high diversity among the unassigned T-RFs in the profile.
Although the scarab beetles P. ephippiata and P. marginata are allopatric species, their larvae share the same type of diet and occupy the same ecological niche. Our study revealed similarities among the bacterial hindgut communities of individual larvae, indicating the presence of bacterial lineages in the gut microbiota that are also shared with other insects. Nevertheless, the differences in the profiles appear to be mostly species specific, with little differences due to diet.
The microbial community of the hindgut
For the larvae of both beetle species, the bacterial species richness in the hindgut is much higher than that in the midgut. These findings are in agreement with previous studies, which reported a reduced abundance of microorganisms in the midgut of Pachnoda spp. (Cazemier et al., 1997; Lemke et al., 2003). This is not surprising because the midgut of insects is considered to be the site for enzymatic digestions (Crowson, 1981; Terra & Ferreira, 1994); the contribution of the gut microbiota to midgut processes in Pachnoda spp. is considered to be minor (Andert et al., 2008). In contrast, the high numbers of microorganisms in the hindgut of many insects signify diverse microbial processes and high concentrations of fermentation products (Odelson & Breznak, 1983; Cruden & Markovetz, 1984; Tholen et al., 1997; Lemke et al., 2003).
Phylogenetic analysis and T-RFLP profiles document a high diversity of the hindgut community in P. ephippiata (Egert et al., 2003; this study). Most of the phylotypes obtained in this study represented lineages of Bacteroidetes, Clostridiales, and Proteobacteria that have been detected previously in intestinal habitats. As in the case of the larvae of the scarab beetles M. melolontha (Egert et al., 2005) and Dermolepida albohirtum (Pittman et al., 2008), the clones from the hindgut of P. ephippiata also clustered mainly with those obtained from the guts of other insects. The dominance of termite gut clones in these apparently insect-specific lineages of gut bacteria is probably caused by their over-representation in the databases. Information on the gut microbiota of other scarab beetle larvae is scarce, but it is likely that – as in the case of termites (Schmitt-Wagner et al., 2003a, Hongoh et al., 2005, Shinzato et al., 2005) – the presence of taxon-specific lineages is indicative of cospeciation between gut microbiota and the host.
In most cases, the Pachnoda-specific lineages do not comprise any cultivated representatives. However, their position among certain higher taxa (Clostridiales, Bacteroidetes) suggests that they represent novel lineages of bacteria with a fermentative metabolism. This is in agreement with the high concentrations of microbial fermentation products, particularly in the hindgut (Andert et al., 2008). Moreover, there are concrete observations of cellulolytic, peptidolytic, and homoacetogenic activities in the hindgut of P. ephippiata (Lemke et al., 2003; Li & Brune, 2007). However, in the absence of any isolates, it would seem premature to draw any conclusions on the particular functions of individual lineages in the digestion of humus.
Differences between individual communities
T-RFLP is frequently used to analyze and compare microbial communities in many environments. Because distinct phylotypes sometimes share the same T-RF, especially in complex communities (Marsh et al., 2000), we compared the patterns obtained for different samples, but did not assign T-RFs to particular clones. This is safe as long as the patterns are different, but it has to be underscored that identical patterns may still represent different phylotypes.
The similarity in the hindgut T-RFLP profiles is caused by the consistent presence of certain OTUs in all larvae tested. Nevertheless, there are significant differences in the bacterial hindgut communities of the two species and also in individuals of one species (see also Fig. 2a and b). It has been shown that congeneric species of soil-feeding termites harbor highly similar gut microbiota in the corresponding gut compartments (Schmitt-Wagner et al., 2003b). On the other hand, sympatric species of wood-feeding termites share considerably more phylotypes than allopatric species (Hongoh et al., 2005), and there are significant differences in the gut microbiota of the same species of termites even at the colony level (Matsuura, 2001; Minkley et al., 2006).
In Pachnoda spp., the community similarity between the hindguts of individual larvae of the same species did not differ significantly from that between species, which is in agreement with the presence of both a constant and a variable component in the bacterial community. The relative proportion of the variable component seems to be higher than that in soil-feeding termites of the genus Cubitermes, where Morisita indices ranged between 50% and 94% for the homologous hindgut segments of different species (Schmitt-Wagner et al., 2003b).
Because the hindgut microbiota of scarab beetle larvae is involved in digestion (e.g. Werner, 1926a; Schlottke, 1945; Cazemier et al., 1997; Andert et al., 2008), changes in the diet might influence the composition of the hindgut microbiota. In crickets and cockroaches, the structure and function of the intestinal microbiota are affected by dietary shifts (Kane & Breznak, 1991; Santo Domingo et al., 1998a, b). In contrast, the results of our analyses suggest that the soil substrate (i.e. diet) had no consistent influence on the composition of the microbiota in the hindgut of Pachnoda spp. (Fig. 2b). Moreover, almost no OTUs were shared between the profiles of ingested soil and hindgut samples, as observed previously in soil-feeding termite Cubitermes orthognathus (Schmitt-Wagner et al., 2003b) and in M. melolontha larvae (Egert et al., 2005), corroborating the gut specificity of the corresponding phylotypes.
We are grateful to Katja Meuser for preparing the clone library and assisting with the T-RFLP analysis. Uwe Deggelmann, keeper of the zoological teaching collection in the Biology Department at the University of Konstanz, kindly provided P. ephippiata larvae.
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