Lignin degradation in wood-feeding insects Scott M. Geib*, Timothy R. Filley†, Patrick G. Hatcher‡, Kelli Hoover*, John E. Carlson§, Maria del Mar Jimenez-Gasco¶, Akiko Nakagawa-Izumiʈ, Rachel L. Sleighter‡, and Ming Tien**†† *Department of Entomology and Center for Chemical Ecology, Pennsylvania State University, University Park, PA 16802; †Department of Earth and Atmospheric Sciences, Purdue University, West Lafayette, IN 47907; ‡Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529; §School of Forest Resources, Department of Horticulture, and The Huck Institutes for Life Sciences, Pennsylvania State University, University Park, PA 16802; ¶Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802; ʈGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; and **Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 Edited by T. Kent Kirk, University of Wisconsin, Deerfield, WI, and approved July 7, 2008 (received for review May 30, 2008) The aromatic polymer lignin protects plants from most forms of the lignin barrier and gain access to the polymer carbohydrates microbial attack. Despite the fact that a significant fraction of all has been a mystery. Herein, we show chemical changes in lignin lignocellulose degraded passes through arthropod guts, the fate of upon passage of undegraded wood through two wood-feeding lignin in these systems is not known. Using tetramethylammonium insect gut systems, one that naturally feeds on living healthy trees hydroxide thermochemolysis, we show lignin degradation by two and a second that typically feeds on dead wood. insect species, the Asian longhorned beetle (Anoplophora glabrip- To determine the fate of lignin in these two systems, we ennis) and the Pacific dampwood termite (Zootermopsis angusti- analyzed the frass of both the Asian longhorned beetle (Ano- collis). In both the beetle and termite, significant levels of propyl plophora glabripennis) and the Pacific dampwood termite (Zoot- side-chain oxidation (depolymerization) and demethylation of ring ermopsis angusticollis). A. glabripennis is a wood-feeding ceram- methoxyl groups is detected; for the termite, ring hydroxylation is bycid beetle that develops in the inner wood of a wide range of also observed. In addition, culture-independent fungal gut com- hardwood trees (14), feeding on living intact wood. Z. angusti- munity analysis of A. glabripennis identified a single species of collis is a lower termite that feeds on coniferous trees, attacking fungus in the Fusarium solani/Nectria haematococca species com- dead timber. Lignin structure of the frass was analyzed by plex. This is a soft-rot fungus that may be contributing to wood unlabeled and 13C-tetramethylammonium hydroxide (TMAH) degradation. These results transform our understanding of lignin thermochemolysis, depolymerizing lignin into its aromatic sub- degradation by wood-feeding insects. units by breaking ␤-O-4 linkages and methylating all ring hydroxyls (15). The resulting product is then analyzed by GC/MS Asian longhorned beetle ͉ Pacific dampwood termite ͉ to determine how the lignin structure has been chemically TMAH thermochemolysis ͉ Anoplophora glabripennis ͉ modified. In undegraded wood, the predominant products from Zootermopsis angusticollis TMAH thermochemolysis are typically 3,4-dimethoxybenzlde- hyde (G4) from guaiacyl (G) lignin (Fig. 1) and 3,4,5- ignin plays a central role in carbon cycling on Earth. Its trimethoxybenzaldehyde (S4) from syringyl (S) lignin. Hard- Lheterogeneous structure imparts plants with structural rigid- wood tree species contain both guaiacyl and syringyl lignin, but ity and also serves to protect cellulose and hemicellulose from coniferous trees only contain guaiacyl lignin. degradation (1). Most of what is known about lignin biodegra- dation is from pure culture studies with filamentous basidiomy- Results cete fungi, known as white-rot and brown-rot decay. Although Studies with fungal lignin degradation show three predominant both white-rot and brown-rot fungal degradation have been reactions: (i) propyl side-chain oxidation/cleavage (reaction 1, characterized, much more is known about the white-rot system Fig. 1), (ii) ring hydroxylation (reaction 2, Fig. 1), and (iii) (2, 3). White-rot fungi simultaneously degrade the three major demethylation (reaction 3, Fig. 1) (15). All these processes can components of the plant cell wall: lignin, cellulose, and hemi- be detected by labeled or unlabeled TMAH analysis (Fig. 1). cellulose. Analysis of white-rot–degraded wood shows that the Side-chain oxidation is a predominant reaction with white-rot reactions in lignin: (i) are oxidative, (ii) involve demethylation fungi resulting in C␣–C␤ cleavage/depolymerization of lignin (or demethoxylation), (iii) include side-chain oxidation at C␣, (16). Previous studies demonstrate that oxidative alteration of and (iv) involve propyl side-chain cleavage between C␣ and C␤ the lignin propyl side chain results in the enhanced production (Fig. 1) (4). In contrast to white-rot fungi, brown-rot fungi are of 3,4-dimethoxybenzoic acid (G6) and 3,4,5-trimethoxybenzoic able to circumvent the lignin barrier, removing the hemicellulose acid (S6) (for hardwoods) (Fig. 2), and increases in G6/G4 and and cellulose with only minor modification to the lignin. Con- S6/S4 ratios indicate a higher degree of side-chain oxidation (16). sequently, lignin remains a major component of the degraded Frass (feces) samples from A. glabripennis that fed on pin oak plant cell wall (5). The remaining lignin is demethylated on aryl (Quercus palustris) (syringyl and guaiacyl lignin) and frass sam- methoxy groups and contains a greater number of ring hydroxyl ples from Z. angusticollis that fed on ponderosa pine (Pinus groups (6). ponderosa) (guaiacyl lignin only) were analyzed by TMAH Little is known about lignin degradation in complex ecosys- tems, such as insect guts, where a consortium of microbes may be involved in degradation rather than just a single species. Author contributions: K.H., J.E.C., M.d.M.J.-G., and M.T. designed research; S.M.G., A.N.-I., and R.L.S. performed research; T.R.F. and P.G.H. contributed new reagents/analytic tools; Although cellulose degradation in insect guts is well documented T.R.F., P.G.H., and M.d.M.J.-G. analyzed data; and S.M.G. and M.T. wrote the paper. (7, 8), the fate of lignin has not clearly been demonstrated (9, 10), The authors declare no conflict of interest. and it is widely accepted that insect gut systems do not have the This article is a PNAS Direct Submission. capacity to degrade lignin (10). Although the majority of pre- Data deposition: The A. glabripennis gut fungal ITS nucleotide sequence has been depos- vious reports suggest that many wood-feeding insects overcome ited in the GenBank database (accession no. EU862818). the lignin barrier by feeding on predegraded wood (11) or ††To whom correspondence should be addressed at: 408 Althouse Laboratory, Department through exosymbiotic relationships with wood-degrading fungi of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, (12, 13), there are species of insects that feed on the inner wood PA 16802. E-mail: [email protected]. of living, healthy trees. How these insects are able to circumvent © 2008 by The National Academy of Sciences of the USA 12932–12937 ͉ PNAS ͉ September 2, 2008 ͉ vol. 105 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805257105 Downloaded by guest on September 28, 2021 O OCH Degraded TMAH A 3 C 3 Undegraded Lignin Products OH G S H 3 CO OCH 3 S6 Lignin 100,000 2 OCH 3 O O OH O OCH 3 n OH C oxidatio chain e TMAH OH 1. Sid 80,000 1 O OCH + Hydrolysis C 3 OCH OCH 3 OCH 3 OH L 3 Acid:Aldehyde O O O OCH 3 L L 60,000 0 2. Ring G6 U D OCH 3 hydroxylatio G6 OCH 3 OH H3CO OCH 3 n CHO CHO O OH L H3CO 40,000 L O OCH 3 S4 3. Ringdemethyla TMAH OCH 3 H 3 CO OCH 3 G4 OCH Hydrolysis 20,000 3 OCH 3 OH OCH 3 H3CO OCH 3 MS Signal Abundance TMAH O O L H3CO Hydrolysis tion S14/15 OH H3*CO 500 520 540 560 580 600 620 OH L H3*CO OC*H 3 CHO O O Retention Time (s) 13 C-TMAH Hydrolysis OCH OH OC*H O OCH3 3 3 C OCH O O B 3 3 L H3*CO G G4 G14/15 100,000 2 G6 OCH3 OCH3 Fig. 1. Lignin biodegradation reactions. Reactions include propyl side-chain O OCH C 3 oxidation/cleavage (reaction 1), ring hydroxylation (reaction 2), and demeth- 80,000 1 ylation (reaction 3). Reaction 1 is characteristic of white-rot fungus, whereas Acid:Aldehyde H CO OCH 60,000 0 3 3 reactions 2 and 3 are characteristic of brown-rot fungus. Application of TMAH U D OCH3 thermochemolysis to each of the degradative reactions is pictured following CHO each reaction, showing chemical structures of compounds G4, G6, G14/15, and 40,000 G4 CHO 13 H3CO OCH3 S14/15 that result from this analysis. Asterisks represent C-labeled carbons OCH3 13 OCH resulting from C-TMAH analysis. 20,000 3 MS Signal Abundance S4 OCH3 S6 thermochemolysis/GC/MS (Fig. 2 A and B). Both digested 500 520 540 560 580 600 620 samples exhibited significant lignin side-chain oxidation (two- Retention Time (s) tailed Student’s t test). For A. glabripennis-digested pin oak (n ϭ 6), relative to undigested samples (n ϭ 6), the G6/G4 ratio Ͻ Fig. 2. Side-chain oxidation and hydroxylation of wood after passage increased from 0.49 to 2.4 (P 0.001) and the S6/S4 ratio through insect guts. Chromatograms show increases in G6 and S6 in wood fed increased from 0.26 to 2.4 (P Ͻ 0.0001) (Fig.