Midgut cysteine protease-inhibiting activity in Trichoplusia ni protects the peritrophic membrane from degradation by plant cysteine proteases

The action of plant cysteine proteases on the midgut peritrophic membrane (PM) of a polyphagous herbivorous lepidopteran, Trichoplusia ni, was studied. Proteins in PMs isolated from T. ni larvae were confirmed to be highly resistant to the serine proteinases trypsin and chymotrypsin, but were susceptible to degradation by plant cysteine proteases, which is consistent with the known molecular and biochemical characteristics of the T. ni PM proteins. However, the PM proteins were not degraded by plant cysteine proteases in larvae or in the presence of larval midgut fluid in vitro. With further biochemical analysis, cysteine protease-inhibiting activity was identified in the midgut fluid of T. ni larvae. The cysteine protease-inhibiting activity was heat resistant and active in the tested pH range from 6.0 to 10.0, but could be suppressed by thiol reducing reagents or reduced by treatment with catalase. In addition to T. ni, cysteine protease-inhibiting activity was also identified from two other polyphagous Lepidoptera species, Helicoverpa zea and Heliothis virescens. In conclusion, results from this study uncovered that herbivorous insects may counteract the attack of plant cysteine proteases on the PM by inhibiting the potentially insecticidal cysteine proteases from plants in the digestive tract. However, the biochemical identity of the cysteine protease-inhibiting activity in midgut fluid has yet to be identified.     

Differential responses of midgut soluble aminopeptidases of Helicoverpa armigera to feeding on various host and non-host plant diets

Differential responses of midgut soluble aminopeptidases were studied in Helicoverpa armigera larvae fed on various host (chickpea and pigeon pea) and non-host (bitter gourd and chili) plant diets. Larval growth was significantly reduced by non-host plant diets. Although the serine proteinase activities were inhibited, aminopeptidase activities were significantly increased in the larvae fed on non-host plant diets. Results were qualitatively and quantitatively confirmed with in vivo and in vitro analyses. It was noted that aminopeptidases had given more preference to ApNA than LpNA on non-host plant diets and vice versa on host plant diets. However, optimum pH for aminopeptidase activity (around pH 7.0–8.0) and susceptibility to inhibitors was similar in the larvae fed on host as well as non-host plant diets. These results suggest that H. armigera regulates digestive enzyme levels to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. A detailed biochemical and molecular analysis of gut aminopeptidases upon exposure of the insect to a particular diet will highlight their specific roles.     

Influence of dietary aconitine and nicotine on the gut microbiota of two lepidopteran herbivores

The gut microbiota plays an important role in pheromone production, pesticide degradation, vitamin synthesis, and pathogen prevention in the host animal. Therefore, similar to gut morphology and digestive enzyme activity, the gut microbiota may also get altered under plant defensive compound-induced stress. To test this hypothesis, Dendrolimus superans larvae were fed either aconitine- or nicotine-treated fresh leaves of Larix gmelinii, and Lymantria dispar larvae were fed either aconitine- or nicotine-treated fresh leaves of Salix matsudana. Subsequently, the larvae were sampled 72hr after diet administration and DNA extracted from larval enteric canals were employed for gut microbial 16S ribosomal RNA gene sequencing (338 F and 806 R primers). The sequence analysis revealed that dietary nicotine and aconitine influenced the dominant bacteria in the larval gut and determined their abundance. Moreover, the effect of either aconitine or nicotine on D. superans and L. dispar larvae had a greater dependence on insect species than on secondary plant metabolites. These findings further our understanding of the interaction between herbivores and host plants and the coevolution of plants and insects.     

Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha-casein, and N-benzoyl-DL-arginine napthylamide-(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p-hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman-Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L-Trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.     

The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse,Tracheoniscus rathkei

Summary The digestive tract of the common woodlouse, Tracheoniscus rathkei Brandt (Isopoda: Oniscoidea), contains digestive enzymes active against -1,4-glucans, which are the chief storage polysaccharides of vascular plants, algae, fungi, and animals, and -1,3-glucans, which are present in algae and fungi. Digestive tract extracts also exhibit significant activity toward xylan and carboxymethyl-cellulose but negligible activity toward microcrystalline cellulose, substrates representative of the major structural polysaccharides of vascular plants. Low activity was detected toward pectin, and no activity was detected toward chitin. Activity toward xylan is due in part to microbial enzymes acquired from the leaf litter which was the isopod's normal food. Although ingested microbial xylanases are stable and active in the gut fluid, they do not make a quantitatively significant contribution to the isopod's ability to assimilate the hemicellulosic component of its diet. However, the assimilation of carbon from labeled plant fiber is enhanced in isopods which have acquired a cellulase by ingestion of leaf litter amended with a commercial preparation of the cellulase complex from the fungus, Penicillium funiculosum. This result demonstrates the potential contribution of acquired enzymes to the digestion of plant fiber in terrestrial detritivores. We urge caution, however, in assigning an important digestive function to ingested enzymes on the basis of evidence that only indicates that such enzymes are present in the gut fluid without additional evidence that their presence results in an enhancement of digestive efficiency.     

The digestion of protein and carbohydrate by the stream detritivore,Tipula abdominalis (Diptera,Tipulidae)

Summary The digestive system of larvae of Tipula abdominalis (Diptera, Tipulidae), a stream detritivore, is poorly adapted for the digestion of the major polysaccharides in its diet, but well adapted for the digestion of protein. These crane fly larvae are unable to digest the major cell wall polysaccharides of higher plants, i.e., cellulose, hemicellulose and pectin. The only polysaccharides toward which the midguts of T. abdominalis exhibited any activity were -amylose and laminarin, indicating that polysaccharide digestion is restricted to -1,4-and -1,3-glucans. The most concentrated source of these two classes of carbohydrates in submerged leaf litter would be associated fungal tissue. The midgut of T. abdominalis is strongly alkaline throughout, with a maximum pH near 11.5 in a narrow zone near the midpoint. Proteolytic activity in the midgut is extraordinarily high, and the pH optimum for midgut proteolytic activity is above 11. We conclude that the high alkalinity and high proteolytic activity observed in T. abdominalis larvae are manifestations of a highly efficient protein-digesting system, a system of crucial importance to a nitrogen-limited organism which must derive its nitrogen from a resource in which much of the limited nitrogen present is in a bound form in complexes of proteins with lignins and polyphenols.     

Gut physicochemistry of grassland grasshoppers

We examined the pH and Eh of the digestive tract of 23 species of mixed-grass prairie grasshoppers, and asked whether these traits were associated with the species breadth and forb composition of their diets. We report that the gut lumen of all grasshoppers was oxidizing and ranged from slightly acid to neutral depending on the gut region and species. Although gut physicochemical conditions differed among species, the differences were of small magnitude. Conditions were fairly uniform along the digestive tract, which suggests little or no regulation of pH or Eh. Gut conditions were independent of diet breadth and the percentage of forbs in the diet. These results suggest that physicochemical conditions of grasshopper guts are not highly regulated and are not influenced by their most recent meal or by broad scale patterns of host-plant use.     

Biochemical composition of Streptocephalus dichotomus Baird Branchinella kugenumaensis (Ishikawa)

Summary Estimations of total nitrogen, non-protein nitrogen protein, amino acids, lipid, carbohydrate, ash and water contents are carried out for the two fairy shrimps Streptocephalus dichotomus and Branchinella kugenumaensis.The mean values for S. dichotomus are: T.N. - 12.13%; N.P.N. - 3.49%; Protein - 54.71%; Lipid - 11.01%; Carbohydrate - 7.43% and ash - 10.44% and for B. kugenumaensis are: T.N. - 12.63%; N.P.N. - 4.92%; Protein - 48.21%; Lipid - 17.14%; Carbohydrate - 8.93% and ash - 9.17% expressed as percentage dry weight.The protein ash contents are higher in S. dichotomus while non-protein nitrogen, lipid and carbohydrate are higher in B. kugenumaensis.These biochemical fractions show no significant difference between males and females in both S. dichotomus and B. kugenumaensis.The work on Streptocephalus dichotomus formed a part of a thesis submitted for the Degree of Doctor of Philosophy to the University of Madras in 1970.     

Food,feeding and digestion in streptocephalus dichotomus baird (Crustacea: Anostraca)

Summary Gut analysis of Streptocephalus dichotomus revealed that the main source of food is phytoplankton. S. dichotomus is a non-selective filter feeder, taking in all the food-items carried in the feeding currents and passed through the midventral groove. The feeding currents are produced by the thoracic limbs.Feeding experiments have shown that the time taken for the passage of food is directly proportional to the number of days of starvation. Food intake of males did not significantly differ from that of females when fresh animals were used, as well as animals used after one day of starvation.Fresh males as well as females took in significantly more food than starved animals.The digestive enzymes such as carbohydrases, proteases and lipases are present in the gut. The optimal pH for amylase ranged between, 5.8 to 6.6, for protease 7.4 to 8.0 and for lipase 5.2 to 6.5.This work formed a part of the thesis submitted to the University of Madras, in 1970 for the award of the Degree of Doctor of Philosophy.     

Gastro‐Intestinal transit time in South American deer

The study of diet and physiological peculiarities of the digestive system of neotropical deer is not well known and the literature shows inconsistencies. To better understand the digestive system of these mammals the difference in the gastro‐intestinal transit time of four species of neotropical deer (Mazama americana, Mazama gouazoubira, Mazama nana, Blastocerus dichotomus), kept in captivity, was evaluated. Four plants (Neonotonia wightii, Morus albans, Medicago sativa, Leucaena leucocephala) were utilized and two variables were measured, mean time of the beginning of the elimination (BE) and mean time of permanence (TP). The results obtained for BE indicated similarity among the deer species, with significant differences between M. gouazoubira (mean=13.62 hr) and M. Americana (mean=19.25 hr). For the plants, the BE was faster with N. wightii and L. leucocephala, and slower for M. sativa. The TP results for B. dichotomus showed longer time when compared to the other species, whereas M. gouazoubira had a lower permanence. Overall, N. wightii had the highest retention time in the digestive tract of all the deer species studied. Associated with this observation, N. wightii had the highest quantity of plant fiber of the plants tested. In a similar fashion M. sativa showed the lowest TP in the digestive tract of the deer and had the lowest quantity of acid detergent fiber. The data from this study showed that, within species, the shape of the excretion curve of the plants was similar when the animals consumed N. wightii or M. sativa. Blastocerus dichotomus and M. gouazoubira had the highest and the lowest gastro‐intestinal transit time, respectively. This suggests that these species characterize different abilities to digest high fiber food, and consequently, represent the two extremities in the morphophysiological adaptation within the deer species evaluated. This information is vital because it is important to know the digestive physiology to define the diet of captive animals, particularly regarding the quantity and quality of fiber. Zoo Biol 0:1–11, 2005. © 2005 Wiley‐Liss, Inc.     

Alkalinity within the midgut of mosquito larvae with alkaline-active digestive enzymes.

Digestive enzyme pH optima were determined in vitro for the larvae of three mosquito spp., Culex pipiens, Aedes aegypti, and Anopheles stephensi. All had optimal amylase activity at about pH 8 and optimal protease activity over a broad range between pH 10 and 12. pH within the digestive tract of intact live larvae was determined from the colours of indicator dyes ingested with kaolin and visible in the gut through the transparent or translucent body tissues. In all three spp. the contents of the anterior midgut were held at a pH just exceeding 10, with an abrupt fall to about pH 7·5 in the posterior third midgut.     

Postprandial changes in pH and enzyme activity from the stomach and intestines of Tilapia rendalli (Boulenger, 1897), Oreochromis mossambicus (Peters, 1852) and Clarias gariepinus (Burchell, 1822)

The ability to utilise dietary components differs among fish species. Digestive enzymes may be used to determine the efficiency of the digestive process. In this study, the activities of the digestive enzymes in Tilapia rendalli, Oreochromis mossambicus and Clarias gariepinus were explored. Protease, amylase, lipase and cellulase activities were measured in different parts of the digestive tract of the three fish species. The pH dynamics along the digestive tract were monitored. In all fish species, the presence of food led to a reduction in stomach pH, whereby pH values of 1.54, 1.58 and 2.01 were recorded 12 h after feeding in O. mossambicus, T. rendalli and C. gariepinus, respectively. Protease and amylase activities were significantly higher (P < 0.05, anova ) in the tilapias than in C. gariepinus. The tilapias may be pre‐adapted to produce more protease and amylase to digest plant material, which is more difficult to digest than animal matter. In all species amylase activity was significantly higher in the proximal intestine than in the other parts of the digestive tract (P < 0.05, anova ). The highest protease activity was recorded in the distal intestines. This is because of the alkaline pH recorded in the proximal and distal intestines, which favours amylase and protease activity, respectively. Lipase activities were significantly higher (P > 0.05) in C. gariepinus than in both tilapias. Marginal cellulase activities were recorded in all species. It is inferred here that phylogeny and not diet may be the main factor influencing enzyme activities, as all fish were fed a similar diet.     

Digestive strategies in the South American subterranean rodent Ctenomys talarum

Ctenomys talarum is a subterranean herbivorous rodent which due to its particular life style is frequently exposed to variations in surface environmental conditions (i.e. food quality and availability, temperature). Thus, unlike other subterranean rodents, C. talarum has to buffer both the surface and burrow challenging environmental conditions. We studied the occurrence of digestive strategies at different levels of C. talarum living in their natural habitat. We determined the dimensions of different parts of the gastrointestinal tract and organs along as the activity of key digestive enzymes (disaccharidase, N-aminopeptidase) in different parts of the gut in individuals seasonally caught. The results show that C. talarum exhibits characteristics in the gut at the biochemical level (high disaccharidase activities in small intestine, high N-aminopeptidase activity in the caecum and large intestine, and a seasonal differential modulation of N-aminopeptidase activity in small and large intestines), which could represent adaptive strategies to face seasonal variations in key environmental factors.     

Promicromonospora pachnodae sp. nov., a member of the (hemi)cellulolytic hindgut flora of larvae of the scarab beetle Pachnoda marginata

Intestinal microorganisms play an important role in plant fiber degradation by larvae of the rose chafer Pachnoda marginata. In the hindgut of the larvae 2.5 to 7.4 × 10⁸ bacteria per ml of gut content with xylanase or endoglucanase activity were found. Bacteria in the midgut were not (hemi)cellulolytic, but the alkaline environment in this part of the intestinal tract functions as a precellulolytic phase, solubilizing part of the lignocellulosic material. Accordingly, the degradation of lignocellulose-rich material in Pachnoda marginata larvae appeared to be a combination of a physico-chemical and microbiological process. A number of different facultative anaerobic and strictly anaerobic bacteria with (hemi)cellulolytic activity were isolated from the hindgut. A dominant (hemi)cellulolytic species was a Gram positive, irregular shaped, facultative aerobic bacterium. Further physiological identification placed the isolate in the genus Promicromonospora. Comparative 16S rDNA analysis and phenotypic features revealed that the isolate represented a new species for which the name Promicromonospora pachnodae is proposed. P. pachnodae produced xylanases and endoglucanases on several plant derived polymers, both under aerobic and anaerobic conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Digestive strategy of the asian colobine genusTrachypithecus

The Asian colobines,Trachypithecus obscurus andT. cirstantus, eat plant-based diets containing 55–80% leaves. The structural polysaccharides in leaves and other plant parts require microbial fermentation before they can be used as an energy source by the monkeys. The major compartments of the gastro-intestinal tract ofTrachypithecus are a voluminous haustrated stomach, a long small itnestine and capacious haustrated hindgut, all of which contribute to the digestive strategy of these two species. Results of digesta marker passage studies indicate there is prolonged retention of digesta for fermentation in both the stomach and haustrated colon. The digestive strategy of these colobines is defined as gastro-colic fermentation, unlike that of other forestomach fermenters in which the hindgut fermentation is of secondary importance.     

Nitrogen Mineralization, Ammonia Accumulation, and Emission of Gaseous NH3 by Soil-feeding Termites

There are numerous reports on the accumulation of ammonia in the mounds of soil-feeding termites. Here, we provided direct evidence for an effective mineralization of nitrogenous soil organic matter in the gut of Cubitermes spp., which gives rise to enormous ammonia concentrations in the intestinal tract. In Cubitermes ugandensis, the ammonia content of the nest material [24.5 μmol (g dry wt.)⁻¹] was about 300-fold higher than that of the parent soil. Large amounts of ammonia were present throughout the intestinal tract, with lowest values in the extremely alkaline gut sections (pH >12) and highest values posterior hindgut [185 μmol (g dry wt.)⁻¹]. Results obtained with other Cubitermes species were similar. Ammonia concentrations in the posterior hindgut of these humivorous species (up to 130 mM) are among the highest values ever reported for soil macroinvertebrates and are matched only by insects feeding on an extremely protein-rich diet (e.g., the sarcophageous larvae of blowflies). Volatilization of ammonia [about 10 nmol (g fresh wt.)⁻¹ h⁻¹], either directly by emission from the termite body or indirectly from their feces, led to NH₃ concentrations in the nest atmosphere of C. ugandensis that were three orders of magnitude above the ambient background – a relative accumulation that is considerably higher than that observed with CH₄ and CO₂. Together with previous results, these observations document that through their feeding activity and due to the physicochemical and biochemical properties of their digestive system, soil-feeding termites effectively catalyze the transformation of refractory soil organic nitrogen to a plant-available form that is protected from leaching by adsorption to the nest soil. Nitrogen mineralization rates of soil-feeding termites may surpass those effected by tropical earthworms and should contribute significantly to nitrogen fluxes in tropical ecosystems.     

Effect of Cultivation Conditions on the Composition of Extracellular Polysaccharide-Containing Substances in the Bacterium Azospirillum brasilense

Maintenance of pH 7.0 during the fermentation period favors accumulation of high molecular weight polysaccharide-containing components called lipopolysaccharide–protein and polysaccharide–lipid complexes in the capsules and culture medium. Increased pH of the culture medium to 8.0 reduced the period of exponential growth and the yield of polysaccharide-containing complexes as compared to optimal conditions. Maintenance of pH 5.5 suppressed the culture growth and polysaccharide production. The polysaccharide–lipid complexes obtained when pH was stabilized at the level of 7.0–8.0 had relatively low molecular weights and included only acidic polysaccharides. The use of potassium gluconate instead of sodium malate as a source of carbon in the culture medium changed the polysaccharide composition and increased the content of glucosamine, which increased the affinity of polysaccharides for wheat germ agglutinin. Prolongation of Azospirillum cultivation to five days introduced new glucose-containing polysaccharide components in the capsule.     

蝙蝠蛾幼虫肠道中的冬虫夏草菌体研究

西藏林芝地区冬虫夏草天然产区野生虫草蝙蝠蛾幼虫的肠道液pH范围为9.69-10.99,体外在此pH下培养冬虫夏草菌长势较差。同时,在该pH的体外模拟中肠液中,虫菌体也会在极短的时间内消亡,而对自然侵染的蝙蝠蛾幼虫的血淋巴、消化道及其“反吐物”（肠液）进行切片和显微镜检却发现,蝙蝠蛾幼虫消化道、呕吐物中均存在与血淋巴中相同的冬虫夏草菌菌体形态,生存状态良好,正常增殖,表明蝙蝠蛾幼虫肠道的生理状态可以维系并支撑冬虫夏草菌菌体某种形态的存在与生长,并证实冬虫夏草菌由口器及消化道入侵蝙蝠蛾幼虫的可能性;肠道虫菌体与血淋巴虫菌体不一定同时存在,表明冬虫夏草菌对蝙蝠蛾幼虫的侵染可能同时存在表皮侵染和肠道侵染两种途径。本文为冬虫夏草菌对蝙蝠蛾幼虫的侵染途径研究提供更加多样化的信息支持。     

胞外多糖产生菌的筛选鉴定及其促生改土作用

【背景】一些微生物具有较强的产胞外多糖潜力,在改良盐碱土壤、促进作物生长方面潜力巨大。【目的】筛选耐盐碱且产胞外多糖的微生物,为开发具有盐碱土壤改良效果的菌剂提供菌种资源。【方法】从滨海盐碱植物根际土中筛选可在盐碱土壤中增殖、产胞外多糖、解有机磷和钾等能力的微生物菌株,并通过形态、生理生化、基于16S rRNA基因序列和gyrB基因的分子鉴定确定菌株类别,分析该菌株的生长、产胞外多糖特性受盐碱胁迫的影响,考察该菌对盐碱土壤不同粒径团聚体比例的影响,并结合盐碱土壤番茄种植试验考察对植物生长指标和土壤指标的影响。【结果】从727株滨海盐碱土壤菌株中筛选出一株产胞外多糖、具有较强盐碱土壤增殖能力且能解有机磷和钾的芽孢杆菌GBW HF-98,确定该菌为枯草芽孢杆菌沙漠亚种(Bacillus subtilis subsp. inaquosorum)。该菌可耐受pH值达10.0和NaCl浓度达110 g/L,在改善轻和中盐度土壤中不同粒径团聚体比例的效果较为显著。该菌的高剂量浓度T2组,在中度盐碱土壤的番茄种植试验中,与对照相比显著增加了壮苗指数、根干重、全株干重、茎粗和株高等指标,增幅分别为33.8%、59.3%、37.2%、12.3%和15.6% (P<0.05);显著降低pH、全盐含量和土壤容重,降幅分别为8.9%、27.9%和17.9%;显著提升土壤速效磷和速效钾指标,增幅分别为36.0%和17.4% (P<0.05)。【结论】GBW HF-98菌株具有较强的胞外多糖产生能力,在中度盐碱土壤中促进番茄生长和改良土壤的效果显著,可用于盐碱土壤改良菌剂的研制。     

Factors affecting calculation of nutritional induces for foliage-fed insects: an experimental approach

To determine how nutritional indices for insects fed leaves are affected by the experimental conditions and the physiology of the plant material, we used larvae of the buckmoth, Hemileuca lucina Hy. Ed. (Saturniidae) and their hostplant Spiraea latifolia Ait. Bork (Rosaceae). Under experimental conditions identical to those used to determine larval nutritional indices, we found that the age of leaves (new versus mature) significantly affected their metabolism and water loss, but simulated herbivory did not directly affect leaf metabolism. Over a 6-day test, nitrogen concentration showed an initial increase followed by a gradual decline, and was higher in new leaves compared to mature leaves. New leaves increased in protein concentration and then gradually returned to the initial level, whereas mature leaves changed little over the 6-day test. These changes in percent nitrogen and protein may largely reflect the disproportional changes in non-nitrogenous materials. Solitary and grouped larvae had similar growth rates on new leaves, but they differed on mature leaves. Deliberate manipulation of larvae during the course of an experiment significantly reduced relative growth rates by increasing duration of the stadium rather than by decreasing biomass gained. Two methods of estimating larval gut contents at mid-stadium were compared: weight of frass produced and weight of digestive tract and contents. After the end of the 4-day test period used to determine nutritional indices, the digestive tracts with food accounted for 10.8% of the larval dry weight. Larval frass produced in 24 h after the end of the test period comprised 9.3% of the larval dry weight. Correction factors for plant metabolism changed nutritional indices by 1 to 8%, while those for larval gut contents altered indices by 2 to 15%.