1157 Cellulase Activity in Higher and Lower Wood-Feeding Termites by Zhi-Qiang Li, Wen-Hui Zeng, Jun-Hong Zhong, Bing-Rong Liu, Qiu-Jian Li & Wei-Liang Xiao1 ABSTRACT Wood-feeding termites have evolved efficient cellulose-decomposing sys- tems. The cellulase activity and distribution of workers were studied in three wood-feeding termites from phylogenetically different lineages; the lower termites, Crypototermes domesticus (Haviland) (Isoptera: Kalotermitidae) and Coptotermes formosanus Skiraki (Rhinotermitidae), and the higher termite, Ahmaditermes sichuanensis Xia et al. (Termitidae). The results showed that the total cellulase activity of the higher termite A. sichuanensis was markedly higher than that of the lower termites. Co. formosanus had the highest activity of endo-β-1,4-glucanase and cellobiohydrolase. The activity of β-glucosidase was not significantly different among the three species. The proportion of cellobiohydrolase was higher in the higher termite. In terms of distributions of cellulolytic activity in the gut, the primary site of endo-β-1,4-glucanase activity was presented in the hindgut of both the lower termites but in the midgut of the higher termite, and the primary site of β-glucosidase activity was restricted to the midgut in the lower termites and the head/foregut in the higher termite. The functions of the gut segments were apparently differ- entiated between the lower and higher termites, with the role of the midgut becoming more important in the higher termite. For the endogenous cellu- lases, the main site of endo-β-1,4-glucanase activity was concentrated in the midgut in both the lower termite and the higher termite, but the main site of β-glucosidase activity was in the head/foregut in the higher termite. The results suggest that characters of cellulase activity could reflect the phylogeny of wood-feeding termites to a certain extent. Key words: Crypototermes domesticus, Coptotermes formosanus, Ahma- ditermes sichuanensis, endo-β-1,4-glucanase, exoglucanase, β-glucosidase, cellobiohydrolase Guangdong entomological institute, 105 Xingang West Road, Guangzhou 510260, China, 1Corresponding author, Email: xwl@gdei.gd.cn 1158 Sociobiolog y Vol. 59, No. 4, 2012 INTRODUCTION Cellulose is the most abundant, widespread and renewable biomass on the earth. Biotic fuels generated from cellulose could replace petroleum, but economic production of cellulosic biotic fuels is dependent on the growth of more efficient and cheaper cellulases that can be applied into both pre- treatment and cellulolytic technologies (Mosier et al. 2005; Emsley 2008). In nature, xylophagous insects have evolved strategies to derive their energ y needs from celluloses with cellulases, of which termites are the most efficient decomposers of celluloses (Sugimoto et al. 2000; Watanabe & Tokuda 2010). Termites are important decomposers in tropical ecosystems (Noble et al. 2009), and cellulase enzymes and cellulase genes in the digestive systems of termites may have potential for cellulosic ethanol production by biological processes (Ni 2007; Warnecke et al. 2007; Li et al. 2009). Termites have specialized cellulose-digesting systems (Nakashima et al. 2002; Tokuda et al. 2007; Zhou et al. 2007). Various cellulases are involved in degradation of celluloses in termites and their symbionts. The related in- vestigation of cellulase activity in termites is an important research field. The three main types of cellulases are endo-β-1,4-glucanases (EGs; EC 3.2.1.4), cellobiohydrolases (CBHs; EC 3.2.1.91), and β-glucosidases (BGs; EC 3.2.1.21), and cellulose degradation requires the synergistic action of the three types of glycoside hydrolases (Lo et al. 2011). Patterns and characters of cellulases in termites and their symbionts have been described extensively (Watanabe and Tokuda 2010; Willis et al. 2010), and cellulase activity and their distributions in the digestive systems were different among diverse ter- mites. Recently, distributions of diverse cellulase activity in each gut segments of termites were mostly studied (Willis et al. 2010), and Tokuda et al. (2004, 2005) found that the expression of the endogenous cellulase genes has shifted from the salivary glands of lower termites to the midgut of higher termites. In addition, differences in total cellulase activity were compared among lower termites (Mo et al. 2004; Lu et al. 2010), and EG expression among differ- ent castes of Hodotermopsis sjostedti (family Termopsidae) and Nasutitermes takasagoensis (family Termitidae) have been reported (Fujita et al. 2008). Recent research suggests that the distributions of cellulase activity in termites are related to their evolutional levels (Tokuda et al. 2004, 2005). 1159 Li, Z.-Q. et al. — Cellulase Activity in Higher and Lower Termites At present, there are approximately 2,600 described termite species around the world (Kambhampati & Eggleton 2000), and comprehensive studies of phylogenetic and taxonomic relationships among termite groups have been published (Donovan et al. 2000; Legendre et al. 2008). However, the cellu- lase activities of only around 16 termite species have been reported (Lu et al. 2010; Lo et al. 2011). Additional research can expand the knowledge of the cellulose-digesting mechanisms of termites, and provide a reference basis for the development of cellulosic ethanol (Rubin 2008) and environmentally- friendly biotermicides (Zhang et al. 2011). Termites feed on dead plant material at all stages of decomposition. Be- sides wood, they feed on soil, leaf litter, fungi, grass, or lichen (Donovan et al. 2000). On the whole, cellulase activity levels in wood-feeding termites are far higher than those found in the fungus-growers and the soil-feeders (Tokuda et al. 2004; Lo et al. 2011). To provide further information about the relationship between cellulolytic activity and evolutionary status in wood- feeding termites from phylogenetically different lineages, the cellulase activ- ity and distribution of workers were studied in three wood-feeding termites from phylogenetically different lineages; the lower termites, Crypototermes domesticus (Haviland) (Isoptera: Kalotermitidae) and Coptotermes formosanus Skiraki (Rhinotermitidae), and the higher termite, Ahmaditermes sichuanensis Xia et al. (Termitidae). MATERIALS AND METHODS Termites Cr. domesticus were collected from two laboratory-maintained colonies. Three Co. formosanus colonies and two A. sichuanensis colonies were collected on Luofu mountain, Guangdong Province in China. The caste of termites used for experiments were healthy adult workers, but pseudoworkers for Cr. domesticus. The workers or pseudoworkers were directly put into liquid nitrogen before enzyme extraction. Preparation of crude enzyme To prepare enzyme extracts, the workers or pseudoworkers were washed with precooling 0.09% normal saline. Fifteen sets per termite colony of heads (including salivary glands) and whole guts were dissected from termites, and 1160 Sociobiolog y Vol. 59, No. 4, 2012 each set was divided into head/foregut, midgut, and hindgut. The three sec- tions and other five bodies of worker per colony were collected in tubes and homogenized in 500 μL of 0.1 M sodium acetate buffer (SAB) ( pH 5.6) on ice. The tubes were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were brought to volume of 500 μL by adding 0.1 M SAB and used as the enzyme extract. The same volume of 0.1 M SAB was used as the control. Assay of cellulase activity Filter paper degrading activity Circular filter paper (3.5 mg per piece) after high temperature sterilization was put into the microtubes with 120μL SAB (pH 5.6), and the crude enzyme (12 μL) was incubated with the filter paper at 37 °C for 60 min. Based on dinitrosalicylic acid method (Eveleigh et al. 2009), 120 μL dinitrosalicylic acid solution was added and boiled for 5min. Then, the tubes were cooled to room temperature rapidly. The glucose production was detected colorimetrically with Victor 3 Multi-label Microplate Reader (Perkin Elmer, US) at 540nm, using glucose as a standard. The protein content of the sample was determined spectrophotomerically at 660nm according to the Coomassie Brilliant Blue G-250 method (Lott et al. 1983), using bovine serum as a standard. One unit (U) of enzyme activity was defined as the amount of enzyme capable of releasing one μmol reducing sugar per minute. Specific activity was expressed as units per mg protein. The enzyme assays were repeated three times. Endo-β-1,4-glucanase and β-glucosidase activity The activity of both EG and BG were determined using 120 μL of 1% sodium carboxymethylcellulose and 120 μL of 1% salicin as the substrates, respectively. Other steps and definitions were the same as the assay of filter paper degrading activity mentioned above. Cellobiohydrolase activity Cellobiohydrolase was assayed using p-nitrophenyl-β-D-cellobioside (pNPC) as the substrate. 120 μL of 0.1 mM pNPC was mixed with 12 μL crude enzyme at 37 °C for 60 min. The incubation was stopped by adding 120 μL of 1 M sodium carbonate, and diluted with SAB to 500 μL. The amount of p-nitrophenyl was measured at 405 nm. 1161 Li, Z.-Q. et al. — Cellulase Activity in Higher and Lower Termites Data analysis The data were analyzed by one-way analysis of variance with least significant difference post-hoc tests (LSD) with SPSS 16.0 for Windows Software. RESULTS Differences in cellulase activity among termites The data (Table 1) of cellulase activity of whole bodies of termite work- ers (or pseudoworkers) showed that the BG activities were not significantly different among the three termites, but activity of both EG and CBH was high in Co. formosanus. Regarding the proportion of different cellulose types within species, EG activity was the principal component in all three termites. However, CBH activity of A. sichuanensis was not significantly different from EG or BG. Filter paper assay Filter paper degradation is directly related to digestibility of naturally occurring cellulose. According to the data of filter paper degrading activity (Table 2), the most evolved species, A. sichuanensis, had the highest activity of filter paper degrading activity in the whole body of three wood-feeding Table 1 Complete cellulase activity of whole bodies in the termite workers tested. Species Whole body activity endo-β-1,4-glucanase β-glucosidase cellobiohydrolase Crypototermes domesticus 0.347±0.028 bA 0.116±0.007 aB 0.016±0.000 bC Coptotermes formosanus 0.780±0.047 aA 0.426±0.057 aB 0.243±0.079 aB Ahmaditermes sichuanensis 0.525±0.122 bA 0.747±0.470 aA 0.015±0.000 bA Mean±S.E. with different small letter means significant difference in the same column, while different capital letter means significant difference in the same line at the 0.05 probability level. The same as below. Table 2 Filter paper degrading activity of different segments and whole body in the termite workers. Species Filter paper degrading activity Whole Body Head/Foregut Midgut Hindgut Crypototermes domesticus 0.167±0.036 b 0.139±0.055aA 0.130±0.057aA 0.155±0.072aA Coptotermes formosanus 0.241±0.031ab 0.131±0.027aB 0.107±0.017aB 0.229±0.022aA Ahmaditermes sichuanensis 0.417±0.102a 0.213±0.087aC 0.326±0.157aA 0.288±0.051aB 1162 Sociobiolog y Vol. 59, No. 4, 2012 termites, but there were no significant differences among filter paper degrading activity of the three termites in each gut segment. Regarding distributions of filter paper degrading activity in guts of the termites, A. sichuanensis showed significantly different filter paper degrading activity among three gut segments, and the midgut was the main segment of filter paper degrading activity in A. sichuanensis. Distribution of EG and BG activities in the gut According to the data from the hindgut (Table 3), it is suggested that flag- ellate-harbouring termites possess a higher percentage of intestinal microbial EG and BG than A. sichuanensis. In addition, activities of both EG and BG were not significantly different in the midgut among the three wood-feeding termites, and neither was BG activity in the hindgut. Co. formosanus had the highest activity of EG in head/foregut and hindgut, and A. sichuanensis had the highest activity of BG in head/foregut. For endogenous endoglucanase and β-glucosidase, EG activities were not significantly different between the head/foregut and midgut of Cr. domesticus, or Co. formosanus. However, the higher termite A. sichuanensis had higher EG activity in the midgut. In contrast, the higher BG activity in A. sichuanensis was in the head/foregut. DISCUSSION Termites play an important role in degradation of cellulosic materials in nature, and special attention is paid to the activity and expression of termite cellulases (Watanabe & Tokuda 2010). Efficient cellulose digestion in termites requires both endogenous and intestinal microbial cellulases (Tokuda 2007; Table 3 Endo-β-1,4-glucanase and β-glucosidase activity of different segments in the termite workers. Cellulases Speices Cellulase acitivity in each gut segment Head/Foregut Midgut Hindgut endo-β-1,4-glucanase Crypototermes domesticus 0.152±0.016bB 0.185±0.073aB 0.499±0.020bA Coptotermes formosanus 0.363±0.016aB 0.295±0.120aB 0.818±0.126aA Ahmaditermes sichuanensis 0.214±0.075bC 0.399±0.085aA 0.289±0.079bB β-glucosidase Crypototermes domesticus 0.080±0.005bC 0.160±0.006aA 0.116±0.007aB Coptotermes formosanus 0.153±0.068bA 0.314±0.107aA 0.200±0.033aA Ahmaditermes sichuanensis 0.363±0.033aA 0.305±0.162aB 0.289±0.103aC 1163 Li, Z.-Q. et al. — Cellulase Activity in Higher and Lower Termites zhou 2007). The present study showed that the hindgut was the primary site of cellulose digestion in lower termites, which was consistent with the report of Tokuda et al. (2005). As for endogenous termite cellulases, Tokuda et al. (2004) proposed that the expression of the endogenous cellulase genes has shifted from the salivary glands of lower termites to the midgut of higher termites. In terms of the dynamic shift in termites, our results showed that activity of EG and filter paper degrading activity were most highly concentrated on the midgut of the higher termite studied than that of the lower termites, which supported the previous studies on EG (Mo et al. 2004; Tokuda et al. 2004, 2005; Fujita et al. 2008; Tokuda et al. 2009; Lo et al. 2011), but the dynamic change of BG was contrary to the related report of Fujita et al. (2008) and Tokuda et al. (2009). They suggested that the main position of BG activity tended to the head/foregut with evolution of wood-feeding termites, and the evolved wood-feeding termites such as the termites of the Rhinotermitidae and Termitidae may have higher BG activity in the head/foregut. As for the comparison of cellulase activities in whole bodies of termites, it was suggested that the degrading activity of filter paper and the percentage value of CBH in complete cellulases were increased with a rise in evolutionary status. The higher termite A. sichuanensis had the highest degrading activity of filter paper and the highest percentage value in complete cellulases. How- ever, a previous study showed that activity in the higher termite Nasutitermes takasagoensis was markedly lower using microcrystalline cellulose as substrate than that of flagellate-harbouring termites (Tokuda et al. 2005).Because of this, Tokuda et al. (2005) considered the cellulase activity of N. takasagoensis was likely to be similar to its requirement for energ y metabolism. In addition, filter paper, microcrystalline cellulose and cotton have been used as cellulase substrates to determine the existence of complete cellulases (Willis et al. 2010), but there is evidence for the degradation of microcrystalline cellulose in insects lacking CBH activity (Scrivener and Slaytor 1994). Co. formosanus is one of the most destructive and invasive pests around the world, and its cellulase activity has been intensively studied on different substrates (Willis et al. 2010). Co. formosanus had the highest activity of EG and BG among termites (Tokuda et al. 2005; Lu et al. 2010). The present study showed that Co. formosanus had the highest degrading activity of not 1164 Sociobiolog y Vol. 59, No. 4, 2012 only EG but also CBH in the three termites studied, but its BG activity was not significantly different from species of the family Kalotermitidae which was consistent with the report of Mo et al. (2004). In addition, In terms of the synthetic cellulase activity in wood-feeding termites, A. sichuanensis and Co. formosanus showed higher activities of EG and CBH. Use of diverse cellulase substrates can help differentiate the specific contribution of specific insect gut areas to the digestion of plant material (Tokuda et al. 2005). For cellulase activity assays, the data are often difficult to compare among studies. Multinomial factors can influence the cellulase activity, such as tem- perature, substrate, detection method and so on (Tokuda et al. 2005; Willis et al. 2010). Furthermore, phylogenetic analysis of cellulase genes from the termite lineages has received special attention (Todaka et al. 2010). The rela- tive expression levels of cellulase genes do not correspond with their activity, which could be affected by different regulators (Fujita et al. 2008). In addition, for the workers of higher termites, the cellulose-digesting division of labor might indirectly influence the determination results (Fujita et al. 2008). ACKNOWLEDGMENTS We thank professor Li-Ying Li for English language revision. This work was supported by the Guangdong Academy of Sciences Foundation for Excellent Young Scientists and Technicians (200901) and the National Natural Science Foundation of China (31172163). 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