Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes

Abstract

Background: Termite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite Reticulitermes flavipes. Our approach consisted of parallel high-throughput sequencing from (i) a host gut cDNA library and (ii) a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing.

Results: Over 10,000 expressed sequence tags (ESTs) were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist) glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450). Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding.

Conclusion: To our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort has been conducted in a single termite species. This sequence database represents an important new genomic resource for use in further studies of collaborative host-symbiont termite digestion, as well as development of coevolved host and symbiont-derived biocatalysts for use in industrial biomass-to-bioethanol applications. Additionally, this study demonstrates that: (i) phenoloxidase activities are prominent in the R. flavipes gut and are not symbiont derived, (ii) expands the known number of host and symbiont glycosyl hydrolase families in Reticulitermes, and (iii) supports previous models of lignin degradation and host-symbiont collaboration in cellulose/hemicellulose digestion in the termite gut. All sequences in this paper are available publicly with the accession numbers FL634956-FL640828 (Termite Gut library) and FL641015-FL645753 (Symbiont library).

Figure 1

Expressed sequence tag (EST) sequencing results. (a) and (c) present sequencing summaries for the host (termite gut tissue) and symbiont cDNA libraries, respectively. (b) and (d) are the taxonomic distributions of top BLASTX hits for putative transcripts from the termite gut and symbiont libraries (respectively). Hits producing an E-value < 10^-5 were considered significant. The charts illustrate that the majority of transcripts sequenced from the termite gut library were homologous to insect sequences, whereas the majority of transcripts obtained from the symbiont library were homologous to genes previously sequenced from unicellular eukaryotes.

Figure 2

Protein neighbor-joining phylogeny of glycoside hydrolase family 11 (GHF11). The tree was rooted with bacterial GHF11 enzyme sequences and showed that all GHF11 transcripts generated during this study clustered with GHF11 genes previously sequenced from termite symbionts (uncultured symbiotic protist (USP)). The transcripts identified in the termite gut (TG) library are shown in blue, whereas the transcript sequences that originated from the termite symbiont (TS) library are shown in red. Numbers (100/100) above the node represent bootstrap support (1000 replicates). For clarity purposes only relevant bootstrap values are indicated.

Figure 3

Distributions of expressed sequence tags (ESTs) encoding glycosyl hydrolase family (GHF) proteins generated from the host gut tissue and hindgut symbiont libraries. (a) A summary of GHF family members with enzyme identities and putative taxonomic origins that were determined based on library of origin and database homology. Shaded rows depict GHF families with representatives from both termite and putative prokaryotic symbionts. CE = cellulase, H = hemicellulase, CH = chitinase, A = α carbohydrolase. (b) Bar graph summarizing the taxonomic distributions of cellulase, hemicellulase, chitinase and α carbohydrolase genes among prokaryotic, protist and host genomes. See Additional file 1 for gene by gene summaries and Additional file 3 for Genbank accession numbers.

Figure 4

Distributions of expressed sequence tags (ESTs) encoding (a) glycosyl transferase family (GTF), (b) carbohydrate esterase (CE), (c) carbohydrate binding modules (CBM), and (d) miscellaneous carbohydrate-active and hydrogen-active proteins generated from the host gut tissue and hindgut symbiont libraries. (a-d) Summaries of GHF family members with protein identities and putative taxonomic origins that were determined based on library of origin and database homology. Shaded rows depict gene families not included in the carbohydrate active enzyme (CAZy) database. (e) Bar graph summarizing the distributions of glycosyl transferase, carbohydrate binding, carbohydrate esterase, Fe-hydrogenase, and ferredoxin oxidoreductase genes among prokaryotic, protist and host genomes. See Additional files 2 and 5 for gene by gene summaries and Additional files 4 and 10 for Genbank accession numbers.

Figure 5

Summary of potential lignase (L), detoxification (D), and antioxidant (A) coding genes identified from the termite gut library. No homologous or functionally similar genes were identified from the symbiont library. Black bars indicate gene families investigated in later functional studies (see Figures 6-8). See Additional file 6 for gene by gene summaries and Additional file 7 for Genbank accession numbers.

Figure 6

Functional analysis of phenoloxidase activity towards the model substrate pyrogallol. (a) pH dependence of pyrogallol oxidation determined using whole guts of worker termites. (b) Distribution of pyrogallol oxidation activity across the termite gut using 14,000 g supernatants from homogenized tissues of the foregut plus salivary gland (FG), midgut (MG), and hindgut (HG). (c) Feeding by live termites for 7 days on papers treated with lignin alkali at concentrations ranging from 0% to 0.625% w/v. (d) Pyrogallol oxidation activity in whole termite guts after feeding on various concentrations as depicted in (c). Bars with the same letters are not significantly different by least significant difference (LSD) t tests (P < 0.05).

Figure 7

Laccase, catalase, RfEst3 esterase, and β actin (as a control) gene expression in Reticulitermes flavipes worker guts as determined from 35 cycles of conventional polymerase chain reaction (PCR) amplification. Shown is a negative image of a representative agarose gel with equal loadings of PCR products for the four genes. Lane labels are as follows: FG, foregut plus salivary gland; MG, midgut; HG, hindgut; and (-), no template control. Two primer sets were tested for the laccase and catalase genes, and one primer set for esterase and β actin. A 100 base pair (bp) low molecular weight ladder (LML) is shown at the left and right. See Additional file 9 for PCR primer sequences.