Carbohydrate fermentation and by-product absorption studied with labelled cellulose in Oryctes nasicornis larvae (Coleoptera:Scarabaeidae)

Isolated gut segments of Oryctes nasicornis larvae were incubated in vitro after injection with ¹⁴C-cellulose. The incubation medium became progressively richer in labelled volatile fatty acids (VFA), demonstrating the process of cellulolysis and the absorption of carbohydrate degradation products. The mesenteron and the proctodeal dilation were both shown to be sites of cellulolysis and VFA absorption. The major acid formed and absorbed was acetic acid which, in the absence of any detectable glucose, would seem to constitute the essential product of carbon metabolism in Oryctes larvae as in ruminant mammals. The cellulolytic capacities of the different intestinal segment were tested and compared to each other.     

HYDROGEN ION CONCENTRATIONS WITHIN THE ALIMENTARY TRACT OF INSECTS

Larvæ of Psychoda and of Chironomus (diptera) maintained in solutions of appropriate indicators show that the typical acidities (pH) prevailing within the several regions of the digestive tract are: esophagus 7.1; cardiac chamber 6.2; mesenteron 7.5; the latter being functionally an intestine. The acidity of the hindgut, pH 6.4, is due to the discharge of the malphigian tubules.     

The effect of volatile fatty acids as a sole carbon source on lipid accumulation by Cryptococcus albidus for biodiesel production

The use of volatile fatty acids (VFAs) for microbial lipid accumulation was investigated in flask cultures of Cryptococcus albidus. The optimum culture temperature and pH were 25 °C and pH 6.0, respectively, and the highest lipid content (27.8%) was obtained with ammonia chloride as a nitrogen source. The lipid yield coefficient on VFAs was 0.167 g/g of C. albidus with a VFAs (acetic, propionic, butyric acids) ratio of 8:1:1, which was in good agreement with a theoretically predicted lipid yield coefficient of the VFAs as a carbon source. The major fatty acids of the lipids accumulated by C. albidus were similar to those of soybean oil and jatropha oil. A preliminary cost analysis shows that VFAs-based biodiesel production is competitive with current palm and soybean based biodiesels. Further process development for lower aeration cost and higher lipid yield will make this process more economical.     

The digestive activity of a phthiracarid mite mesenteron

The pH within the alimentary canal of the mite ranges from 5·4 in the caecae to 6·6 in the colon and post-colon. Microchemical tests indicate the presence of protease, lipase, and carbohydrase activity in the mesenteron. The hydrolysis of carbohydrates is particularly vigorous but no cellulase activity or cellulolytic gut symbionts are apparent.A brush border epithelium lines the mesenteron but no specialized secretory or excretory regions of the gut are evident. Morphological evidence for the endocytosis of material from the lumen is confirmed with the aid of food treated by the addition of ferritin tracer, which is readily identified under the electron microscope. Lysosomes are identified in the wall of the mesenteron using the histochemical localization of acid phosphatase and cholinesterase activity within membrane-bounded organelles. The existence of an intracellular mechanism for digestion is postulated, which could account for the hydrolysis of protein and, possibly, small particles of cellulose, but polysaccharides are probably broken down at the brush-border and in the gut lumen.The cells of the gut wall may also ingest material from the haemocoel, as indicated by the purely morphological evidence of invaginations in the external wall of the gut. It is suggested that this process may involve the intracellular digestive system of the gut wall in a ‘retinculoedothelial’ mechanism.     

酸碱调控污泥厌氧发酵实现乙酸累积及微生物种群变化

摘要：【目的】通过对污泥厌氧发酵pH调控,研究挥发性脂肪酸的累积、产酸微生物种群变化及产氢产乙酸菌群对乙酸产生的贡献。【方法】测定不同pH条件下污泥厌氧发酵过程中挥发性脂肪酸的累积;分别应用末端限制性片段长度多态性（T-RFLP）和荧光原位杂交技术（FISH）分析产酸系统中微生物种群结构的变化及产氢产乙酸菌的数量。【结果】 pH为10.0时,有机酸和乙酸的产率在发酵结束时分别达到652.6 mg COD/g-VS和322.4 mg COD/g-VS,显著高于其它pH条件。T-RFLP结果表明,pH值为12     

Volatile fatty acids distribution during acidogenesis of algal residues with pH control

The anaerobic acidification of protein-rich algal residues with pH control (4, 6, 8, 10) was studied in batch reactors, which was operated at mesophilic(35 °C) condition. The distribution of major volatile fatty acids (VFAs) during acidogenesis was emphasized in this paper. The results showed that the acidification efficiency and VFAs distribution in the acid reactor strongly depended on the pH. The main product for all the runs involved acetic acid except that the proportion of butyric acid acidified at pH 6 was relatively higher. The other organic acids remained at lower levels. The VFAs yield reached the maximum value with about 0.6 g VFAs/g volatile solid (VS) added as pH was 8, and also the content of total ammonia nitrogen (TAN) reached the highest values of 9,629 mg/l. Low acidification degrees were obtained under the conditions at pH 4 and 10, which was not suitable for the metabolism of acidogens. Hydralic retention time (HRT) required for different conditions varied. As a consequence, it was indicated that pH was crucial to the acidification efficiency and products distribution. The investigation of acidogenesis process, which was producing the major substrates, short-chain fatty acids, would play the primary role in the efficient operation of methanogenesis.     

The Role of Salivary and Intestinal Complement System Inhibitors in the Midgut Protection of Triatomines and Mosquitoes

Saliva of haematophagous arthropods contain biomolecules involved directly or indirectly with the haematophagy process, and among them are encountered some complement system inhibitors. The most obvious function for these inhibitors would be the protection of the midgut against injury by the complement. To investigate this hypothesis, Triatoma brasiliensis nymphs were forced to ingest human serum in conditions in which the protection of midgut by the inhibitors is bypassed. In these conditions, the anterior midgut epithelium was injured by the complement, causing cell death. Once some insects such as Aedes aegypti have no salivary inhibitors, we hypothesized the existence of intestinal inhibitors. The inhibitory activity was investigated in the intestine of A. aegypti as well as in the saliva and intestine of other three triatomine species (T. brasiliensis, T. infestans and Rhodnius prolixus) using an immunological method able to determine the level of deposition of some complement factors (C1q, C3b, or C4b) on the surface of complement activator molecules linked to microplates. This methodology permitted to identify which points along the activation phase of the complement cascade were inhibited. As expected, soluble contents of A. aegypti''s intestine was capable to inhibit C3b deposition by the classical and alternative pathways. Saliva or soluble intestinal contents, obtained from triatomines were unable to inhibit C1q deposition by the classical pathway. C4b deposition by the classical pathway was inhibited by the intestinal contents from the three triatomines. On the other hand, only T. brasiliensis saliva inhibited C4b deposition. Both, saliva and intestinal contents from all triatomines were able to inhibit C3b deposition in the classical and alternative pathways. None of the material extracted from the intestinal cell membranes from the triatomines inhibited C3b deposition in the classical pathway. The existence of complement inhibitors may have important biological consequences which are discussed in detail.     

Peritrophic membrane structure and formation of larvalTrichoplusia niwith an investigation on the secretion patterns of a PM mucin

Peritrophic membrane or matrix (PM) secretion and formation patterns were examined in the cabbage looper larvae (Trichoplusia ni[Hubner]) by transmission and scanning electron microscopy (SEM). PM first became visible in the lumen between tips of the microvilli and the stomodeal valves as a single layered fibrous structure that became more compact in appearance in the middle and posterior mesenteron. In the anterior mesenteron, nascent PM was visible within the brush border as a fibrous linear structure that contained both the major PM matrix protein, invertebrate intestinal mucin (IIM) and chitin-containing structures. Even though delamination events were confined to the anterior mesenteron, IIM was secreted by columnar epithelial cells throughout the length of the mesenteron. SEM of the midgut epithelium revealed PM covering individual epithelial cells.     

Production of microbial lipid by Cryptococcus curvatus on rice straw hydrolysates

Microbial biolipids/biodiesels derived from volatile fatty acids (VFAs) can be a valuable alternative to plant oils if optimum fermentation conditions are determined. VFAs were used for cell mass and microbial lipid production by Cryptococcus curvatus. The lipid content in the cells increased up to 48% and 28% in batch cultures with the use of 20 g/L glucose and 6 g/L of VFAs as the carbon source, respectively. In this study, C. curvatus used VFAs as a carbon source via anaerobic digestion of rice straw hydrolysates. VFAs produced from rice straw resulted in yield of 0.43 g VFAs/g substrate and 40% higher specific growth rate(0.305 h⁻¹) than synthetic VFAs. The highest fatty acid composition observed was C18:1, was obtained using glucose and VFAs as the carbon source to yield a cetane number of 56–59, which is suitable for biodiesel production. The cost of microbial lipids was estimated to be 0.30–1.15 USD/L given 0–150 USD/ton of VFAs cost for a yield of 0.17 g/g of lipids. Thus, VFAs can be a suitable carbon source for economical biodiesel production.     

Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

Changes in amount of bacteria during gut passage of leaf litter and during coprophagy in three species of<Emphasis Type="Italic">Bibionidae (Diptera)</Emphasis> larvae

To elucidate the interaction between bacteria and saprophagous Diptera larvae, the amounts of bacteria in leaf litter, individual gut compartments, and feces of three species of Bibionidae (Bibio pomonae, Bibio marci, and Penthetria holosericea), feeding either directly on leaf litter or on fecal pellets produced from leaf litter by larvae of the same species, were assessed by determining total direct counts and viable counts on solid media at different pH. In P. holosericea, the effect of various cultivation temperatures on direct counts of bacteria in individual compartments was also demonstrated. In all species, the amount of bacteria in the anterior mesenteron was lower than in the consumed food, regardless of whether the larvae were feeding on leaf litter or feces, and increased again in the posterior part of the gut. The amount of bacteria in these compartments was generally higher in larvae feeding on feces than in those feeding on leaf litter, whereas the amount of bacteria found in the ceca varied. In B. marci, the amount of bacteria in the mesenteron sections able to grow on alkaline medium (pH 9) was higher than that of bacteria able to grow on slightly acidic medium (pH 5.5) during both the first and the second gut passage. In B. pomonae and P. holosericea, this increase was observed only during the second gut passage. The effect of gut passage in P. holosericea on changes in direct counts of bacteria was more pronounced when the larvae were fed at 5 degrees C as compared to 20 degrees C. Radiolabeled bacteria were digested in the gut and utilized as a source of energy and nutrients by the larvae; digested bacteria represented up to 10% of the material assimilated by the larvae. Lysozyme activity in whole-gut extracts of P. holosericea had a pH optimum of at pH 7, indicating a low in situ activity in the alkaline mesenteron. Proteinase activity, however, had an optimum at pH > 12, suggesting that the digestion of bacteria in the bibionid gut is caused by a combination of digestive proteinases and alkaline pH in the anterior mesenteron.     

Hydrogen and volatile fatty acids production from marine macroalgae by anaerobic fermentation

Hydrogen and volatile fatty acids (VFAs) were coproduced from marine macroalgae by anaerobic fermentation using a microbial community. The hydrogen and VFAs production were characterized based on inoculum heat-treatment, methanogen inhibitor addition, operating temperature, and in-situ extraction of VFAs. Maximum hydrogen of 179 mL/g-VS and VFAs concentration of 9.8 g/L were produced from 35 g/L of S. japonica within 5 days of anaerobic fermentation. Hydrogen and VFAs yields were well-correlated with carbohydrate content of substrate. Inoculum heat-treatment significantly improved hydrogen production while the VFAs productivity was affected adversely. The addition of methanogen inhibitors also enhanced the hydrogen production, but the effect on VFAs production was dependent on the type of inhibitor used. Low temperature (25°C) was found to be favorable for high hydrogen and VFAs yield, while high temperature (40°C) and programmed-temperature (35 ~ 45°C) were more favorable for hydrogen and VFAs productivity. Clostridium sp. content was found to be the most abundant at 25°C. An extractive fermentation with anion-exchange resin was tested to recover the VFAs and to control the pH during the anaerobic fermentation.     

Action sur le mésentéron dePieris brassicae L. de la toxine de l'inclusion parasporale deBacillus thuringiensis Berliner

Summary Ingestion of the toxin of the parasporal inclusion ofB. thuringiensis byP. brassicae causes digestive disturbances which are first manifested by cessation of feeding and a decrease in the pH of the stomodeal and mesenteral content. The physiopathological condition of individuals so poisoned shows that this alteration of the intestinal medium can cause lesions of the mesenteric epithelium, which can be so serious that all the cellular digestive elements are destroyed. It was not possible to link the origin of these alterations with the established mortality rate, but it was suggested that the acidification of the intestinal medium could be the cause. This suggestion seems to have been verified after forced citric acid ingestion. The morphological modifications of the mesenteron cannot explain alone the death of the treated individuals; on the other hand, having shown a proliferation of the intestinal bacterial flora, one is lead at the present stage of research to follow the opinion ofVago on the intoxication-septicaemia chain. In fact, all the phenomena which can be described as resulting from theB. thuringiensis method of poisoning, specially ofP. brassicae, namely lesions of the mesenteric epithelium, proliferation of the intestinal bacterial flora, only appear to be secondary manifestations.      

On the organogenesis of Seuratum cadarachense Desportes, 1947 (Nematoda Seuratoidea) and the cellular reactions of the intermediate insect host Locusta migratoria (author's transl)]

The adaptation of primitive heteroxenous nematodes, parasites of vertebrates, to the intermediate host, is detailed by studying the organogenesis of the first larval stages of the Seuratoid nematode Seuratum cadarachense and an histological approach of the cellular reactions caused by the penetration and the development of the parasite in the insect Locusta migratoria. The larval organization of Seuratum at hatching is similar to that of Subuluridae and characterized by a large number of initial cells: among them, eight cells alined in a row forming the intestinal primordia, and two mesenchymal cells. In Seuratum, as in Subulurid in general, the first larval stage has a slow development. The larval migration occurs in the wall of the mesenteron. Hemocytic capsules are observed in the hemocoel in contact with the muscle fibers of the mesenteron. Some of them show necrotic features which bear witness to an imperfect adaptation of the nematode to the intermediate host.     

Isolation of proctolin,a myotropic peptide,from Periplaneta americana

The isolation of a myotropic substance, proctolin, associated with the innervation of the viscera of the cockroach, Periplaneta americana, is reported. Proctolin, previously described as a neurotransmitter or neurohormone, is a small basic peptide with an estimated molecular weight of 500 to 700. The proctodeal (hindgut) muscles of P. americana contract at threshold levels of about 0·5 ng/ml, equivalent to about 10⁻⁹ M proctolin. Approximately 180 μg proctolin were isolated from 125 kg of whole cockroaches. The isolated peptide was homogeneous on paper and thin-layer chromatography and on high-voltage paper electrophoresis at pH 3·5 and 6·4.     

Determination of pH and oxygen status in the guts of lower and higher termites

The pH of the gut was determined in vitro in six species of termite by means of indicator dyes and a pH electrode. In the lower termite Zootermopsis nevadensis the pH was close to neutrality throughout, ranging 6.0–7.5, but in Reticulitermes lucifugus, acid conditions (pH 5.5–6.0) occurred in the crop and paunch. In the higher termites Nasutitermes costalis, Microcerotermes arboreus, Cubitermes severus and Procubitermes aburiensis, there was a common trend of incresing pH from the crop, which was slightly or moderately acidic, to the first proctodaeal segment (P1) where moderately (N. costalis) and strongly (M. arboreus, C. severus and P. aburiensis) alkaline conditions prevailed. A pH of 10.4 was measured in C. severus, equalling the highest recorded in any insect. In the posterior regions of the hindgut there was a return towards neutral or acidic conditions. When termite guts were homogenized with air-saturated Ringer's solution, the dissolved O₂ content of the Ringer's was reduced. This was shown to be largely attributable to an oxygen deficit generated within the gut in situ. The combined effects of strongly alkaline conditions and reduced oxygen tension on digestive processes and intestinal micro-organisms are discussed.     

Intestinal disaccharidase activities in the chick

1. Disaccharidase activities of the small and large intestines of the chick were studied. 2. Homogenates of the small intestine readily hydrolysed maltose, sucrose and palatinose (6-O-α-d-glucopyranosyl-d-fructose), hydrolysed lactose slowly and did not hydrolyse trehalose and cellobiose. 3. Within the small intestine the disaccharidases were located mainly in the intestinal wall; the activity in the contents accounted for less than 5% of the total activity. 4. The disaccharidases were non-uniformly distributed along the small intestine, the activities being greatest in the middle section. 5. The disaccharidase activities increased with age between 1 and 43 days. 6. Homogenates of the large intestine and contents readily hydrolysed maltose, sucrose, palatinose and lactose and hydrolysed cellobiose and trehalose slowly. 7. The large-intestinal disaccharidases were located mainly in the contents. 8. Similar K_m and pH optimum values were found for the maltase, sucrase and palatinase activities of the large and small intestines. 9. The lactase activity of the large intestine was markedly affected by diet and had different K_m and pH values from the small intestinal lactase. 10. Low activities of intestinal disaccharidase were found in 12-day-old embryos and marked increases in the intestinal disaccharidases of the developing embryo occurred 2–3 days before hatching.     

Microbial synergists pathogenic to Lymantria dispar: Chitinolytic and fermentative bacterial interactions

A chitinolytic bacterium isolated from a healthy gypsy moth, Lymantria dispar, larva was shown to be pathogenic to larvae when administered per os after growth on chitin broth. However, the chitin hydrolase produced by this isolate had a pH optimum for activity of 5.5 and the high alkalinity in the mesenteron of L. dispar larvae severely limited chitinolysis of the midgut lining. Fermentative, nonpathogenic, acid-producing bacteria isolated from healthy gypsy moth larvae effectively lowered larval mesenteron pH when administered per os and the combination of fermentative isolates with a crude culture aliquot of the chitinolytic strain produced a synergistic increase in mortality over either dose administered by itself. Increased mortality was also observed for most fermentative strains when they were combined with crude supernatants of centrifuged cultures of the chitinolytic strain, although these combinations proved less effective than when fermenters were added to the whole-culture aliquots of the chitinolytic strain. In vitro studies showed that other bacteria isolated from environments foreign to that of the gypsy moth could ferment carbohydrates with acid production at an alkaline pH; however, in vivo studies demonstrated that these bacteria were incapable both of poising larval midgut pH and of enhancing mortality when added to chitinolytic bacteria.     

Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp.)

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (Termitidae: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.     

High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasutitermes spp.)

Higher termites are characterized by a purely prokaryotic gut microbiota and an increased compartmentation of their intestinal tract. In soil-feeding species, each gut compartment has different physicochemical conditions and is colonized by a specific microbial community. Although considerable information has accumulated also for wood-feeding species of the genus Nasutitermes, including cellulase activities and metagenomic data, a comprehensive study linking physicochemical gut conditions with the structure of the microbial communities in the different gut compartments is lacking. In this study, we measured high-resolution profiles of H(2), O(2), pH, and redox potential in the gut of Nasutitermes corniger termites, determined the fermentation products accumulating in the individual gut compartments, and analyzed the bacterial communities in detail by pyrotag sequencing of the V3-V4 region of the 16S rRNA genes. The dilated hindgut paunch (P3 compartment) was the only anoxic gut region, showed the highest density of bacteria, and accumulated H(2) to high partial pressures (up to 12 kPa). Molecular hydrogen is apparently produced by a dense community of Spirochaetes and Fibrobacteres, which also dominate the gut of other Nasutitermes species. All other compartments, such as the alkaline P1 compartment (average pH, 10.0), showed high redox potentials and comprised small but distinct populations characteristic for each gut region. In the crop and the posterior hindgut compartments, the community was even more diverse than in the paunch. Similarities in the communities of the posterior hindgut and crop suggested that proctodeal trophallaxis or coprophagy also occurs in higher termites. The large sampling depths of pyrotag sequencing in combination with the determination of important physicochemical parameters allow cautious conclusions concerning the functions of particular bacterial lineages in the respective gut sections to be drawn.