Prospecting for cellulolytic activity in insect digestive fluids

Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quantitative studies comparing cellulase activity among insect taxa constrains identification of candidate species for more targeted identification of effective cellulolytic systems. We describe quantitative determinations of the cellulolytic activity in gut or head-derived fluids from 68 phytophagous or xylophagous insect species belonging to eight different taxonomic orders. Enzymatic activity was determined for two different substrates, carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), approximating endo-β-1,4-glucanase and complete cellulolytic activity, respectively. Highest CMC gut fluid activities were found in Dictyoptera, Coleoptera, Isoptera, and Orthoptera, while highest MCC gut fluid activities were found in Coleoptera, Hymenoptera, Lepidoptera, and Orthoptera. In most cases, gut fluid activities were greater with CMC compared to MCC substrate, except in Diptera, Hymenoptera, and Lepidoptera. In contrast, cellulolytic activity levels in most head fluids were greater on the MCC substrate. Our data suggests that a phylogenetic relationship may exist for the origin of cellulolytic enzymes in insects, and that cellulase activity levels correlate with taxonomic classification, probably reflecting differences in plant host or feeding strategies.     

The origin and distribution of cellulase in the termites,Nasutitermes exitiosus and Coptotermes lacteus

The cellulase of the higher termite Nasutitermes exitiosus was located in the foregut (19%), the midgut (59%), the mixed segment (14%) and in the hindgut (8%). Removal of the gut flora by feeding tetracycline or by starving the termite did not affect the activity of the enzymes indicating that the termite secretes its own cellulase and is not dependent on its gut flora for the digestion of cellulose. The cellulase of the lower termite Coptotermes lacteus was distributed through the foregut (19%), midgut (32%) and hindgut (49%). Removal of the gut flora and fauna, left the specific activity of the cellulase of the fore- and mid-gut largely unaffected, but led to a 20% decrease in the specific activity of the cellulase in the hindgut. In starved C. lacteus the distribution of cellulase activity was foregut, 44%; midgut, 30% and hindgut, 26%. These results indicate that C. lacteus synthesises its own cellulase in addition to using the gut protozoa for cellulose digestion.     

Screening and characterization of lipase producing bacteria isolated from the gut of a lepidopteran larvae,Samia ricini

Lipolytic enzymes are an important group of hydrolases that have found immense industrial application in biotechnology. In this study, the ability of gut bacteria isolated from the gut of the Eri silkworm, Samia ricini, to produce lipolytic enzymes was evaluated through qualitative and quantitative assays. The results of lipase screening showed that 28 isolates had lipolytic activity. The results of 16S ribosomal RNA sequencing indicated that the genus Bacillus comprised majority of the lipolytic bacterial isolates (71%) followed by Pseudomonas (15%); whilst Acinetobacter, Enterobacter and Enterococcus comprised 11%. Lipolytic activity was found in bacteria isolates identified from all the three gut compartments of S. ricini larvae with significant activity from isolates extracted from the foregut and midgut. The lipolytic index among the bacterial isolates ranged between 0.63 and 2.81 on Rhodamine B medium, and all isolates exhibited significant lipolytic activity with p-nitrophenyl butyrate (PNPB) with specific activity ranging from 0.52 to 0.82 μmol/min/mg. The effect of pH and temperature showed that lipase activity was optimum at 37 °C and pH 7–9. A phylogenetic relationship of lipase producing gut bacteria indicated high cluster stability for isolates from different stages (>50%) suggesting that the isolates persist across developmental stages of the host. The Eri silkworm is reared for its silk and the knowledge of its gut bacteria with the ability to produce lipases lies in the significance as far as boosting production of this insect via development of probiotics to enhance commercial Eri rearing. In addition, this insect may be a good resource for profiling novel lipolytic microbes for commercial production of lipases as lipases from microbial origin have assumed a great deal of importance as industrial enzymes due to their potential for use in biotechnology.     

Photoperiodic effect on some enzymes and metabolites in diapausingAntheraea mylitta pupae andPhilosamia ricini larvae during development

The variation in acid phosphatase (EC 3.1.3.2) activity inAntheraea mylitta was similar in all light and dark groups exposed to different photophases (LD =0:24, 24:0, 18:6, 14:10, 10:14 and 12:12 h) maintaining all along a higher activity than its alkaline counterpart. The highest activity was recorded on day 82 in LD group 10:14 h. The non-diapausingPhilosamia ricini larvae registered highest activity in LD group 0:24 h on day 5. Alkaline phosphatase (EC 3.1.3.1) activity was low all through metamorphosis in both the lepidopterans, although significantly elevated activity was observed on day 5 in larvae of allPhilosamia ricini LD regimens and on day 82 inAntherae mylitta. Photoperiodic effect on Phosphorylase (EC 2.3.1.1) activity, glycogen and inorganic phosphates content have also been studied. Exposure to LD 10:14, 14:10 and 18:6 h provoked early diapause termination inAntheraea mylitta. The non-diapausingPhilosamia ricini was unaffected in moth emergence but the emerged adults of LD 24:0 and 0:24 h groups were unhealthy, small and did not mate or oviposit.     

Cellulose digestion by the firebrat Thermobia domestica

• 1.1. A highly efficient cellulose digestion could be demonstrated in a primitive insect species, Thermobia domestica (Thysanura:Lepismatidae), by the application of a uniformly ¹⁴C-labelled substrate.
• 2.2. Gut extracts exhibit distinct hydrolytic activities toward different cellulosic substrates (cellobiose, sodium carboxymethylcellulose and microcrystalline cellulose). Therefore, the complete cellular complex must be present.
• 3.3. Besides cellulases, several other carbohydrates occur in the digestive juice, thus reflecting the omnivorous feeding habits of the insect.
• 4.4. The crop was found to be the main site of carbohydrate digestiopn, also including cellulolysis.
• 5.5. It is very likely that the cellulolytic enzymes derive from the gut tissues of the firebrat.

     

椰子织蛾幼虫肠道细菌的初步分离鉴定及功能分析

[目的] 研究椰子织蛾幼虫肠道微生物的种类和功能,以揭示其消化利用寄主老叶的机制。[方法] 采用传统微生物分离培养技术分离培养肠道细菌,用16S rRNA基因序列分析的方法鉴定菌株,采用透明圈染色法对所得菌株进行功能性验证。[结果] 基因序列检测对比鉴定得到9种可培养细菌菌株,主要属于变形菌门和厚壁菌门以及放线菌门;功能性验证结果表明,伯克霍尔德氏菌、解淀粉芽孢杆菌、贝莱斯芽孢杆菌、蜡样芽孢杆菌菌株具有纤维素降解酶,寒气玫瑰单胞菌、解淀粉芽孢杆菌含木聚糖降解酶。[结论] 椰子织蛾肠道中存在可培养的具有降解纤维素及木聚糖能力的细菌,这些细菌可能有助于椰子织蛾取食消化椰子等老叶,研究所获得的肠道微生物菌株也为后续研究该虫与环境的关系及相关菌株应用于农业、能源、环保价值的探索提供帮助。     

Is a urea cycle present in insects?

1. The presence of appreciable activity of the urea-cycle enzymes in the tissues of Sarcophaga ruficornis, a carnivorous dipteran insect, all through its life-cycle appears significant in view of their total absence barring arginase (L-arginine ureohydrolase, EC 3.5.3.1) in the phytophagous lepidopteran eri silkwork Philosamia ricini at any stage of development. Further, the variation of all these enzymes all through its development suggests the possibility of the operation of the Krebs-Henseleit urea cycle in this carnivorous insect. 2. The almost parallel behaviour of arginase and ornithine delta-transaminase (L-ornithine-2-oxo acid aminotransferase, EC 2.6.1.13) in both the insects suggests another important role of the former in proline biosynthesis in insects. 3. High proteolytic activity accompanied with significant protein depletion and simultaneous increase in arginine is suggestive of the degradation of proteins and peptides.     

Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii.     

Microbial Symbionts in Insects Influence Down-Regulation of Defense Genes in Maize

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.     

Characterization of crystal prototoxin and an activated toxin from Bacillus thuringiensis var. entomocidus

Toxin crystals from Bacillus thuringiensis var. entomocidus were lysed by proteases present in gut juice from larval Philosamia ricini (Lepidoptera) with the release of a prototoxin and an activated toxin. Some of the toxic activity of the lysate was complexed with a pheophytinlike pigment and this complex was retarded on filtration through Sephadex gels. A method is described for the removal of the pheophytin from larval protease preparations. The prototoxin has a molecular weight greater than 200,000, determined by its exclusion from Sephadex G-200 and on activation produces a toxin of molecular weight about 50,000. Isoelectric focusing of crystal lysates gave pI values of 4.5 and 6.4 for the prototoxin and toxin, respectively. The antigenic composition of the prototoxin and of the toxin are compared and the significance of antigen h as an indicator of activation is discussed.     

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.     

Effect of malathion and acetylcholine on the developing larvae ofPhilosamia ricini (Lepidoptera: Saturniidae)

Feeding of malathion induces accumulation of acetylcholine inPhilosamia ricini developing larvaevia inhibition of acetyl cholinesterase activity. The insecticide also causes depletion of all nutrients, loss in weight, under-development of silk glands resulting in reduced silk production, lack of oviposition and high mortality among the insects. Acetylcholine however, while fed during the same period of development, tones up their nutritional status, induces better growth, weight gain, improved silk production and oviposition and significantly lower mortality than in the control group of insects maintained on castor leaves. This improved status of insects has been attributed to choline, the insect vitamin released from acetylcholine. Acetylcholine has also been noted to protect the insects to some extent from the poisonous effect of malathion on exposure to it after a dose of acetylcholine during the preceding instar stadium     

Thermal properties for digestive enzymes of a sub-antarctic beetle,hydromedion sparsutum (coleoptera,perimylopidae) compared to those in two thermophilic insects

• 1.1. Proteolytic, lipolytic, amylolytic and cellulolytic activities were studied in adults of the phytophagous beetle, Hydromedion sparsutum, indigenous to the sub-Antarctic island of South Georgia.
• 2.2. Gastric enzyme activities were measured at experimental temperatures of 5–40°C and results were compared with those obtained from two thermophilic insects, Gryllus bimaculatus and Tenebrio molitor.
• 3.3. Protease and lipase activities in Hydromedion were 10–15 times lower than in Gryllus and Tenebrio.
• 4.4. In the temperature range of 5–15°C, α-amylase activity from Hydromedion was only slightly lower than that from Gryllus.
• 5.5. Hydromedion gut homogenates exhibited a distinct cellulolytic activity, even at a low temperature of 5°C.
• 6.6. Cellulolytic activity in the digestive tract of Hydromedion was confirmed by the evolution of ¹⁴CO₂ after consumption of labelled cellulose.
• 7.7. The thermal properties of digestive enzymes agree well with the role of Hydromedion as primary decomposer in its ecosystem.

     

Influence of lignin in Reticulitermes santonensis: symbiotic interactions investigated through proteomics

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting together and with the insect host. Their likely ability to degrade ligno-cellulosic compounds could lead to improvements in second generation biofuels production. Lignin elimination represents a critical point as this polymer significantly interferes with industrial process of cellulose. Although host produces its own lignin-degrading enzymes, some symbionts may participate in digestion of lignin and its degradation products in termite gut. Here, we compared gut proteomes from R. santonensis after rearing on artificial diets composed of cellulose with and without lignin. The effect of lignin in artificial diets on different parts of the digestive tract was compared through liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) experiments. Enzymatic assays were performed to characterize activities present in R. santonensis digestive tract after feeding on artificial diets. Microscopic observations of microbial communities provided some information on population balances after feeding experiment.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Genome Sequence of the Cellulolytic Gliding Bacterium Cytophaga hutchinsonii

The complete DNA sequence of the aerobic cellulolytic soil bacterium Cytophaga hutchinsonii, which belongs to the phylum Bacteroidetes, is presented. The genome consists of a single, circular, 4.43-Mb chromosome containing 3,790 open reading frames, 1,986 of which have been assigned a tentative function. Two of the most striking characteristics of C. hutchinsonii are its rapid gliding motility over surfaces and its contact-dependent digestion of crystalline cellulose. The mechanism of C. hutchinsonii motility is not known, but its genome contains homologs for each of the gld genes that are required for gliding of the distantly related bacteroidete Flavobacterium johnsoniae. Cytophaga-Flavobacterium gliding appears to be novel and does not involve well-studied motility organelles such as flagella or type IV pili. Many genes thought to encode proteins involved in cellulose utilization were identified. These include candidate endo-β-1,4-glucanases and β-glucosidases. Surprisingly, obvious homologs of known cellobiohydrolases were not detected. Since such enzymes are needed for efficient cellulose digestion by well-studied cellulolytic bacteria, C. hutchinsonii either has novel cellobiohydrolases or has an unusual method of cellulose utilization. Genes encoding proteins with cohesin domains, which are characteristic of cellulosomes, were absent, but many proteins predicted to be involved in polysaccharide utilization had putative D5 domains, which are thought to be involved in anchoring proteins to the cell surface.     

New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis

β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.     

Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes

This research focused on digestive beta-glucosidases from glycosyl hydrolase family (GHF) 1 from the gut of the lower termite Reticulitermes flavipes. In preceding studies on R. flavipes, we characterized beta-glucosidase activity across the gut and its inhibition by carbohydrate-based inhibitors, and subsequently we identified two partial beta-glucosidase cDNA sequences from a host gut cDNA library. Here, we report on the full-length cDNA sequence for one of the R. flavipes beta-glucosidases (RfBGluc-1), the expression of its mRNA in the salivary gland and foregut, the production of recombinant protein using a baculovirus–insect expression system, optimal recombinant substrate specificity profiles and parameters, and significant inhibition by the established beta-glucosidase inhibitor cellobioimidazole. We also report the partial cDNA sequence for a second gut beta-glucosidase (RfBGluc-2), and show that like RfBGluc-1 its mRNA is localized mainly in the salivary gland. Other results for RfBGluc-1 showing activity against laminaribose, a component of microbial cell walls, suggest that RfBGluc-1 may serve dual functions in cellulose digestion and immunity. These findings provide important information that will enable the testing of hypotheses related to collaborative host–symbiont lignocellulose digestion, and that contributes to the development of next-generation termiticides and novel biocatalyst cocktails for use in biomass-to-bioethanol applications.     

Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii

Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that rapidly digests crystalline cellulose in a contact-dependent manner. The different roles of various predicted glycoside hydrolases and the detailed mechanism used by C. hutchinsonii in cellulose digestion are, however, not known. In this study, an endoglucanase belonging to glycoside hydrolase family 5 (GH5) named as ChCel5A was isolated from the outer membrane of C. hutchinsonii. The catalytic domain of ChCel5A exhibited typical endoglucanase activity and was capable of hydrolyzing insoluble cellulose with cellobiose and cellotriose as the predominant digestion products. Site-directed mutagenesis identified two aromatic amino acids in ChCle5A, W61 and W308, that dramatically decreased its hydrolytic activity toward filter paper while causing only a slight decrease in carboxymethylcellulase (CMCase) activity. Disruption of chu_1107 encoding ChCel5A caused no drastic effect on the growth parameters on cellulose for the resulting mutant strain with negligible reduction in the specific CMCase activities for intact cells. The demonstration of targeted gene inactivation capability for C. hutchinsonii has provided an opportunity to improve understanding of the details of the mechanism underlying its efficient utilization of cellulose.