Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

Enhanced Nematicidal Activity of Organic and Inorganic Ammonia-Releasing Amendments by Azadirachta indica Extracts

The nematicidal activities of ammonium sulfate, chicken litter and chitin, alone or in combination with neem (Azadirachta indica) extracts were tested against Meloidogyne javanica. Soil application of these amendments or the neem extracts alone did not reduce the root galling index of tomato plants or did so only slightly, but application of the amendments in combination with the neem extracts reduced root galling significantly. Soil analysis indicated that the neem extract inhibited the nitrification of the ammonium released from the amendments and extended the persistence of the ammonium concentrations in the soil. In microplot experiments, tomato plants were grown in pots filled with soils from the treated microplots. The galling indices of tomato plants grown in soil treated with ammonium sulfate or chicken litter in combination with the neem extract or a chemical nitrification inhibitor were far lower than those of plants grown in the control soil or in soil treated with chicken litter, neem extract or nitrification inhibitor alone. However, plants grown in the microplots showed only slight reductions in galling, probably because the soil amendments were inadequately mixed compared to their application in the pot experiments. The extended exposure of nematodes to ammonia as a result of nitrification inhibition by the neem extracts appeared to be the cause of the enhanced nematicidal activity of the ammonia-releasing amendments.     

Effects of antibiotics on hydrogen production and gut symbionts in the Formosan subterranean termite Coptotermes formosanus (Isoptera: Rhinotermitidae)

Abstract Symbiotic microorganisms that inhabit the gut of Coptotermes formosanus enable this termite to degrade lignocelluloses and further produce hydrogen as an important intermediate to be recycled in its hindgut or as a byproduct to be emitted to the atmosphere. Both symbiotic protists and prokaryotes in the guts of termites demonstrated some different roles with respect to hydrogen production. In this study, the effects of two antibiotics, ampicillin and tetracycline, on hydrogen emission and the gut symbionts of C. formosanus were investigated. Hydrogen emission from termite guts was significantly enhanced when termites fed on wood diets treated with either ampicillin or tetracycline. The greatest H₂ emission rates, 2 519 ± 74 and 2 080 ± 377 nmol/h/g body weight, were recorded with the treatments of ampicillin and tetracycline, respectively, which showed 6–7 times more H₂ production than that of controls. Antibiotic‐treated diets negatively affected the prokaryotic communities and reduced their abundances, particularly on those ectosymbionts inhabiting the gut walls or in the gut fluid of C. formosanus, such as spirochetes. However, no significant reductions in the counts of gut cellulolytic protists, Pseudotrichonympha grassii and Holomastigotoids hartmanni, were recorded; and with a further observation by confocal laser scanning microscopy, the endosymbionts inhabiting P. grassii generally survived the antibiotic treatments. These results suggest that some prokaryotes may serve as the main hydrogen consumers, while P. grassii, together with its endosymbionts, may function as the main contributors for hydrogen production in the hindgut of C. formosanus.     

Leaf extract of neem (Azadirachta indica) alleviates adverse effects of drought in quinoa (Chenopodium quinoa Willd.) plants through alterations in biochemical attributes and antioxidants

The influence of varying concentrations (0, 1, 3, 4, 5, 6, 8, and 10 % v/v) of neem (Azadirachta indica) leaf extract on drought stressed (40 % field capacity) quinoa (Chenopodium quinoa Willd.) plants was assessed. During the current study two cultivars of quinoa (V7 and V9) were used. This study revealed that water stress adversely affects the fresh and dry weight of shoots and roots as well as chlorophyll pigments (a and b) of both quinoa cultivars. In contrast, drought stress enhanced glycinebetaine (GB), free proline, phenolic content, hydrogen peroxide (H₂O₂), activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzymes, and relative membrane permeability (RMP). However, application of neem leaf extract improved the accumulation of key osmoprotectants like proline, GB and activities of enzymatic antioxidants. Our findings showed 5 % neem leaf extract is an effective treatment in counteracting the oxidative damage caused by water stress, thereby improving overall plant growth. Of both cultivars of quinoa, the response of cv. V9 to stress as well as foliar applied neem was relatively more promising.     

SYSTEMIC INSECTICIDAL ACTION OF AZADIRACHTIN, NEEM SEED AND CHINABERRY SEED EXTRACTS APPLIED AS SOIL DRENCHES TO POTTED PLANTS

Abstract  The systemic insecticidal activities of: 1) azadirachtin, a potent insect antifeedant and growth regulator isolated from seeds of the neem tree, Azadirachta indica , 2) a neem seed extract containing 0. 30 quantitative fraction of azadirachtin, and 3) a methanolic extract of seeds from the chinaberry tree, Melia azedarach , a close relative of neem were determined. Soil drenches of azadirachtin or the neem seed extract at 1 and 2. 5 mg to potted potato ( Solanum tuberosum ) and tomato ( Lycopersicon esculentum ), remarkably retarded the growth of Manduca sexta larvae and strongly disrupted the pupation of Leptinotarsa decemlineata larvae, respectively. However, five to ten times as much azadirachtin or neem seed extract was needed to reduce the amount of tomato and potato foliage consumed. On sorghum ( Surghum biocolor ), much higher quantity (≥25 mg) of azadirachtin or the neem seed extract was necessary to slow the population growth of Schizaphis graminum , and prolong the survival of treated plants. But methanolic extracts of chinaberry seeds ( M. azedarach ) from two sources (Guangdong, PRC and Hurricane, Utah, USA) at 150 mg per pot were ineffective in reducing aphid population growth and preventing plant damage.     

Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora

The importance of the gut microorganisms in the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding them with antibiotics. With N. exitiosus, antibiotics which killed both the bacteria and the spirochaetes (ampicillin, kanamycin, chloramphenicol, erythromycin, cephaloridine, tetracycline) reduced the life span of the termite from 250 days to about 13 days, whereas antibiotics which had little effect on the flora (penicillin, methicillin) did not greatly reduce the life span of the termite. The essential role of the spirochaetes in N. exitiosus was shown by feeding metronidazole, or exposing the termites to pure oxygen. Both treatments killed the spirochaetes, but not the bacteria, resulting in a life span for the termite of 13–22 days. Acid fuchsin did not kill the spirochaetes. Fungi were not essential for N. exitiosus. In C. lacteus all treatments, except that with acid fuchsin, killed the protozoa, thereby reducing the life span of the termite from 69 days to 6–29 days.     

The Integrated Effect of the Nematode-Bacteria Complex and Neem Plant Extracts Against Plutella Xylostella (L.) Larvae (Lepidoptera: Yponomeutidae) on Chinese Cabbage

The compatibility of the nematode-bacteria complex of Steinernema carpocapsae, Steinernema abbasi, and Heterorhabditis bacteriophora with neem plant extracts (seeds and leaves) to control diamondback moth, Plutella xylostella (L.) on Chinese cabbage plants was investigated under laboratory conditions. The initial treatment of Chinese plant leaves with neem plant extracts resulted in more susceptibility of P. xylostella to neem seeds extract than neem leaves extract. The combination studies resulted in a higher synergistic effect of nematode-bacteria complex of H. bacteriophora, S. abbasi and S. carpocapsae with neem seeds extract compared with that of S. abbasi with neem leaves extract. In contrast, the combination of nematode-bacteria complex of S. carpocapsae and H. bacteriophora with neem leaves extract gave an antagonistic effect. On the other hand, the time-mortality response was more reduced with neem seeds extract than with neem leaves extract, besides it was a concentration dependant factor. We conclude from these investigations that the combination of entomopathogenic nematodes with neem plant extracts could be of success in controlling P. xylostella on Chinese cabbage plants in the field.     

Effect of Host Diet and Hindgut Microbial Composition on Cellulolytic Activity in the Hindgut of the American Cockroach, Periplaneta americana

Cellulase activity measured as filter paper digesting activity (FPase) and carboxymethyl cellulase (CMCase) was demonstrated in hindgut extracts of the cockroach Periplaneta americana. The highest activities measured amounted to 0.89 and 0.12 U · ml^-1 for CMCase and FPase, respectively. The cellulolytic capacity of the hindgut population increased dramatically when protozoa were present, and the activities were found to vary depending on the feeding regimen. Cellulose-rich diets induced high protozoal numbers, resulting in a high cellulase activity. A close correlation was found between the number of Nyctotherus ovalis organisms, the major protozoans in the hindgut, and both FPase and CMCase activity. Since the numbers of this protozoan also correlated with the methane production of the insect, it appears that N. ovalis is responsible for the major part of cellulolytic and methanogenic activity found in the hindgut of P. americana.     

Isolation,identification and synthesis of locustamyotropin II,an additional neuropeptide of Locusta migratoria: Member of the cephalomyotropic peptide family

An eight residue neuropeptide (Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-NH₂) has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata-suboesophageal ganglion complexes of Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae. The peptide designated as locustamyotropin II, or Lom-MT II according to Raina and Gäde (Insect Biochem.18, 785–787, 1988), has a Phe-X-Pro-Arg-Leu-NH₂ carboxyl-terminal in common with the previously identified locustamyotropin I. Locustamyotropin II is also related to leucopyrokinin (Lem-PK), a blocked myotropic neuropeptide isolated from cockroach heads. Both peptides have identical carboxyterminal pentapeptide sequences. The constituent amino acids of this C-terminal are important for biological activity on the Leucophaea hindgut. Lom-MT II differs from Lem-PK in the first three aminoterminal residues. In contrast to Lem-PK and like Lom-MT I, the novel locust peptide is not N-terminally blocked. Lom-MT II has a stimulatory effect on the motility of the oviduct of Locusta but not on the hindgut.     

Neem seed oil inhibits aphid transmission of potato virus Y to pepper*

Transmission of potato vims Y to sweet pepper by the green peach aphid, Myzus persicae (Sulzer), was inhibited by foliar applications of 1.0% or 2.0% neem seed oil to infected source plants or to uninfected recipient plants. Neem seed oil interfered with virus acquisition and inoculation in a manner comparable to that of a commercial horticultural oil, while an oil-free neem seed extract did not reduce rates of transmission compared with controls. The finding that neem seed oil inhibits virus transmission, while oil-free neem seed extract does not, suggests that the presence of the oil rather than biologically active limonoids such as azadirachtin interfere with virus transmission. None of the treatments affected rates of infection when potato virus Y was transmitted mechanically, or the resulting virus titre and symptom expression. In addition to direct control of insect pests, formulated neem oils may help reduce or delay the spread of non-persistent plant viruses.     

Histological changes in the Dengue vector,Aedes aegypti (Diptera: Culicidae) larvae treated with neem oil loaded niosomes

Mosquitoes are one of the greatest threats to human health around the globe. They act as vectors for common diseases like Malaria, Dengue, Chikungunya, etc. Niosomes encapsulated with neem oil showed a significant mortality rate against Aedes aegypti larvae when treated for 24 h. In this study, the histological changes that led to the mortality of the Aedes aegypti mosquito larvae were studied. Organs of the late III-instar stage larvae such as head, optic lobes, cuticle, adipose tissue, midgut region, haemolymph were investigated. Several histological alterations such as disorientation of the brain and antenna in the head part, damage in the optic lobe and microvilli were observed. Total disruption was seen in the inner and outer retractor muscles of the larval body. The midgut and hindgut regions were disintegrated due to the damage to the fat bodies in the region. A Series of such histological changes in the body of mosquito larvae compared to the control larvae hindered metabolic functions leading to death. The results suggested that the neem oil loaded niosomes could be used as a biocontrol agent against the Dengue vector, Aedes aegypti larvae.     

Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle

Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective  The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods  In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results  Follistatin treatment resulted in smaller muscles after 3 months denervation ( p  = 0.019) and reduced force after 3 months sham denervation ( p  < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p  = 0.066). Follistatin increased satellite cell counts after denervation ( p  < 0.001) but reduced satellite cell counts after sham denervation ( p  = 0.037). Conclusion  Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.     

Azadirachta indica (neem) leaf dietary effects on the immunity response and disease resistance of Asian seabass,Lates calcarifer challenged with Vibrio harveyi

The present study was aimed to address the possible evaluation of Azadirachta indica (neem) leaf-supplemented diets on innate immune response in Asian seabass, Lates calcarifer fingerlings against Vibrio harveyi infection. Fish were fed for two weeks diets containing six graded levels of neem leaf at 0 g, 1 g, 2 g, 3 g, 4 g and 5 g per kg feed. Fish fed neem leaf-supplemented diet displayed significant differences (p < 0.05) in weight gain, specific growth rate (SGR) and feed conversion ratio (FCR) compared to the control group fed without neem leaf-supplemented diet. Various innate immune parameters were examined pre-challenge and post-challenge. Fish was injected intraperitoneally with a lethal dose of V. harveyi containing 10⁸ cells mL^?1. Supplementation of neem leaf diet significantly increased phagocytic activity, superoxide anion production, serum lysozyme, serum bactericidal activity, serum anti-protease activity throughout the experimental period when compared with the control group. Dietary doses of neem leaf diet significantly influenced the immune parameters, haematological parameters and blood biochemical indices of treated fish. The results suggested that fish fed neem leaf-supplemented diet improved the immune system and increased survival rate in L. calcarifer fingerlings against V. harveyi infection.     

Studies on the population dynamics and integrated management of Fusarium oxysporum f. sp. psidi (Fop), a cause of guava (Psidium guajava) wilt

To assess the impact of non-host crops intercropping, bioagents and oil cakes, on population dynamics of Fusarium oxysporum f. sp. psidii (Fop) and wilt of guava. Lowest population of Fop was recorded in garlic followed by intercropping with marigold with reduction of 84.9 and 83.9%, respectively. Bioagents viz; Aspergillus niger, Trichoderma viride and Trichoderma harzianum reduced the Fop population significantly, with the lowest in T. harzianum followed by A. niger. Garlic bulb extract exhibited maximum inhibition of Fop growth (2.7 cm) followed by marigold (2.4 cm), respectively, over control. Neem cake significantly reduced population of Fop, closely followed by mahua cake, over control. Integration of neem cake + T. harzianum + garlic reduced the Fop population significantly, over control, followed by neem cake + T. harzianum + marigold but neem cake + T. harzianum + marigold reduced wilt disease significantly in comparison to neem cake + T. harzianum + garlic inter-cropping.     

Screening and design of anti-diabetic compounds sourced from the leaves of neem (Azadirachta indica)

Diabetes Mellitus is affecting people of all age groups worldwide. Many synthetic medicines available for type 2 diabetes mellitus in the market. However, there is a strong requirement for the development of better anti-diabetes compounds sourced especially from natural sources like medicinal plants. The extracts from the leaves of neem (Azadirachta indica) is traditionally known to have anti-diabetes properties. Therefore, there is an increased interest to identify potential compounds identified from neem leaf extracts showing predicted binding property with the known diabetes mellitus type 2 protein enzyme target phosphoenol-pyruvate carboxykinase(PEPCK). The structure data for compounds found in the leaf extract of neem was screened against PEPCK using molecular docking simulation and screening techniques. Results show that the compound 3-Deacetyl-3-cinnamoyl-azadirachtin possesses best binding properties with PEPCK. This observation finds application for further consideration in in vitro and in vivo validation.     

Tetranortriterpenoids from the seeds of Azadirachta indica

Six new tetranortriterpenoids have been isolated from the extract of neem seeds. The C-7 benzoates of tetranortriterpenoids occurring in Azadirachta indica are described for the first time. The structures of all new compounds were determined by spectroscopical methods.     

Induction and expression of peroxidase in tomato is age-dependent

Peroxidases (POXs) are actively synthesised under stress conditions and induced by biotic/abiotic elicitors. Their age-regulated expression in plants has been a matter of investigation during recent times. The present study focuses on studying the expression of POX at different stages of growth and variation in its inducibility by aqueous extract of Azadirachta indica (neem) fruits. Tomato plants at 6, 8, 10 and 12 weeks were chosen for the study. The third nodal leaf of each plant was treated with neem fruit extract alone or in combination with Pseudomonas syringae pv. tomato. Sampling was performed at an interval of 24 h up to five days and after two weeks of treatment, from both the treated and distal untreated nodes of the plants. POX activity and its isoforms were analysed. The results demonstrated that POX expression and its inducibility by the biotic inducer (neem fruit extract) varies with age of the plant.     

Integrated management of fusarial wilt of tomato with implementation of soil solarisation

Fusarial wilt of tomato (Lycopersicon esculentum Mill.) is a very common and severe disease occurring in most of the vegetable fields in West Bengal, India. Potenciation and formulation of different fungicidal chemicals and phytoextracts were evaluated against the growth of the pathogen wherein carbendazim (bavistin) and leaf extracts of Azadirachta indica (neem) were recorded to be most effective. Combined treatment with 4 ml neem leaf extract and 1 ml captan (0.01%) or with 4 ml garlic bulb extract and 1 ml captan (0.01%) exhibited 100% growth inhibition of the pathogen. Integrated control of the pathogen with phytoextracts, fungicide and biocontrol agents was carried out. Among the treatments, a combination with extracts of neem, captan (0.01%) and metabolites of Trichoderma harzianum was proved to be superior over the other. Field experiment with three fungicides at 0.5% concentration was carried out in randomised block design where application of bavistin showed up to 62.27% reduction of wilt infection in tomato plants. Soil solarisation of tomato field showed 62.50 and 66.69% reduction of infection during the trial years. However, integration of soil solarisation with the applications of T. harzianum, captan (0.01%) and neem resulted in 100% reduction of infection and thus it was recorded as the most effective treatment in reducing the incidence of the disease.     

Side Effects of Neem Oil on the Midgut Endocrine Cells of the Green Lacewing Ceraeochrysa claveri (Navás) (Neuroptera: Chrysopidae)

We described the ultrastructure of Ceraeochrysa claveri (Navás) midgut endocrine cells in larva, pupa, and adult, and evaluated the side effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree (Azadirachta indica), on these cells. During the larval period, C. claveri were fed (ad libitum) Diatraea saccharalis (F.) eggs treated with neem oil at concentrations of 0.5%, 1%, or 2%. Transmission electron microscopy showed that two subtypes of endocrine cells, namely granular and vesicular, occurred in the midgut epithelium during the three stages of the life cycle. Both cell types did not reach the midgut lumen and were positioned basally in the epithelium. The endocrine cells did not show extensive infoldings of the basal plasma membrane, and there were numerous secretory granules in the basal region of the cytoplasm. In the granular endocrine cells, the granules were completely filled with a dense matrix. In the vesicular endocrine cells, the main secretory products consisted of haloed vesicles. Ultrastructural examination indicated that only the granular endocrine cells exhibited signs of morphologic changes of cell injury present in all life cycle stages after the larvae were chronically exposed to neem oil by ingestion. The major cellular damage consisted of dilatation and vesiculation of the rough endoplasmic reticulum and the development of smooth endoplasmic reticulum and mitochondrial swelling. Our data suggest that cytotoxic effects on midgut endocrine cells can contribute to a generalized disruption of the physiological processes in this organ due to a general alteration of endocrine function.