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Keywords:

  • insects;
  • Coleoptera;
  • gut microbiota;
  • microbial diversity;
  • host specificity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

T-RFLP analysis

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.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

image

Figure 1.  Schematic presentation of all T-RFs represented in the normalized T-RFLP profiles of the samples analyzed in this study (black, average peak height; white, SD): S, food soil; Mm, midgut of Pachnoda marginata (n=3); Me, midgut of Pachnoda ephippiata (n=5); Hm, hindgut of P. marginata (n=8); He, hindgut of P. ephippiata (n=9); Fm, fecal pellets of P. marginata (n=3). The 16S rRNA genes were amplified with general Bacteria primers and digested with MspI. T-RFs representing <1% of the total T-RF peak height were excluded; to faciliate viewing, not all gaps are shown.

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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).

Table 1.   Species richness and Simpson diversity of the bacterial communities in the midgut and hindgut of larvae of Pachnoda marginata and Pachnoda ephippiata
 Species richnessSimpson diversity
MidgutHindgutMidgutHindgut
  1. All values are means ± SD of the indices obtained for the T-RFLP profiles (Pachnoda ephippiata midgut, n=5; hindgut, n=8; Pachnoda marginata midgut, n=3; hindgut, n=9).

P. ephippiata11 ± 2.725 ± 4.40.79 ± 0.110.93 ± 0.021
P. marginata11 ± 3.517 ± 4.60.82 ± 0.120.87 ± 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).

image

Figure 2.  (a) Ordination plot using nonparametric multidimensional scaling on the dissimilarity matrix among bacterial assemblages in the hindgut (black symbols) and midgut (gray symbols) of Pachnoda ephippiata (circles) and Pachnoda marginata (squares). Each symbol represents the T-RFLP profile of an individual gut compartment. (b) Ordination plot as above, but using only the hindgut samples. The shading of the symbols here indicates the batch of soil used as a substrate: soil I (gray), soil II (black), and soil III (unfilled). In both ordinations, the indicated stress values show that the plots provide a reliable representation of the original matrix.

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Table 2.   Morisita index of community similarity among bacterial assemblages in the larval midgut and hindgut of Pachnoda marginata and Pachnoda ephippiata
 Morisita index (mean ± SD)
MidgutHindgut
  1. T-RFLP profiles (Pachnoda ephippiata midgut, n=5; hindgut, n=8; Pachnoda marginata midgut, n=3; hindgut, n=9) were generated using general Bacteria primers and restriction digestion with MspI.

P. ephippiata : P. ephippiata0.26 ± 0.240.62 ± 0.10
P. marginata : P. marginata0.11 ± 0.180.35 ± 0.21
P. ephippiata : P. marginata0.14 ± 0.150.35 ± 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.

image

Figure 3.  Phylogenetic tree of Bacteroidetes showing the relationship of the clones from the hindgut of Pachnoda ephippiata with bacterial 16S rRNA gene sequences from other intestinal environments and the closest cultivated representatives of the phylum. The clones obtained in this study are shown in bold. The analysis is based on 1337 unambiguously aligned base positions. Node support of the maximum-likelihood analysis was confirmed by maximum-parsimony analysis (1000 bootstraps; •>90%, ○>75%). The tree was rooted using selected sequences from other phyla as an outgroup. Numbers in parentheses refer to putative T-RFs determined by in silico restriction.

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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.

image

Figure 4.  Phylogenetic tree of Firmicutes showing the relationship of the clones from the hindgut of Pachnoda ephippiata with bacterial 16S rRNA gene sequences from other intestinal environments and the closest cultivated representatives of the phylum. The analysis is based on 1160 unambiguously aligned base positions. For other details, see Fig. 3.

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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).

image

Figure 5.  Phylogenetic tree of Proteobacteria showing the relationship of the clones from the hindgut of Pachnoda ephippiata with bacterial 16S rRNA gene sequences from other intestinal environments and the closest cultivated representatives of the phylum. The analysis is based on 1340 unambiguously aligned base positions. For other details, see Fig. 3.

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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.

image

Figure 6.  T-RFLP profile of the bacterial community in the hindgut sample of Pachnoda ephippiata subjected to clonal analysis. The T-RFs labeled in bold were represented in the 16S rRNA gene clone library prepared from the same DNA extract. The phylogenetic position of the corresponding clones (Bact, Bacteroidetes; Clostr, Clostridiales; Beta, Betaproteobacteria; Delta, Deltaproteobacteria; Pla, Planctomycetes) can be found in the respective trees (Figs 3–5).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Anderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Austral Ecol 66: 3246.
  • Andert J, Geissinger O & Brune A (2008) Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae). J Insect Physiol 54: 105113.
  • Ashelford KE, Chuzhanova NA, Jones AJ & Weightman AJ (2006) New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl Environ Microb 72: 57345741.
  • Blackwood CB, Hudleston D, Zak DR & Buyer JS (2007) Interpreting ecological diversity indices applied to T-RFLP data: insights from simulated microbial communities. Appl Environ Microb 73: 52765283.
  • Brune A (2006) Symbiotic associations between termites and prokaryotes. The Prokaryotes: Symbiotic Associations, Biotechnology, Applied Microbiology, Vol. 13rd edn (DworkinM, FalkowS, RosenbergE, SchleiferK-H & StackebrandtE, eds), pp. 439474. Springer, New York.
  • Brune A (2009) Symbionts aiding digestion. Encyclopedia of Insects, 2nd edn (ReshVH & CardéRT, eds), pp. 978983. Academic Press, New York.
  • Cazemier AE, Op den Camp HJM, Hackstein JHP & Vogels GD (1997) Fibre digestion in arthropods. Comp Biochem Physiol 118A: 101109.
  • Crowson RA (1981) The Biology of the Coleoptera. Academic Press, London.
  • Cruden DL & Markovetz AJ (1984) Microbial aspects of the cockroach hindgut. Arch Microbiol 138: 131139.
  • Dunbar J, Ticknor LO & Kuske CR (2001) Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Appl Environ Microb 67: 190197.
  • Egert M, Wagner B, Lemke T, Brune A & Friedrich MW (2003) Microbial community structure in midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl Environ Microb 69: 66596668.
  • Egert M, Marhan S, Wagner B, Scheu S & Friedrich MW (2004) Molecular profiling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae). FEMS Microbiol Ecol 48: 187197.
  • Egert M, Stingl U, Dyhrberg Bruun L, Wagner B, Brune A & Friedrich MW (2005) Structure and topology of microbial communities in the major gut compartments of Melolontha melolontha larvae (Coleoptera: Scarabaeidae). Appl Environ Microb 71: 45564566.
  • Felsenstein J (1989) PHYLIP, phylogeny inference package version 3.57c. Cladistics 5: 164166.
  • Henckel T, Friedrich M & Conrad R (1999) Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl Environ Microb 65: 19801990.
  • Hongoh Y, Deevong P, Inoue T, Moriya S, Trakulnaleamsai S, Ohkuma M, Vongkaluang C, Noparatnaraporn N & Kudo T (2005) Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl Environ Microb 71: 65906599.
  • Kane MD & Breznak JA (1991) Effect of host diet on production of organic acids and methane by cockroach gut bacteria. Appl Environ Microb 57: 26282634.
  • Krebs CJ (1989) Ecological Methodology. Harper and Row Publishers, New York, 654pp.
  • Krebs CJ (1994) Ecology. The Experimental Analysis of Distribution and Abundance. Harper Collins College Publisher, New York.
  • Lande R (1996) Statistics and partitioning of species diversity, and similarity among multiple communities. Oikos 76: 513.
  • Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acids Techniques in Bacterial Systematics (StackebrandtE & GoodfellowM, eds), pp. 115175. Wiley, Chichester.
  • Lemke T, Stingl U, Egert M, Friedrich MW & Brune A (2003) Physicochemical conditions and microbial activities in the highly alkaline gut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl Environ Microb 69: 66506658.
  • Li X & Brune A (2007) Transformation and mineralization of soil organic nitrogen by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Plant Soil 301: 233244.
  • Ludwig W, Bauer SH, Bauer M, Held I, Kirchhof G, Schulze R, Huber I, Spring S, Hartmann A & Schleifer KH (1997) Detection and in situ identification of representatives of a widely distributed new bacterial phylum. FEMS Microbiol Lett 153: 181190.
  • Ludwig W, Strunk O, Westram R et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32: 13631371.
  • Marsh TL, Saxmann P, Cole J & Tiedje J (2000) Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl Environ Microb 66: 36163620.
  • Matsuura K (2001) Nestmate recognition mediated by intestinal bacteria in a termite, Reticulitermes speratus. Oikos 92: 2026.
  • Minkley N, Fujita A, Brune A & Kirchner WH (2006) Nest specificity of the bacterial community in termite guts (Hodotermes mossambicus). Insect Soc 53: 339344.
  • Odelson DA & Breznak JA (1983) Volatile fatty acid production by the hindgut microbiota of xylophagous termites. Appl Environ Microb 45: 16021613.
  • Oksanen J, Kindt R, Legendre P, O'Hara B, Simpson GL, Solymos P, Stevens MHH & Wagner H (2009) vegan: Community Ecology Package. R package version 1.16-12. Available at http://vegan.r-forge.r-project.org/ (accessed November, 2009).
  • Olsen GJ, Matsuda H, Hagstrom R & Overbeek R (1994) fastDNAmL: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci 10: 4148.
  • Pester M, Friedrich MW, Schink B & Brune A (2004) pmoA-based analysis of methanotrophs in a littoral lake sediment reveals a diverse and stable community in a dynamic environment. Appl Environ Microb 70: 31383142.
  • Pittman GW, Brumbley SM, Allsopp PG & O'Neill SL (2008) Assessment of gut bacteria for a paratransgenic approach to control Dermolepida albohirtum larvae. Appl Environ Microb 74: 40364043.
  • Pruesse E, Quast C, Knittel K, Fuchs B, Ludwig W, Peplies J & Glöckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35: 71827196.
  • Ricke P, Kolb S & Braker G (2005) Application of a newly developed ARB software-integrated tool for in silico terminal restriction fragment length polymorphism analysis reveals the dominance of a novel pmoA cluster in a forest soil. Appl Environ Microb 71: 16711673.
  • Rössler ME (1961) Ernährungsphysiologische Untersuchungen an Scarabaeidenlarven (Oryctes nasicornis L., Melolontha melolontha L.). J Insect Physiol 6: 6274.
  • Santo Domingo JW, Kaufman MG, Klug MJ & Tiedje JM (1998a) Characterization of the cricket hindgut microbiota with fluorescently labeled rRNA-targeted oligonucleotide probes. Appl Environ Microb 64: 752755.
  • Santo Domingo JW, Kaufman MG, Klug MJ, Holben WE, Harris D & Tiedje JM (1998b) Influence of diet on the structure and function of the bacterial hindgut community of crickets. Mol Ecol 7: 761767.
  • Schloss PD & Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microb 71: 15011506.
  • Schlottke E (1945) Über die Verdauungsfermente im Holz fressender Käferlarven. Allg Zool Physiol Tiere 61: 88140.
  • Schmitt-Wagner D, Friedrich MW, Wagner B & Brune A (2003a) Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp). Appl Environ Microb 69: 60076017.
  • Schmitt-Wagner D, Friedrich MW, Wagner B & Brune A (2003b) Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.). Appl Environ Microb 69: 60186024.
  • Shinzato N, Muramatsu M, Matsui T & Watanabe Y (2005) Molecular phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus. Biosci Biotech Bioch 69: 11451155.
  • Terra WR & Ferreira C (1994) Insect digestive enzymes: properties, compartmentalization and function. Comp Biochem Physiol 109B: 162.
  • Tholen A, Schink B & Brune A (1997) The gut microflora of Reticulitermes flavipes, its relation to oxygen, and evidence for oxygen-dependent acetogenesis by the most abundant Enterococcus sp. FEMS Microbiol Ecol 24: 137149.
  • Werner E (1926a) Die Ernährung der Larve von Potosia cuprea. Z Morph Ökol Tiere 6: 150206.
  • Werner E (1926b) Der Erreger der Zelluloseverdauung bei der Rosenkäferlarve (Potosia cuprea Fbr.) Bacillus cellulosam fermentans n. sp.. Zbl Bakt II 67: 297330.
  • Wiedemann JF (1930) Die Zelluloseverdauung bei Lamellicornierlarven. Z Morph Ökol Tiere 19: 228258.
  • Yang H, Schmitt-Wagner D, Stingl U & Brune A (2005) Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis). Environ Microbiol 7: 916932.