Cloning, Expression, and Immunological Characterization of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Arginine Kinase.

Several parts of the world regularly consume termites. Arthropod arginine kinase proteins often cross-react with human immunoblobulin E (IgE) antibodies and they are considered pan-allergens. The Formosan subterranean termite Coptotermes formosanus (C. formosanus (Shiraki) [Isoptera: Rhinotermitidae]), along with cockroaches, belong to the order Blattodea and they are common household pests in tropical and subtropical parts of the world. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band migrating at approximately 37 kDa in C. formosanus termite extracts cross-reacted with IgE from five cockroach allergic patient samples by immunoblot. Liquid chromatography-mass spectrometry analysis of gel slices from the corresponding region of a gel indicated several peptides from the excised region were identical to the American cockroach arginine kinase allergen, Per a 9. The sequence of the full-length C. formosanus arginine kinase gene indicates the protein it encodes is 96% identical to American cockroach Per a 9, 94% identical to German cockroach Bla g 9, and 82-84% identical to shrimp arginine kinase proteins Pen m 2, Lit v 2, and Cra c 2. Full-length C. formosanus arginine kinase was fused to a glutathione S-transferase tag and recombinantly expressed and purified from Escherichia coli by affinity chromatography. The recombinant protein was recognized by IgE from 11 of 12 cockroach or shrimp allergic samples, but did not cross-react with dust mite allergic or peanut/tree nut allergic samples. The results of this study indicate the C. formosanus arginine kinase cross-reacts with cockroach and shrimp allergic IgE, and if consumed would likely act as an allergen.

Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris.

BACKGROUND: Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet. RESULTS: A mutant RsEGm (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEGm, with optimum pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEGm against Carboxymethyl Cellulose (CMC) were determined, with K m and V max of 7.6 mg/ml and 5.4 µmol/min•mg respectively. Co2+, Mn2+ and Fe2+ enhanced the activity of RsEGm by 32.0, 19.5 and 11.2% respectively, while Pb2+ and Cu2+ decreased its activity by 19.6 and 12.7% separately. CONCLUSIONS: RsEGm could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals.

Enzyme Activities at Different Stages of Plant Biomass Decomposition in Three Species of Fungus-Growing Termites.

Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites.IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished.

Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the characterization of a multimodular ß-1,4-xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular ß-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50 °C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Phenoloxidase activity in the infraorder Isoptera: unraveling life-history correlates of immune investment.

Within the area of ecological immunology, the quantification of phenoloxidase (PO) activity has been used as a proxy for estimating immune investment. Because termites have unique life-history traits and significant inter-specific differences exist regarding their nesting and foraging habits, comparative studies on PO activity can shed light on the general principles influencing immune investment against the backdrop of sociality, reproductive potential, and gender. We quantified PO activity across four termite species ranging from the phylogenetically basal to the most derived, each with their particular nesting/foraging strategies. Our data indicate that PO activity varies across species, with soil-dwelling termites exhibiting significantly higher PO levels than the above-ground wood nester species which in turn have higher PO levels than arboreal species. Moreover, our comparative approach suggests that pathogenic risks can override reproductive potential as a more important driver of immune investment. No gender-based differences in PO activities were recorded. Although termite PO activity levels vary in accordance with a priori predictions made from life-history theory, our data indicate that nesting and foraging strategies (and their resulting pathogenic pressures) can supersede reproductive potential and other life-history traits in influencing investment in PO. Termites, within the eusocial insects, provide a unique perspective for inferring how different ecological pressures may have influenced immune function in general and their levels of PO activity, in particular.

Metatranscriptomic profiles of Eastern subterranean termites, Reticulitermes flavipes (Kollar) fed on second generation feedstocks.

BACKGROUND: Second generation lignocellulosic feedstocks are being considered as an alternative to first generation biofuels that are derived from grain starches and sugars. However, the current pre-treatment methods for second generation biofuel production are inefficient and expensive due to the recalcitrant nature of lignocellulose. In this study, we used the lower termite Reticulitermes flavipes (Kollar), as a model to identify potential pretreatment genes/enzymes specifically adapted for use against agricultural feedstocks. RESULTS: Metatranscriptomic profiling was performed on worker termite guts after feeding on corn stover (CS), soybean residue (SR), or 98% pure cellulose (paper) to identify (i) microbial community, (ii) pathway level and (iii) gene-level responses. Microbial community profiles after CS and SR feeding were different from the paper feeding profile, and protist symbiont abundance decreased significantly in termites feeding on SR and CS relative to paper. Functional profiles after CS feeding were similar to paper and SR; whereas paper and SR showed different profiles. Amino acid and carbohydrate metabolism pathways were downregulated in termites feeding on SR relative to paper and CS. Gene expression analyses showed more significant down regulation of genes after SR feeding relative to paper and CS. Stereotypical lignocellulase genes/enzymes were not differentially expressed, but rather were among the most abundant/constitutively-expressed genes. CONCLUSIONS: These results suggest that the effect of CS and SR feeding on termite gut lignocellulase composition is minimal and thus, the most abundantly expressed enzymes appear to encode the best candidate catalysts for use in saccharification of these and related second-generation feedstocks. Further, based on these findings we hypothesize that the most abundantly expressed lignocellulases, rather than those that are differentially expressed have the best potential as pretreatment enzymes for CS and SR feedstocks.

The scent of royalty: a p450 gene signals reproductive status in a social insect.

Cooperation requires communication; this applies to animals and humans alike. The main communication means differ between taxa and social insects (ants, termites, and some bees and wasps) lack the cognitive abilities of most social vertebrates. Central to the regulation of the reproductive harmony in insect societies is the production of a royalty scent which signals the fertility status of the reproducing queen to the nonreproducing workers. Here, we revealed a central genetic component underlying this hallmark of insect societies in the termite Cryptotermes secundus. Communication between queens and workers relied upon the expression of a gene, Neofem4, which belongs to the cytochrome P450 genes. We inhibited Neofem4 in queens by RNA interference. This resulted in the loss of the royalty scent in queens and the workers behaved as though the queen were absent. The queen's behavior was not generally affected by silencing Neofem4. This suggests that the lack of the royalty scent lead to workers not recognizing her anymore as queen. P450 genes are known to be involved in the production of chemical signals in cockroaches and their expression has been linked to a major fertility regulator, juvenile hormone. This makes P450 genes, both a suitable and available evolutionary substrate in the face of natural selection for production of a queen substance. Our data suggest that in an organism without elaborate cognitive abilities communication has been achieved by the exploitation of a central gene that links the fertility network with the chemical communication pathway. As termites and social Hymenoptera seem to share the same class of compounds in signaling fertility, this role of P450 genes might be more widespread across social insects.

EFFECTS OF FIVE DIVERSE LIGNOCELLULOSIC DIETS ON DIGESTIVE ENZYME BIOCHEMISTRY IN THE TERMITE Reticulitermes flavipes.

Termites have recently drawn much attention as models for biomass processing, mainly due to their lignocellulose digestion capabilities and mutualisms with cellulolytic gut symbionts. This research used the lower termite Reticulitermes flavipes to investigate gut enzyme activity changes in response to feeding on five diverse lignocellulosic diets (cellulose filter paper [FP], pine wood [PW], beech wood xylan [X], corn stover [CS], and soybean residue [SB]). Our objectives were to compare whole-gut digestive enzyme activity and host versus symbiont contributions to enzyme activity after feeding on these diets. Our hypothesis was that enzyme activities would vary among diets as an adaptive mechanism enabling termites and symbiota to optimally utilize variable resources. Results support our "diet-adaptation" hypothesis and further indicate that, in most cases, host contributions are greater than those of symbionts with respect to the enzymes and activities studied. The results obtained thus provide indications as to which types of transcriptomic resources, termite or symbiont, are most relevant for developing recombinant enzyme cocktails tailored to specific feedstocks. With regard to the agricultural feedstocks tested (CS and SB), our results suggest endoglucanase and exoglucanase (cellobiohydrolase) activities are most relevant for CS breakdown; whereas endoglucanase and xylosidase activities are relevant for SB breakdown. However, other unexplored activities than those tested may also be important for breakdown of these two feedstocks. These findings provide new protein-level insights into diet adaptation by termites, and also complement host-symbiont metatranscriptomic studies that have been completed for R. flavipes after FP, PW, CS, and SB feeding.

The complexities of hydrolytic enzymes from the termite digestive system.

The main challenge in second generation bioethanol production is the efficient breakdown of cellulose to sugar monomers (hydrolysis). Due to the recalcitrant character of cellulose, feedstock pretreatment and adapted hydrolysis steps are needed to obtain fermentable sugar monomers. The conventional industrial production process of second-generation bioethanol from biomass comprises several steps: thermochemical pretreatment, enzymatic hydrolysis and sugar fermentation. This process is undergoing continuous optimization in order to increase the bioethanol yield and reduce the economic cost. Therefore, the discovery of new enzymes with high lignocellulytic activity or new strategies is extremely important. In nature, wood-feeding termites have developed a sophisticated and efficient cellulose degrading system in terms of the rate and extent of cellulose hydrolysis and exploitation. This system, which represents a model for digestive symbiosis has attracted the attention of biofuel researchers. This review describes the termite digestive system, gut symbionts, termite enzyme resources, in vitro studies of isolated enzymes and lignin degradation in termites.

cDNA cloning and heterologous expression of an endo-ß-1,4-glucanase from the fungus-growing termite Macrotermes barneyi.

Major ß-glucosidase (BG) and endo-ß-1,4-glucanase (EG) activities were localized to the midgut of the fungus-growing termite Macrotermes barneyi. Previously, we obtained the endogenous BG gene (MbmgBG1) from the midgut of M. barneyi. Here, we report the cDNA cloning of another endogenous cellulase, the EG protein MbEG1. This cellulase was partially purified from crude extract of the midgut of worker termites using zymogram analysis. Based on the N-terminal amino acid sequence and using rapid amplification of cDNA ends (RACE), a full-length cDNA of 1,843 base pairs was obtained. This encoded 448 amino acids and the sequence was similar to that of the members of glycoside hydrolase family 9. The MbEG1 transcript was detected primarily in the midgut using quantitative real-time polymerase chain reaction (PCR). To confirm functional activity of MbEG1, heterologous expression was conducted in both Escherichia coli and Pichia pastoris expression systems. Results indicated that MbEG1 could be functionally expressed in P. pastoris. This study provides the information that may facilitate understanding of cellulolytic systems in fungus-growing termites.

Trehalase activity in fungus-growing termite, Odontotermes feae (Isoptera: Termitideae) and inhibitory effect of validamycin.

Trehalase is the hydrolytic enzyme that catalyzed the hydrolysis of trehalose to glucose. In this study, trehalase activity in the fungus-growing termite, Odontotermes feae Wasmann had been examined. Trehalase activity in digestive tract and carcass of O. feae was higher than that in wood-feeding termite, Coptotermes gestroi Wasmann. The intestinal tract of worker caste of O. feae was the main source of trehalase compared with that in salivary, fat body, and carcass. In particular, the highest activity was found in the midgut and hindgut parts. More specifically, the contents of midgut and hindgut had higher enzyme activity compared with that trehalase prepared from their epithelial tissue. The enzyme activity of gut trehalase in three different termite castes, worker, soldier, and reproductive, had been determined. The result showed that female alate had the highest activity, followed by worker, male alate, and soldier castes. Trehalose concentration in the reproductive caste was at lowest level, while soldier and worker contained the high trehalose concentration. This study indicates that high trehalase activity locates in the midgut and hindgut contents and change in trehalase activity in fungus-growing termite is caste-specific. Validamycin inhibited trehalase activity of O. feae in vivo and caused high mortality, indicating that this trehalase inhibitor is valuable tools for termite control.

MALDI-TOF MS analysis of cellodextrins and xylo-oligosaccharides produced by hindgut homogenates of Reticulitermes santonensis.

Hindgut homogenates of the termite Reticulitermes santonensis were incubated with carboxymethyl cellulose (CMC), crystalline celluloses or xylan substrates. Hydrolysates were analyzed with matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS). The method was first set up using acid hydrolysis analysis to characterize non-enzymatic profiles. Commercial enzymes of Trichoderma reesei or T. longibrachiatum were also tested to validate the enzymatic hydrolysis analysis. For CMC hydrolysis, data processing and visual display were optimized to obtain comprehensive profiles and allow rapid comparison and evaluation of enzymatic selectivity, according to the number of substituents of each hydrolysis product. Oligosaccharides with degrees of polymerization (DPs) ranging from three to 12 were measured from CMC and the enzymatic selectivity was demonstrated. Neutral and acidic xylo-oligosaccharides with DPs ranging from three to 11 were measured from xylan substrate. These results are of interest for lignocellulose biomass valorization and demonstrated the potential of termites and their symbiotic microbiota as a source of interesting enzymes for oligosaccharides production.

A GHF7 cellulase from the protist symbiont community of Reticulitermes flavipes enables more efficient lignocellulose processing by host enzymes.

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7-3, GHF7-5, and GHF7-6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full-length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus-insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7-3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and ß-glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell-1), beta-glucosidase (ß-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and ß-glu. Finally, GHF7-3, Cell-1, and ß-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only ß-glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7-3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.

Character of cellulase activity in the guts of flagellate-free termites with different feeding habits.

Cellulose digestion in termites (Isoptera) is highly important for ecological reasons and applications in biofuel conversion. The speciose Termitidae family has lost flagellates in the hindgut and developed diverse feeding habits. To address the response of cellulase activity to the differentiation of feeding habits, a comparative study of the activity and distribution of composite cellulases, endo-ß-1,4-glucanase, and ß-glucosidase was performed in seven common flagellate-free termites with three feeding habits: the humus-feeding termites Sinocapritermes mushae (Oshima et Maki), Malaysiocapritermes zhangfengensis Zhu, Yang et Huang and Pericapritermes jiangtsekiangensis (Kemner); the fungus-growing termites Macrotermes barneyi Light and Odontotermes formosanus (Shiraki); and the wood-feeding termites Nasutitermes parvonasutus (Shiraki) and Havilanditermes orthonasus (Tsai et Chen). The results showed that in diverse feeding groups, the wood-feeding group had the highest total composite cellulase and endo-ß-1,4-glucanase activities, while the fungus-growing group had the highest ß-glucosidase activity. In terms of the distribution of cellulase activity in the alimentary canals, the cellulase activities in wood-feeding termites were concentrated in the midgut, but there was no significant difference between all gut segments in humus-feeding termites. As for the fungus-growing termites, the main site of composite cellulase activity was in the midgut. The endo-ß-1,4-glucanase activity was restricted to the midgut, but the primary site of ß-glucosidase activity was in the foregut and the midgut (Mac. barneyi). The functions of the gut segments apparently differentiated between feeding groups. The results suggest that the differentiation of feeding habits in flagellate-free termites was characterized by the distribution of cellulases in the gut rather than by variations in cellulase activity.

High-resolution structures of Neotermes koshunensis ß-glucosidase mutants provide insights into the catalytic mechanism and the synthesis of glucoconjugates.

NkBgl, a ß-glucosidase from Neotermes koshunensis, is a ß-retaining glycosyl hydrolase family 1 enzyme that cleaves ß-glucosidic linkages in disaccharide or glucose-substituted molecules. ß-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in ß-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve ß-glucosidases for the enzymatic synthesis of oligosaccharides.

Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant ß-glucosidase from the termite Nasutitermes takasagoensis.

Termites are well-known cellulose decomposers and can give researchers insights into how to utilize lignocellulosic biomass in the actual scenario of energy consumption. In this work, an endogenous ß-glucosidase from the midgut of the higher termite Nasutitermes takasagoensis was purified to homogeneity by Ni(2+) affinity chromatography and its properties were characterized. This ß-glucosidase (G1mgNtBG1), which belongs to glycoside hydrolase family 1, is a homotrimer in its native form, with a molecular mass of 169.5 kDa, as demonstrated by gel filtration chromatography. The enzyme displayed maximum activity at pH 5.5 and had broad substrate specificities toward several saccharides, including cellobiose. G1mgNtBG1 showed a relatively high temperature optimum of 65°C and one of the highest levels of glucose tolerance among several ß-glucosidases already characterized, with a K(i) of 600 mM glucose. To examine the applicability of G1mgNtBG1 in biomass conversion, we compared the thermostability and glucose tolerance of G1mgNtBG1 with those of Novozym 188. We found that G1mgNtBG1 was more thermostable after 5 h of incubation at 60°C and more resistant to glucose inhibition than Novozym 188. Furthermore, our result suggests that G1mgNtBG1 acts synergistically with Celluclast 1.5 L in releasing reducing sugars from Avicel. Thus, G1mgNtBG1 seems to be a potential candidate for use as a supplement in the hydrolysis of biomass.

Exploration and characterization of genes involved in the synthesis of diterpene defence secretion in nasute termite soldiers.

Nasutitermes takasagoensis soldiers defend their colonies using characteristic diterpenes. Diterpenes are thought to be synthesized in the frontal gland cells surrounding the gland reservoir. To identify the genes involved in diterpene synthesis, a cDNA library was prepared from the frontal gland cells and exhaustively sequenced using a 454 pyrosequencer (GS Junior; Roche, Branford, CT, USA). A total of 50,290 clean sequences were assembled into 1111 contigs, which were grouped into 774 genes (isogroups). Based on sequence similarity with known proteins, we identified seven genes encoding the following four enzymes associated with diterpene synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS), HMG-CoA reductase (HMGR), farnesyl diphosphate synthase, and geranylgeranyl diphosphate synthases. The expression levels of two enzymes, HMGS and HMGR, involved in the mevalonate pathway were examined, assuming that the site of the defensive terpenoid synthesis strongly activates the mevalonate pathway, which produces a precursor of terpenoids. Real-time quantitative reverse-transcriptase PCR confirmed significantly higher expression of HMGS and HMGR in the heads of soldiers. We then divided the head into three parts and found that the expression levels of HMGS and HMGR were significantly higher in the part containing class 1 secretory cells of the frontal gland. Overall, the results suggested that the mevalonate pathway for diterpene synthesis occurs in class 1 cells around the frontal gland reservoir.

Molecular cloning and characterization of an endogenous digestive ß-glucosidase from the midgut of the fungus-growing termite Macrotermes barneyi.

ß-glucosidase from the midgut of the fungus-growing termite Macrotermes barneyi was first cloned and characterized to gain a better understanding of cellulolytic systems in fungus-growing termites. ß-glucosidase activity was proven to present primarily in the midgut of M. barneyi and two ß-glucosidases were partially purified from the midgut. Based on the N-terminus sequence of one of the ß-glucosidases, a full-length cDNA fragment of 1708 bp was obtained. This sequence encodes a 493 amino acid protein belonging to glycoside hydrolase family 1. Quantitative real-time PCR analysis proved that the ß-glucosidase gene was primarily expressed in the midgut. ß-glucosidase was expressed heterologously and biochemically characterized. Results indicate that ß-glucosidase is an endogenous, midgut-origin termite digestive enzyme. It may have applications in understanding the mechanism of lignocellulose degradation in fungus-growing termites.

Carbohydrate-active enzymes revealed in Coptotermes formosanus (Isoptera: Rhinotermitidae) transcriptome.

Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 ß-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.