Application of 13C NMR to investigate the transformations and biodegradation of organic materials by wood- and soil-feeding termites, and a coprophagous litter-dwelling dipteran larva

Solid-state 13C nuclear magnetic resonance spectroscopy has been used to characterize the C in samples of the food (wood), gut contents and faeces from the wood-feeding termite, Microcerotermes parvus; soil in the guts and mound material from the soil-feeding termite, Thoracotermes macrothorax; and the food and faeces from the litter-feeding, coprophagous larvae of the dipteran fly, Bibio marci. Spectra from the wood-feeding termite indicated preferential loss of polysaccharide and accumulation of lignin with some modification to the O-aromatic-C and methoxyl-C (O-methyl-C) components during passage through the gut. Spectra for the soil-feeding termite indicated little change in the distribution of 13C between resonances following passage through the gut, except for some evidence of preferential polysaccharide loss. Interpretation of the spectra from these organisms was restricted by the relatively low C content of the soils and mound material, and by the large contribution to the NMR spectra from the gut tissue rather than the gut contents. Spectra for the litter-feeding dipteran larvae indicated preferential feeding on the polysaccharide-rich component of the litter and then overall loss of polysaccharide-C and accumulation of both aromatic-C and methoxyl-C in the gut. These changes were greater for the second passage than for the first passage through the gut, suggesting that principally mechanical and physical changes occurred initially and that chemical digestion was prevalent during the second passage.     

Mobilization of soil phosphorus during passage through the gut of larvae of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

Soil macrofauna play important roles in the mobilization of soil nutrients. The influence of gut passage on soil phosphorus availability was investigated using the larvae of the scarabaeid beetle Pachnoda ephippiata. Gut passage significantly increased the levels of available P in the feces over that of the soil consumed. ³¹P-NMR recorded the changes in the nature of organic P in alkaline extracts from gut content. Alkaline phosphatase activity was high in the alkaline midgut, but low in the hindgut and soil. Ferric iron (Fe³⁺) in the soil was reduced to ferrous iron (Fe²⁺) in the gut. The amount of Ca²⁺ was lower in the gut than in the soil. Iron reduction and the decrease of Ca²⁺ concentration in the gut would reduce P sorption. We think that the following effects are responsible for increased availability of P during the gut passage: alkaline extraction of soil organic matter; hydrolysis of organic phosphate esters with alkaline phosphatase; digestion by the resident microorganisms; and changes in the concentration of metal ions during gut passage. This study suggests that the feeding activities of humivorous larvae would affect the amount of soil P available to plant growth.     

Assessment of the role of a terrestrial isopod in the survival of a genetically modified pseudomonad and its detection using the polymerase chain reaction

Abstract The effects of a terrestrial isopod, Porcellio scaber , on the survival of a genetically modified pseudomonad were studied. Pseudomonas fluorescens KTG was inoculated onto ash leaf litter and supplied to populations of P. scaber . Plate counts were lower in fresh faeces than the ash leaf litter for P. fluorescens KTG, and higher counts were detected in the faeces for the total bacterial population. When faeces were aged by incubation for up to 7 days at 15–17°C, plate counts for P. fluorescens KTG increased during the first day to a level similar to those in the corresponding ash leaf litter, and remained relatively constant thereafter. The total bacterial population in the faeces continued to increase steadily over the 7 days, whilst remaining at a constant level in the ash leaf litter during the same period. Counts of bacteria in faecal material showed that P. fluorescens KTG was present for 6 days after the isopods had fed on inoculated litter although transit times of food through the gut were as little as 5 h. The implications for GEMMO dispersal of bacterial retention in the gut is considered. The polymerase chain reaction was utilised in the detection of the inserted DNA. Positive amplification of the inserted DNA sequence of P. fluorescens KTG was achieved in ash leaf litter, fresh faeces, and faeces from animal which were supplied uninoculated litter for one day after feeding on the inoculated litter. However, plate counts were more sensitive than the polymerase chain reaction in detecting P. fluorescens KTG in the faeces. Our findings suggest that when the GEMMO is ingested by the woodlouse it can survive within the guts and faeces. This has implications for risk assessment of genetically modified bacteria in terrestrial environments.     

Hostplant,larval age,and feeding behavior influence midgut pH in the gypsy moth (Lymantria dispar)

Summary The midgut pH of late instar gypsy moth (Lymantria dispar L.) larvae is strongly alkaline, and varies with diet, larval stadium, and time since feeding. Midgut pH rises with time since feeding, and does so more quickly, reaching greater maximum values, on some diets than others. Leaf tissues of 23 tree species resist increases in alkalinity differentially; this trait and differing initial leaf pH may explain the impact of diet on gut pH. Third instar larvae may have gut conditions favorable for tannin-protein binding shortly after ingesting certain foods, but with time midgut alkalinity becomes great enough to dissociate tannin-protein complexes. Older instars rarely exhibit gut pHs low enough to permit tannin activity. Alkaline gut conditions may explain the gypsy moth's ability to feed on many tanniniferous plant species, especially in later instars. Consequences for pathogen effectiveness are discussed.     

Volatile fatty acids and methane production in relation to anaerobic carbohydrate fermentation in Oryctes nasicornis larvae (Coleoptera: Scarabaeidae)

Physico-chemical investigations into the conditions of cellulose digestion by Oryctes nasicornis larvae show that the intestinal contents constitute a reducing alkaline medium. The mesenteron presents facultative conditions and high pH values whereas in the proctodeum anaerobiosis is strict and the pH approaches neutrality. No cellulase activity is detected either in the gut walls or the contents. Among the products of polysaccharide fermentation, volatile fatty acids (VFA) and methane are identified. The VFAs are 3 or 4 times more concentrated in the mesenteron than in the proctodeal dilation and acetic acid predominates. The VFAs, which are also present in the haemolymph, must cross the intestinal barrier. Methane formation takes place exclusively in the proctodeal dilation from which it is released to the exterior.     

Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae)

Humivorous scarab beetle larvae can thrive exclusively on soil organic matter. Feeding experiments have revealed that the larva of Pachnoda ephippiata mineralizes all major humus components except the polyphenolic fraction. High proteolytic activity in the alkaline midgut fluid and an enormous ammonia production during gut passage suggested that peptidic soil components are an important dietary resource for the larva. By comparing acid-hydrolyzable amino acids in food soil and feces, we showed that a significant fraction of the peptides in soil are removed during gut passage. This agrees well with the high concentrations of free amino acids found the midgut section. Incubation experiments revealed the presence of substantial particle-associated proteolytic activity also in the hindgut, most probably due to microbial activity. High rates of ammonia formation in hindgut homogenates and the conversion of radiolabeled amino acids to acetate and propionate indicated that microbial fermentations of soil peptides play an important role in the hindgut. This was corroborated by viable counts of amino-acid-fermenting bacteria, which formed a substantial fraction of the hindgut microbiota. A complete inventory of organic and inorganic nitrogen species before, during, and after gut passage revealed the formation of nitrite and nitrate in midgut and hindgut, and a substantial nitrogen deficit in the feces, suggesting that part of the ammonia formed by mineralization is subjected to oxidation and subsequent denitrification to N2. Together, the results strongly support the hypothesis that peptidic soil components form a major energy and nutrient source for humivorous insects, supplying the animal with microbial fermentation products and essential amino acids.     

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food,gut and excrement of saprophagous larvae of<Emphasis Type="Italic">Penthetria holosericea</Emphasis> (<Emphasis Type="Italic">Diptera: Bibionidae</Emphasis>): a pilot study

Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare the bacterial communities of the food, the gut sections (ceca, anterior and posterior midgut, hindgut) and the excrement of the litter feeding bibionid larvae of Penthetria holosericea. For universal eubacterial primers ARDRA patterns were complex with only minor differences among samples. Taxon specific primers were also applied to characterize the samples. Fragment composition was transformed to presence/absence binary data and further analyzed. Cluster analysis revealed that bacterial communities of gut highly resembled each other with the exception of the ceca. ARDRA patterns of consumed leaves clustered together with the intact leaves but differed from those of the excrement. ARDRA results were compared with microbial community structure based on phospholipid fatty acid (PLFA) fingerprints. The cluster analysis of PLFA (presence/absence binary) data resulted in a pattern similar to the ARDRA data. The PCA analysis of PLFA relative content separated microbial communities into five groups: (1) anterior and posterior midgut, (2) hindgut, (3) ceca, (4) consumed and intact litter, (5) excrement. Both methods indicated that conditions in the larval gut result in formation of a specific microbial community which differs from both the food and excrement ones. Particularly ceca--(blind appendages, harbor very specific microbial community) are divided from the rest of the gut by perithropic membrane.     

Survival of yogurt bacteria in the human gut

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.     

肠道细菌通过竞争生态位和保护肠道内壁降低小菜蛾对Bt的敏感性

【目的】肠道微生物可能在介导昆虫宿主对苏云金芽孢杆菌Bacillus thuringiensis(Bt)抗性方面具有重要作用。本研究拟通过探究肠道细菌影响Bt对小菜蛾Plutella xylostella杀虫活性的效应,分析肠道细菌在宿主保护方面的作用机制。【方法】检测小菜蛾3龄幼虫分别取食无菌人工饲料与含肠道总菌群、肠杆菌Enterobacter sp. IAE5 (EbPXG5)、Bt菌株Bt8010、Bt8010+肠道总菌群和Bt8010+EbPXG5的人工饲料,以及分别取食无菌人工饲料与含EbPXG5上清、EbPXG5菌体破碎液、Bt8010+EbPXG5上清、Bt8010+EbPXG5菌体破碎液和Bt8010的人工饲料不同时间后的存活率,分析肠道细菌对小菜蛾Bt敏感性的影响;利用平板培养技术,测定分别取食含EbPXG5, Bt8010和Bt8010+EbPXG5人工饲料的小菜蛾3龄幼虫肠道和血淋巴中EbPXG5和Bt8010的丰度以及EbPXG5对Bt8010的体外抑制效应,分析肠道细菌对Bt8010在小菜蛾肠道中增殖以及入侵血腔的影响;利用扫描电镜观察小菜蛾3龄幼虫无菌肠道组织的内壁形态以及EbPXG5, Bt8010和EbPXG5+Bt8010分别处理的肠道组织的内壁形态,揭示肠道细菌对肠道内壁的保护功能。【结果】与取食无菌人工饲料的对照组相比,取食含肠道总菌群饲料和含EbPXG5饲料的小菜蛾3龄幼虫存活率没有显著差异,但取食含Bt8010+EbPXG5和含Bt8010+肠道总菌群饲料的3龄幼虫在24, 36, 48和60 h时存活率均显著高于取食含Bt8010人工饲料的;取食含EbPXG5上清和含EbPXG5菌体破碎液饲料的3龄幼虫存活率与对照组相比无差异,取食含Bt8010+EbPXG5上清和含Bt8010+EbPXG5菌体破碎液饲料的3龄幼虫存活率与取食含Bt8010饲料的相比也无差异。分别取食含EbPXG5和Bt8010+EbPXG5人工饲料的小菜蛾3龄幼虫肠道中EbPXG5菌株的丰度均没有显著差异,但取食含Bt8010+EbPXG5饲料24, 36和48 h的小菜蛾3龄幼虫肠道内Bt8010丰度显著低于取食含单一Bt8010饲料;血淋巴中,取食含Bt8010+EbPXG5饲料的小菜蛾,36 h和48 h的EbPXG5丰度高于取食含单一EbPXG5饲料的,同时取食含Bt8010+EbPXG5饲料的血淋巴中Bt8010丰度显著低于取食含单一Bt8010饲料的。牛津杯抑菌圈试验表明小菜蛾肠道细菌EbPXG5在体外对Bt8010菌株无抑制作用。扫描电镜观察结果发现,Bt8010会破坏小菜蛾幼虫肠道形成孔洞,同时介导Bt8010及其他细菌穿越肠道屏障进入血淋巴;肠杆菌EbPXG5能定殖于小菜蛾肠腔内壁,减弱Bt8010对肠道内壁的破坏,降低Bt8010在小菜蛾肠道和血淋巴中的丰度。【结论】肠道细菌EbPXG5在保护小菜蛾,降低其对Bt敏感性方面起一定作用,推测该菌通过竞争生态位和保护肠道内壁等方式减弱病原体的定殖和入侵,从而降低宿主对Bt的敏感性。该结果对于促进小菜蛾的生物防治和综合治理具有重要的参考意义。     

Fate of Bacillus sphaericus 2362 spores following ingestion by nontarget invertebrates.

Elimination of Bacillus sphaericus spores ingested by midge larvae, snails, and oysters was most rapid among midge larvae. Spores remained in oysters up to 21 days and in snails up to 49 days. Viable spores were recovered in snail and oyster feces for these same periods. There was no indication of actively growing B. sphaericus in the animals. Passage through oyster gut detoxified the B. sphaericus mosquito larval toxin, but there was a 33% retention of toxicity following snail gut passage. Midge larvae reared to adults in spore-containing water carried spores in/on the adult body. This suggests that these animals could carry the bacteria to sites beyond the application area.     

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.     

取食不同食物对小菜蛾幼虫肠道细菌多样性的影响

【目的】植食性昆虫肠道细菌的组成与其食物密切相关。本研究旨在探究小菜蛾Plutella xylostella幼虫肠道细菌多样性与其取食食物之间的关系以及它们之间相互适应的过程。【方法】本研究选取小菜蛾人工饲料品系(S)及其转寄主到结球甘蓝Brassica oleracea var. capitata、结球白菜Brassica rapa subsp. pekinensis和花椰菜Brassica olerocea var. botrytis饲养后第1代(分别为G1C, G1CC和G1WC)和第3代(分别为G3C, G3CC和G3WC)的4龄幼虫,提取小菜蛾肠道细菌基因组DNA,利用Illumina MiSeq二代高通量测序技术,分析其肠道细菌多样性和丰度。【结果】α多样性指数分析发现,取食不同食物的小菜蛾4龄幼虫肠道细菌多样性高低顺序为G1WC>G1CC>S>G1C。在菌群组成上,以人工饲料为食的S样品肠道细菌主要由厚壁菌门(Firmicutes)组成,转寄主植物后的G1C, G1CC和G1WC肠道中厚壁菌门(Firmicutes)相对丰度显著下降,G1C和G1CC小菜蛾肠道中变形菌门(Proteobacteria)相对丰度显著上升成为优势菌群,G1WC肠道中拟杆菌门(Bacteroidetes)成为优势菌群。在寄主植物上连续饲养3代后,与第1代相比,小菜蛾肠道细菌α多样性指数没有显著性改变,但在结球甘蓝和结球白菜上小菜蛾肠道菌群结构却发生了变化,相比G1C,G3C肠道中芽孢杆菌目(Bacillales)的相对丰度显著下降;相比G1CC, G3CC肠道中放线菌门(Proteobacteria)、芽单胞菌门(Gemmatimonadetes)和硝化螺旋菌门(Nitrospirae)的相对丰度均显著上升。【结论】取食人工饲料和不同寄主植物的小菜蛾幼虫肠道细菌多样性和群落构成存在显著差异,寄主植物对小菜蛾肠道微生物的结构组成具有重要的影响,且小菜蛾肠道微生物对寄主植物可能存在一个长期适应的过程。本研究为进一步探讨影响小菜蛾肠道细菌变化的因素,以及后续研究肠道细菌与寄主植物之间的互作奠定了良好的基础。     

Survival of Yogurt Bacteria in the Human Gut

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.     

Gut passage times in Gammarus pulex (Crustacea,Amphipoda) and aspects of summer feeding in a stony stream

Gut passage times in Gammarus pulex (Crustacea, Amphipoda) were calculated by counting and measuring voided faecal pellets. Following feeding on elm leaves conditioned in a stream, or summer fine detritus from the same source, calculated gut passage times were usually short, about 2 h or less at 15 °C. Using harder oak and beech leaves, not previously conditioned in the stream, apparent gut passage times were much longer, 8–48 h, but since intermittent feeding occurred these figures are not comparable to the others.In connection with summer feeding of the animal in the stream, the microbiological status of the fine detritus there was studied. It contained fungal hyphae, which were mostly empty, viable fungal spores of terrestrial derivation, and bacteria, all largely carried on fragments of vascular plant tissues. In experiments, such fungal spores (of Aureobasidium pullulans and of Mucor sp.), incorporated into fine detritus, were not digested by the animal. The bacterium Bacillus cereus was present in the fine detritus and when the latter was consumed it survived passage through the gut of the animal. Using B. cereus as a proportion marker it was concluded that other unicellular bacteria in the fine detritus were not digested either. In other experiments, not immediately related to the summer season, nutrient extraction by the animal from hyphae of the fungus Nectria lugdunensis was examined; it probably occurs through sub-microscopic pores connecting the cells. This mode of extraction may apply when the animal consumes vascular plant tissues.     

Variation in the pH of experimental diets affects the performance of Lymantria dispar asiatica larvae and its gut microbiota

To study dietary pH effects on Lymantria dispar asiatica larvae and provide a theoretical basis for its control in different forests, phosphate buffers (PBs) of pH 6, 7, and 8 were used to prepare experimental diets. The diet prepared with pH 6 PB was named as DPB6, with pH 8 PB as DPB8, and with pH 7 PB as DPB7 (control). The dietary pH was 5.00 in DPB6, 6.05 in control, and 6.50 in DPB8. After feeding on the diets with different pH values for 84 hr, fourth-instar caterpillars were randomly collected. Growth and various physiological traits were determined and 16S recombinant DNA sequencing was performed using the intestinal microflora of surviving larvae. Results showed that the mortality was 30% in DPB6, and 10% in DPB8, while no mortality was observed in control. The partial least squares discriminant analyses suggested that diets prepared with PB of different pH resulted in different food intake, amount of produced feces, weight gain, digestive enzyme activities, and antioxidant enzyme activities in larvae. Interestingly, both the highest weight gain and the lowest total antioxidant capacities were seen in control larvae. Results also showed that the larval gut microbiota community structure was significantly affected by dietary pH. Moreover, linear discriminant analysis effect size suggested that the family Acetobacteraceae in control, genus Prevotella in DPB8, and genus Lactococcus, family Flavobacteriaceae, family Mitochondria, and family Burkholderiaceae in DPB6 contributed to the diversity of the larval gut microbial community.     

Host plant urease in the hemolymph of the silkworm, Bombyx mori

Urease activity was detected in the hemolymph of the silkworm, Bombyx mori from the beginning of spinning to the pharate adult stage if the larvae were reared on mulberry leaves throughout the 5th-instar (the last larval instar). In contrast, no urease activity was detected in the hemolymph of insects fed artificial diets, resulting in accumulation of urea during the spinning stage. To identify the hemolymph urease, the enzyme was highly purified from the hemolymph of the spinning larvae that had been reared on mulberry leaves and the properties of the purified enzyme were compared with those of the mulberry leaf urease. Four out of six monoclonal antibodies raised against jack bean seed urease cross-reacted equally with the silkworm hemolymph urease and the mulberry leaf urease. Under reducing conditions, the hemolymph urease and the mulberry leaf urease migrated at 90.5 kDa on SDS-PAGE gels. The first 20 N-terminal sequence of the hemolymph urease revealed complete identity with that of the leaf urease. The optimum pH for activity and Km value for urea were almost the same for the two enzymes. In conclusion, these two ureases are very likely identical, strongly suggesting that the mulberry leaf urease passes through the larval gut wall into the hemolymph without being digested. In addition, oral administration of mulberry leaf urease just before spinning induced considerable urease activity in the hemolymph of the larvae, but the same treatment did not induce enzyme activity in the hemolymph of the larvae three days before the onset of spinning. These results suggest that the silkworm larvae acquire the host plant urease specifically at the end of the feeding stage in order to degrade urea accumulated in the hemolymph.     

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.     

Ecosystem consequences of selective feeding of an insect herbivore: palatability–decomposability relationship revisited

1. The relationship between leaf palatability and litter decomposability is critical to understanding the effects of selective feeding by herbivores on decomposition processes, and several studies have reported that there is a positive relationship between them. 2. However, palatability is not always positively correlated with decomposability, because of species‐specific feeding adaptation of herbivores to host plants. Moreover, the effects of selective feeding by herbivores on soil decomposition processes should be understood in terms of the inputs of leaf litter and excrement. 3. The present study examined the relationships between leaf palatability and the decomposability of litter and frass, using Lymantria dispar Linnaeus and 15 temperate deciduous tree species. 4. Larvae of L. dispar exhibited a clear feeding preference, and subsequently the excreted frass mass differed among tree species. Litter and frass decomposability also differed among tree species, and frass was more rapidly decomposed than litter. There were no positive or negative correlations between palatability and decomposability of litter and frass. 5. These results indicate that L. dispar larvae may accelerate the decomposition process in temperate deciduous forests through selective feeding on plants with relatively low litter decomposability and the production of frass with higher decomposability than the litter.     

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera: Plutellidae)

Larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics (rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P. xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL significantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.