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.     

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.     

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.     

Metazoan cellulase genes from termites: intron/exon structures and sites of expression.

Endogenous endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) cDNAs were cloned from representatives of the termite families Termitidae and Rhinotermitidae. These EGases are all composed of 448 amino acids and belong to glycosyl hydrolase family 9 (GHF9), sharing high levels of identity (40-52%) with selected bacterial, mycetozoan and plant EGases. Like most plant EGases, they consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. Using a PCR-based strategy, the entire sequence of the coding region of NtEG, a gene putatively encoding an EGase from Nasutitermes takasagoensis (Termitidae), was determined. NtEG consists of 10 exons interrupted by 9 introns and contains typical eukaryotic promoter elements. Genomic fragments of EGase genes from Reticulitermes speratus (Rhinotermitidae) were also sequenced. In situ hybridization of N. takasagoensis guts with an antisense NtEG RNA probe demonstrated that expression occurs in the midgut, which contrasts to EGase expression being detected only in the salivary glands of R. speratus. NtEG, when expressed in Escherichia coli, was shown to have in vitro activity against carboxymethylcellulose.     

Marked variations in patterns of cellulase activity against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, wood-feeding termites

Abstract.  Throughout the history of studies on cellulose digestion in termites, carboxymethyl-cellulose has been preferably used as a substrate for measuring cellulase activity in termites due to its high solubility. However, carboxymethyl-cellulose degradation is not directly related to digestibility of naturally occurring cellulose because many noncellulolytic organisms can also hydrolyse carboxymethyl-cellulose. To address this issue, a comparative study of microcrystalline cellulose digestion is performed in diverse xylophagous termites, using gut homogenates. For those termites harbouring gut flagellates , the majority of crystalline cellulose appears to be digested in the hindgut, both in the supernatant and the pellet. For Nasutitermes takasagoensis , a termite free of gut flagellates, crystalline cellulose is degraded primarily in the midgut supernatant, and partially in the pellet of the hindgut. The fungus-growing termite Odontotermes formosanus , which also does not possess intestinal flagellates, shows only a trace of crystalline cellulose hydrolysis throughout the gut. Comparison of levels of activity against crystalline cellulose with previously reported levels of activity against carboxymethyl-cellulose in the gut of each termite reveals significant differences between these activities. The results suggest that the hindgut flagellates produce commonly cellobiohydrolases in addition to endo-β-1,4-glucanases, which presumably act synergistically to digest cellulose. Preliminary evidence for the involvement of bacteria in the cellulose digestion of N. takasagoensis is also found.     

长江口安氏白虾与日本沼虾营养成分比较

本文对长江口安氏白虾和日本沼虾的营养成分和营养品质进行了比较.结果表明:安氏白虾的水分和粗灰分含量显著低于日本沼虾 (P<0.05),而粗脂肪和粗蛋白含量显著高于日本沼虾(P<0.05);安氏白虾和日本沼虾的氨基酸组成基本一致,均含有17种氨基酸,必需氨基酸指数(EAAI)分别为52.77和52.67,其构成比例符合联合国粮农组织/世界卫生组织(FAO/WHO)的标准;安氏白虾脂肪酸中二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)的总量明显高于日本沼虾,分别为28.32%±0.49%和14.89%±0.63%;矿物元素中常量和微量元素含量均较丰富,微量元素之间的比例较合理,其中,安氏白虾的常量元素含量较高,而日本沼虾的微量元素含量较高,而日本沼虾的微量元素含量较高,从以上结果可见,安氏白虾和日本沼虾均含有较丰富的各种营养成分,但从EPA和DHA的含量来看,脂肪酸的营养价值安氏白虾高于日本沼虾;从Fe、Cu、Zn和Se的含量来看,微量元素营养价值日本沼虾高于安氏白虾.     

Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

Studies on the mode of action of cholesterol oxidase on insect midgut membranes

Cholesterol oxidase (EC 1.1.3.6.) is an insecticidal protein known to have potent activity against the boll weevil, milder activity against a number of lepidopteran species, and virtually no activity against other insects. Several factors that could explain its species-dependent differential activity were examined. We compared cholesterol concentrations and rates of cholesterol oxidation in the midgut membranes from larvae of boll weevil (Anthonomus grandis grandis Boheman), southern corn rootworm (Diabrotica undecimpunctata howardi Barber), tobacco budworm (Heliothis virescens Fabricius), and yellow mealworm (Tenebrio molitor Linnaeus). Results showed that cholesterol concentration alone could not account for the differences in insecticidal activity and that midgut brush-border membranes of all species tested were generally susceptible to oxidation by cholesterol oxidase in vitro. We also demonstrated that cholesterol oxidase stability in the midgut environment was similar for the species tested and thus could not account for the differential activity. However, comparison of the pH of the insect midgut fluids with the pH optimum of cholesterol oxidase indicated that the lower sensitivity of lepidopteran larvae to the enzyme may be partially due to the alkaline nature of their midgut environments. In some species, oxidation caused significant changes in the activities of brush-border membrane alkaline phosphatase, and these changes did correlate with the susceptibility of the insect to cholesterol oxidase. Arch. Insect Biochem. Physiol. 34:429–442, 1997. © 1997 Wiley-Liss, Inc.     

Galactolipase, phospholipase and triacylglycerol lipase activities in the midgut of six species of lepidopteran larvae feeding on different lipid diets

Galactolipase, phospholipase and triacylglycerol lipase activities were measured from the midgut of six species of lepidopteran larvae, two folivores, Epiphyas postvittana (Tortricidae) and Helicoverpa armigera (Noctuidae); two granivores, Plodia interpunctella (Pyralidae) and Ephestia kuehniella (Pyrallidae); a presumptive carnivore, Galleria mellonella (Pyralidae); and a keratinophage, Tineola bisselliella (Tineidae). Galactolipase has not been previously reported in insects. Galactolipase and phospholipase activities were high in the folivores and triacylglycerol lipase activity was low, matching the high galactolipid content of leaves. Conversely, galactolipase and phospholipase activities were low, but not absent, and triacylglycerol lipase activity high in the four other non-folivorous species, matching the high acylglycerol content of their diets. These data suggest the utility of reclassification, for evolutionary studies, of phytophagous lepidoptera into two feeding classes; folivore and granivore, the latter having similarity to the fungivore line of feeders in terms of its lipase activities and ability to retrieve essential polyunsaturated long chain fatty acids from their diets. All the digestive lipases have alkaline pH optima for activity, matching the pH of the lepidopteran midgut and their amino acid content show modifications likely to stabilize the proteins in that environment.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

Histochemical changes in the midgut of two ixodid tick speciesBoophilus microplus andRhipicephalus appendiculatus during digestion of the blood meal

The changes in the midgut epithelia of two ixodid tick species,Boophilus microplus andRhipicephalus appendiculatus, have been studied using several histochemical techniques. It was revealed that there is an accumulation of RNA at the time of tick attachment to the host and prior to the arrival of the blood meal, indicating that the midgut digest cell is furnished with the machinery characteristic of a synthetic cell. There appears to be a synchrony in the appearance of granules with peroxidase activity and the uptake of haemoglobin into the midgut digest cells. Alkaline phosphatase activity was observed in the midgut epithelia of all ticks except in a few of the long-starved ticks, and was concentrated in the apical plasma membrane regions of those digest cells involved in absorption and the intracellular digestion of haemoglobin. The presence of these enzymes suggests that the midgut digest cell is a multifunctional cell capable of both secretory and digestive activities. The colloidal material in the midgut lumen was found to result from the accretion of several products both secreted and excreted by the midgut epithelial cells and exhibited different staining reactions depending on which component dominated. The nature of the material suggests that in addition to its digestive function it may serve as a sink to bind all the by-products of digestion and thereby facilitate their excretion.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

Trypanosoma cruzi: effects of infection on cathepsin D activity in the midgut of Rhodnius prolixus

Cathepsin D activity was estimated in midgut homogenates from Rhodnius prolixus, uninfected and experimentally infected with Trypanosoma cruzi, at different times after blood ingestion. No enzyme activity was found in the anterior midgut and rectum. In the posterior midgut, enzyme activity was found both in lumen and wall. In starved uninfected insects, in lumen and wall, cathepsin D activity was high, decreasing to a constant rate at 1-15 days after feeding. In insects infected with T. cruzi cathepsin D activity increased 1 and 3 days after blood meal. We suggest that these changes in cathepsin D activity in R. prolixus posterior midgut are due to the establishment of T. cruzi infection.     

Localization of symbiotic clostridia in the mixed segment of the termite Nasutitermes takasagoensis (Shiraki)

Phylogeny and the distribution of symbiotic bacteria in the mixed segment of the wood-eating termite Nasutitermes takasagoensis (Shiraki) were studied. Bacterial 16S rRNA genes (rDNA) were amplified from the mixed segment of the gut by PCR, and two kinds of sequences were identified. The phylogenetic tree was constructed by neighbor-joining and maximum parsimony methods to identify symbionts harbored in the mixed segment. They are classified as low-G+C-content gram-positive bacteria and are most closely related to the genus Clostridium. The distribution of these bacteria throughout the whole gut was examined by PCR using specific primers, which suggested that they are confined to the mixed segment despite the presence of bacteria throughout the gut. In situ hybridization indicated that the symbiotic bacteria were localized to the ectoperitrophic space between the midgut wall and the peritrophic membrane in the mixed segment. Electron microscopy revealed the close association between these bacteria and the mesenteric epithelium, suggesting that they have some interactions with the gut tissue of termites.     

Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs

Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-beta-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5-96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-beta-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20-30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.     

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.     

Post-engorgement dynamics of haemagglutination activity in the midgut of phlebotomine sandflies

Abstract. Haemagglutination activity (HA) was studied in gut extracts of both sexes of adults of six sandfly species. HA was sex dependent, with the activity in males more than 50 times lower man that of unfed females. In females, high HA was demonstrable in both the thoracic and abdominal midgut but not in the hindgut. In unfed flies the activity was similar in the midgut wall and the gut contents whereas, in fed females, a high increase was seen in the midgut contents. After blood-feeding, HA was elevated, reaching peak titres 2 days later and then falling to the base level or less immediately after defaecation. The magnitude of the HA response differed according to species, ranging from 2-fold in Lutzomyia carmelinoi up to 16-fold in Phlebotomus duboscqi. Quantitative differences between sandflies in their HA response may influence their ability to support the development of Leishmania spp.     

家蚕BmlncR2036调控P25基因启动子活性及参与中肠发育调控

[目的]长链非编码RNA (long non-coding RNA,lncRNA)对家蚕Bombyx mori发育具有重要调控作用.我们在前期研究中发现一个位于家蚕丝素蛋白基因P25附近的lncRNA BmlncR2036.本研究旨在进一步探索BmlncR2036调控家蚕P25基因表达的分子机制.[方法]qPCR检测B...     

Proteinase, amylase, and proteinase-inhibitor activities in the gut of six cockroach species

Representative species, two from each of the cockroach families Blattidae, Blattellidae, and Blaberidae, have similar morphology of the digestive tract but differ in the physiology of digestion. The pH of crop and along the midgut varies in different species from 5.9 to 9.0 and the redox parameter from 10.1 to 12.9. Activities of proteinases and amylases in comparable gut regions differ among the species up to 100 times. Proteolytic activity is high in the midgut and moderate in the crop of Blattidae; in the other species, it is very low in the crop and increases to a moderate level in the posterior half of midgut (PM). The level of amylolytic activity is similar in the examined gut compartments of Blattidae and Blattellidae but low in the PM of Blaberidae. Blaberidae are also characterized by a high potential of the salivary glands, crop, and midgut to inhibit subtilisin, trypsin, and chymotrypsin. Inhibition of these proteinases by the extracts of salivary glands and gut is several orders of magnitude lower and often undetectable in the representatives of Blattidae and Blattellidae.