Dual cellulose-digesting system of the wood-feeding termite,Coptotermes formosanus Shiraki

The distribution of endo-beta-1,4-glucanase (EG) components in the digestive system of the wood-feeding termite, Coptotermes formosanus Shiraki, was investigated by zymogram analysis using polyacrylamide gel electrophoresis, followed by N-terminal protein sequencing. EG components similar to glycoside hydrolase family (GHF) 9 members were restricted to the salivary glands, the foregut, and the midgut, whereas components similar to GHF7 members were confined to the hindgut where numerous cellulolytic flagellates were harbored. RT-PCR experiments revealed that five GHF9 EG mRNAs (1348 bp) homologous to other termite EGs were expressed in the salivary glands and the midgut. The crude extract prepared from the midgut as well as that from the hindgut produced glucose from crystalline cellulose. These data suggest that C. formosanus has two independent cellulose-digesting systems: one in the midgut where cellulose digestion is accomplished by endogenous cellulases and the other in the hindgut which makes use of other cellulases possibly from symbiotic flagellates.     

Hidden cellulases in termites: revision of an old hypothesis

The intestinal flagellates of termites produce cellulases that contribute to cellulose digestion of their host termites. However, 75% of all termite species do not harbour the cellulolytic flagellates; the endogenous cellulase secreted from the midgut tissue has been considered a sole source of cellulases in these termites. Using the xylophagous flagellate-free termites Nasutitermes takasagoensis and Nasutitermes walkeri, we successfully solubilized cellulases present in the hindgut pellets. Zymograms showed that the hindguts of these termites possessed several cellulases and contained up to 59% cellulase activity against crystalline cellulose when compared with the midgut. Antibiotic treatment administered to N. takasagoensis significantly reduced cellulase activity in the hindgut, suggesting that these cellulases were produced by symbiotic bacteria.     

Symbiotic "Archaezoa" of the primitive termite Mastotermes darwiniensis still play a role in cellulase production

The relictual Mastotermes darwiniensis is one of the world's most destructive termites. Like all phylogenetically basal termites, it possesses protozoa in its hindgut, which are believed to help it digest wood. L. Li, J. Frohlich, P. Pfeiffer, and H. Konig (Eukaryot. Cell 2:1091-1098, 2003) recently cloned the genes encoding cellulases from the protozoa of M. darwiniensis; however, they claimed that these genes are essentially inactive, not contributing significantly to cellulose digestion. Instead, they suggested that the protozoa sequester enzymes produced by the termite in its salivary glands and use these to degrade cellulose in the hindgut. We tested this idea by performing gel filtration of enzymes in extracts of the hindgut, as well as in a combination of the salivary glands, foregut, and midgut. Three major cellulases were found in the hindgut, each of which had a larger molecular size than termite-derived salivary gland enzymes. N-terminal amino acid sequencing of one of the hindgut-derived enzymes showed that it was identical to the putative amino acid sequence of one mRNA sequence isolated by Li et al. (Eukaryot. Cell 2:1091-1098, 2003). The overall activity of the hindgut cellulases was found to be of approximately equal magnitude to the termite-derived cellulases detected in the mixture of salivary gland, foregut, and midguts. Based on these results, we conclude that, contrary to Li et al. (Eukaryot. Cell 2:1091-1098, 2003), the hindgut protozoan fauna of M. darwiniensis actively produce cellulases, which play an important role in cellulose digestion of the host termite.     

Does correlation of cellulase gene expression and cellulolytic activity in the gut of termite suggest synergistic collaboration of cellulases?

Termites play an important role in degradation of dead plant materials in nature. Over the last century, many researchers have investigated the mechanisms of their lignocellulose digesting system. A recent publication by Zhou et al. (Zhou, X., Smith, J.A., Oi, F.M., Koehler, P.G., Bennett, G.W., Scharf, M.E., 2007. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes. Gene 395, 29-39) dealt with the cellulolytic system of the flagellate-harboring termite R. flavipes and suggested "the presence of a single unified cellulose digestion system" in the termite, as an alternative hypothesis of a "dual (i.e. endogenous and symbiotic) cellulose digesting system" proposed by Nakashima et al. (Nakashima, K., Watanabe, H., Saitoh, H., Tokuda, G., Azuma, J.-I., 2002. Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki. Insect Biochem. Mol. Biol. 32, 777-784). Here we show that their results actually support a dual cellulose digesting system rather than "a single unified cellulose digestion system". In addition, potential problems with their results are highlighted.     

Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis

Unlike lower termites, xylophagous higher termites thrive on wood without the aid of symbiotic protists. In the higher termite Nasutitermes takasagoensis, both endogenous endo-β-1,4-glucanase and β-glucosidase genes are expressed in the midgut, which is believed to be the main site of cellulose digestion. To further explore the detailed cellulolytic system in the midgut of N. takasagoensis, we performed immunohistochemistry and digital light microscopy to determine distributions of cellulolytic enzymes in the salivary glands and the midgut as well as the total cellulolytic activity in the midgut. Although cellulolytic enzymes were uniformly produced in the midgut epithelium, the concentration of endo-β-1,4-glucanase activity and luminal volume in the midgut were comparable to those of the wood-feeding lower termite Coptotermes formosanus, which digests cellulose with the aid of hindgut protists. However, the size of ingested wood particles was considerably larger in N. takasagoensis than that in C. formosanus. Nevertheless, it is possible that the cellulolytic system in the midgut of N. takasagoensis hydrolyzes highly crystalline cellulose to a certain extent. The glucose produced did not accumulate in the midgut lumen. Therefore, the present study suggests that the midgut of the higher termite provides the necessary conditions for cellulolysis.     

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood-feeding termite, Reticulitermes speratus (Kolbe): possible digestion of symbiont bacteria transferred by trophallaxis

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood‐feeding termite, Reticulitermes speratus (Kolbe) (Isoptera, Rhinotermitidae) were studied to examine the possibility that termites digest symbiont bacteria transferred by trophallaxis. Total lysozyme activity was found predominantly in the salivary gland and to a minor extent in the digestive tracts. However, specific lysozyme activity was high in the foregut as well as in the salivary gland. The similarity of the lysozyme pH profile of the salivary gland and of the foregut suggested that the foregut lysozyme came from the salivary gland. Major protease activity having the optimum pH of 7.5 was found in the midgut. Total free amino acid amount and concentration in the midgut was higher than elsewhere in the digestive tract. The possibility that lysozyme secreted from the salivary gland into the foregut digests hindgut bacteria transferred by trophallaxis was discussed.     

高效降解木质纤维素的白蚁肠道微生物组

木食性白蚁是自然界木质纤维素的高效降解者,在长期进化过程中白蚁与其肠道微生物组协同作用发展出不同的纤维素降解机制。木食性白蚁具有分别来源于白蚁和共生微生物的两套纤维素酶系统。在低等白蚁中,木质颗粒经过白蚁前、中肠分泌的内源性酶初步消化后,在后肠共生鞭毛虫中被降解为乙酸、二氧化碳和氢。高等木食性白蚁在进化中丢失了鞭毛虫,木质颗粒经白蚁自身分泌的酶初步消化后,在后肠大量共生细菌的帮助下被有效降解。培菌类白蚁利用其菌圃中的蚁巢伞菌和肠道微生物协同作用降解木质纤维素。共生微生物在白蚁的氮素固定与循环、中间产物代谢及纤维素降解等过程中发挥了重要作用。学习和模拟白蚁高效降解木质纤维素的体系,对生物质能源的产业化发展具有积极的意义。     

Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.     

Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus

Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218–221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences. Received: July 10, 2000 / Accepted: August 15, 2000     

Impact of oxygen on metabolic fluxes and in situ rates of reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes

The symbiotic digestion of lignocellulose in the hindgut of the wood-feeding termite Reticulitermes flavipes is characterized by two major metabolic pathways: (i) the oxidation of polysaccharides to acetate by anaerobic hydrogen-producing protozoa; and (ii) the reduction of CO2 by hydrogenotrophic acetogenic bacteria. Both reactions together would render the hindgut largely homoacetogenic. However, the results of this study show that the situation is more complex. By microinjection of radiolabelled metabolites into intact agarose-embedded hindguts, we showed that the in situ rates of reductive acetogenesis (3.3 nmol termite(-1) h(-1)) represent only 10% of the total carbon flux in the living termite, whereas 30% of the carbon flux proceeds via lactate. The rapid turnover of the lactate pool (7.2 nmol termite(-1) h(-1)) consolidates the previously reported presence of lactic acid bacteria in the R. flavipes hindgut and the low lactate concentrations in the hindgut fluid. However, the immediate precursor of lactate remains unknown; the low turnover rates of injected glucose (< 0.5 nmol termite(-1) h(-1)) indicate that free glucose is not an important intermediate under in situ conditions. The influence of the incubation atmosphere on the turnover rate and the product pattern of glucose and lactate confirmed that the influx of oxygen via the gut epithelium and its reduction in the hindgut periphery have a significant impact on carbon and electron flow within the hindgut microbial community. The in situ rates of reductive acetogenesis were not significantly affected by the presence of oxygen or exogenous H2, which is in agreement with a localization of homoacetogens in the anoxic gut lumen rather than in the oxic periphery. This adds strong support to the hypothesis that the co-existence of methanogens and homoacetogens in this termite is based on the spatial arrangement of the different populations of the gut microbiota. A refined model of metabolic fluxes in the hindgut of R. flavipes is presented.     

Termite gut symbiotic archaezoa are becoming living metabolic fossils

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.     

Molecular and biochemical markers for monitoring dynamic shifts of cellulolytic protozoa in Reticulitermes flavipes

Lower termites rely on cellulolytic protozoa to aid in the digestion of their wood-based diet. However, despite the major contribution of protozoa to the lower termite digestive system, few techniques have been developed to monitor shifts in protozoan populations. This study investigated whether quantitative real-time PCR (qRT-PCR) and/or cellulase enzyme assays can be used to monitor changes of cellulolytic protozoan populations in the lower termite, Reticulitermes flavipes (Kollar). Previously developed cellulase primer sets were used to test for changes in cellulase gene expression, while three different cellulase enzyme assays were used to assess changes in cellulase enzyme activity. The results from this study indicate that qRT-PCR is a reliable method to monitor shifts in cellulolytic protozoan populations. Specifically, qRT-PCR can serve as a useful monitoring technique during high-throughput screening of novel termite control agents such as cellulase inhibitors, and help to answer questions relating to whether or not such control agents impact cellulolytic protozoan populations.     

The scarab gut: A potential bioreactor for bio-fuel production

Abstract Cellulose and hemicelluloses are the most prevalent sources of carbon in nature. Currently many approaches employ micro-organisms and their enzyme products to degrade plant feedstocks for production of bioenergy. Scarab larvae are one such model. They consume celluloses from a variety of sources including plant roots, soil organic matter and decaying wood, and are able to extract nutrients and energy from these sources. In this paper, we review the physicochemical properties of the scarab larval gut, the diversity and digestive role that microflora play in the scarab gut and discuss the potential for applying these digestive processes in bioreactors for improving bio-fuel production. Scarab larvae are characterised by their highly alkaline midgut which is dominated by serine proteinase enzymes, and a modified hindgut which harbors the majority of the intestinal microbiota under anaerobic conditions. Evidence suggests that digestion of recalcitrant organic matter in scarab larvae likely results from a combination of endogenous gut proteinases and cellulolytic enzymes produced by symbiotic micro-organisms. Most of the easily digestible proteins are mobilized and absorbed in the midgut by endogenous proteinases. The hindgut contents of scarab larvae are characterized by high concentrations of volatile fatty acids, the presence of fermenting bacteria, and typical anaerobic activities, such as methanogenesis. The hindgut typically contains a wide diversity of micro-organisms, some of which appear to be obligate symbionts with cellulolytic potential. As a result, the scarab larval gut can be regarded as a small bioreactor resembling the rumen of sheep or cattle, where solid food particles composed of cellulose, hemicellulose, pectin and polysaccharides are degraded through enzymatic and fermentation processes. Together these observations suggest scarab larvae have potential to assist the bio-fuel industry by providing new sources of (hemi)cellulolytic bacteria and bacterial (hemi)cellulolytic enzymes.     

Evidence for the presence of a cellulase gene in the last common ancestor of bilaterian animals

Until recently, the textbook view of cellulose hydrolysis in animals was that gut-resident symbiotic organisms such as bacteria or unicellular eukaryotes are responsible for the cellulases produced. This view has been challenged by the characterization and sequencing of endogenous cellulase genes from some invertebrate animals, including plant-parasitic nematodes, arthropods and a mollusc. Most of these genes are completely unrelated in terms of sequence, and their evolutionary origins remain unclear. In the case of plant-parasitic nematodes, it has been suggested that their ancestor obtained a cellulase gene via horizontal gene transfer from a prokaryote, and similar suggestions have been made about a cellulase gene recently discovered in a sea squirt. To improve understanding about the evolution of animal cellulases, we searched for all known types of these enzymes in GenBank, and performed phylogenetic comparisons. Low phylogenetic resolution was found among most of the sequences examined, however, positional identity in the introns of cellulase genes from a termite, a sea squirt and an abalone provided compelling evidence that a similar gene was present in the last common ancestor of protostomes and deuterostomes. In a different enzyme family, cellulases from beetles and plant-parasitic nematodes were found to cluster together. This result questions the idea of lateral gene transfer into the ancestors of the latter, although statistical tests did not allow this possibility to be ruled out. Overall, our results suggest that at least one family of endogenous cellulases may be more widespread in animals than previously thought.     

Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

粉尘螨消化系统的形态学观察

光镜下观察了粉尘螨Dermatophagoides farinae消化系统结构,其组成包括：口前腔、前肠、中肠、后肠、肛门和唾液腺。口前腔由颚体围绕而成;前肠包括一个肌肉的咽和食道,食道从脑中穿过;中肠分为前中肠（包括一对盲肠）和后中肠,中肠的上皮细胞呈现多种形态; 后肠包括相对大的结肠和狭窄的直肠;消化腺为不规则形,位于脑前方。本文阐述了消化道的分支情况、显微结构及细胞形态。