The effect of neem allelochemicals on nutritional physiology of larval Spodoptera litura

Neem allelochemicals azadirachtin, salannin, nimbinene and nimbin were administered to different larval instars of the tobacco armyworm, Spodoptera litura orally in artificial diet, topically or via injection. Nutritional analyses revealed strong antifeedant and growth regulatory effects of azadirachtin which were independent of each other. While salannin and nimbinene induced concentration dependent feeding deterrence only; nimbin was inactive to the 1000 ppm level against this insect species. One of the causes of the reduced growth rate of azadirachtin treated insects was due to an increase in the costs associated with growth. This was relative to a drastic reduction in the activity of gut trypsin. Salannin and nimbinene, however, did not interfere with the trypsin activity of the gut. These results and those from nutritional studies suggest that salannin and nimbinene have no toxicity mediated effects on S. litura larvae and antifeedant activity is a result of the effects on deterrent and other chemoreceptors. The fact that azadirachtin directly or indirectly inhibits the secretion of trypsin by the enzyme-secreting cells of the gut is discussed.     

Systemic antifeedant effects of azadirachtin on the peach-potato aphidMyzus persicae

The electrical penetration graph (EPG) method was used to analyse the feeding behaviour of apterous, adultMyzus persicae (Sulzer) (Homoptera: Aphididae) onNicotiana clevelandii (Gray) seedlings, treated systemically with azadirachtin. A preliminary experiment showed that the effects of tethering aphids for EPG recording were minimal. The percentage of the 9 h recording period devoted to non-penetration activities, and to stylet pathway patterns increased as the azadirachtin concentration in the root treatment increased. The number of probes initiated, and the numbers of sieve tube penetrations also increased with increased azadirachtin concentration. The mean time elapsing between the initiation of the first probe to reach a sieve element and contact with this tissue was not significantly altered by azadirachtin treatment. However, azadirachtin treatment significantly reduced the percentage of probes that reached sieve elements and increased non-penetration activity before and after the first perid of ingestion from the sieve elements. The percentage of the recording period spent in the EPG pattern associated with sieve tube penetration was significantly reduced by an azadirachtin concentration of 300 ppm, and the duration of each individual penetration was significantly reduced by an azadirachtin concentration of 100 ppm. When the total EPG was split into 3 h periods, significant interactions were seen between time period and azadirachtin concentration for the duration of non-penetration, pathway, and sieve tube penetration patterns.     

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.     

Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus

Growth and survival responses were determined for black field crickets Teleogryllus commodus (Walker) (Orthoptera: Gryllidae) on artificial diets containing a range of levels of dietary protein, and protease inhibitors (PI's) at 0.33% (weight volume, w:v). The effect on cricket gut enzyme activities of adding PI's to a high protein diet was measured. All PI's which had in vitro binding activity against either trypsin or elastase (the two major cricket gut endopeptidases) reduced growth, but those which bound to both enzymes had the greatest effect. None of the PI's acted as a source of nutritional protein. Cricket growth rate increased with the addition of casein up to 3% w:v, but not with a similar addition of wheatgerm. The impact of PI's on growth was greatest on a 1.5% casein diet. On a high protein (3% casein) diet, the gut activity of trypsin was increased by potato proteinase inhibitors 1 and 2 while the activity of elastase and leucine amino peptidase were increased by soybean trypsin inhibitor and potato proteinase inhibitor 2. Increasing dietary casein up to 3.3% improved cricket survival. The potential of PI's as plant resistance factors against crickets was confirmed.     

A trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis> seeds: isolation,characterization and activity against <Emphasis Type="Italic">Pieris rapae</Emphasis>

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.     

Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants

Leaf disc choice test bioassay demonstrated that formulated neem seed extracts were highly deterrent and growth regulatory to rose aphid,Microsiphum rosae (L.) and Chrysanthemum aphid,Macrosiphoniella sanbornii (Gillete). Effective concentrations to produce 50% feeding deterrence was 0.80 and 0.84% respectively for 2nd instar nymphs irrespective of bioassay duration. The disruption of aphid feeding was related to the presence of azadirachtin concentration in the extract. The toxicity on contact from the leaf surface or via topical application due to azadirachtin was significantly different and topical treatment was at least 7 times more effective for both species. Thus growth regulatory effects of azadirachtin were influenced by the host plant and the stage of treatment. Field evaluation with formulated neem extracts revealed the effect to be more of growth regulatory nature thereby showing that azadirachtin is a physiological toxin for aphid species. Neem seed extracts reduced the population of aphid on respective host plants significantly, EC₅₀ values being 0.88 and 0.96% forM. rosae andM. sanbornii respectively.     

THE INFLUENCE OF A 21 kDa KUNITZ‐TYPE TRYPSIN INHIBITOR FROM NONHOST MADRAS THORN,Pithecellobium dulce,SEEDS ON H. armigera (HÜBNER) (LEPIDOPTERA: NOCTUIDAE)

A trypsin inhibitor purified from the seeds of the Manila tamarind, Pithecellobium dulce (PDTI), was studied for its effects on growth parameters and developmental stages of  Helicoverpa armigera. PDTI exhibited inhibitory activity against bovine trypsin (～86%; ～1.33 ug/ml IC₅₀). The inhibitory activity of PDTI was unaltered over a wide range of temperature, pH, and in the presence of dithiothreitol. Larval midgut proteases were unable to digest PDTI for up to 12 h of incubation. Dixon and Lineweaver–Burk double reciprocal plots analysis revealed a competitive inhibition mechanism and a K_i of ～3.9 × 10^?8 M. Lethal dose (0.50% w/w) and dosage for weight reduction by 50% (0.25% w/w) were determined. PDTI showed a dose‐dependent effect on mean larval weight and a series of nutritional disturbances. In artificial diet at 0.25% w/w PDTI, the efficiency of conversion of ingested food, of digested food, relative growth rate, and growth index declined, whereas approximate digestibility, relative consumption rate, metabolic cost, consumption index, and total developmental period were increased in larvae. This is the first report of antifeedant and antimetabolic activities of PDTI on midgut proteases of  H. armigera.     

The expression of a mammalian proteinase inhibitor,bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae

The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3 and 5 changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.     

DNA synthesis in the imaginal wing discs of the American bollwormHelicoverpa armigera (Hübner)

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instarHelicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Underin vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fernCheilanthes farinosa enhanced DNA synthesis     

Oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and egg development in mosquitoes

Injection of partially purified oostatic hormone (0.7 μg) into female Aedes aegypti inhibited egg development, proteolytic enzyme activity, and blood digestion in the midgut, whereas control injections of saline or insulin chain A (0.7 μg) did not affect these processes. Oostatic hormone given by enema, on the other hand, did not inhibit proteolytic enzyme activity, indicating that the hormone acts outside the midgut. A single injection of oostatic hormone (0.7 μg) caused a 1.7–1.5-fold reduction in activity of trypsinlike enzymes during blood digestion, with a 10-h delay in peak activity. Using [1,3-³H]diisopropylfluorophosphate (DFP) in the presence of 8 mM tosylamide-2-phenylethyl chloromethyl ketone, the synthesis of trypsinlike derivatives was followed in the midgut of female A. aegypti. A 4-fold reduction in [1,3-³H]diisopropylphosphoryl-trypsinlike derivatives was noted after oostatic hormone treatment. Several isozymes that are normally synthesized were absent in the presence of DFP, as assessed by polyacrylamide gel electrophoresis. Injection of oostatic hormone into decapitated and ovariectomized females that did not synthesize ecdysteroids inhibited trypsinlike enzyme synthesis and blood digestion in the midgut, indicating that oostatic hormone inhibits the midgut cells and not the ovary or the brain's endocrine system. Comparison between oostatic hormone and soybean trypsin inhibitor indicated that the former inhibited trypsin synthesis whereas the latter inhibited trypsin activity. A. aegypti oostatic hormone is not species specific and injections of the hormone into Culex quinquefasciatus, Culex nigripalpus, and Anopheles albimanus caused inhibition of egg development, blood digestion, and synthesis of trypsinlike enzymes. A direct relation between oostatic hormone synthesis and the regulation of trypsinlike activity in the midgut is proposed.     

Lethal and sublethal effects of Embelia ribes and two commercial pesticides on a generalist predator

Even as new substances show promise as biopesticides for controlling pests due to their natural properties and high effectiveness in inhibiting pests, their side effects on non‐target organisms must nevertheless be evaluated before they can be included into integrated pest management systems. In this study, a crude extract from dried leaves of Embelia ribes was evaluated together with two commercial pesticides: azadirachtin (a natural product) and amitraz (a synthetic acaricide). We examined both lethal and sublethal effects on the predatory potential of the lynx spider Oxyopes lineatipes, which is among the most dominant predator in tropical agricultural agroecosystems. We found that the spider's mortality increased with rising concentration of both commercial products, azadirachtin and amitraz, but not with rising concentration of the extracts from E. ribes. The greatest mortality occurred when amitraz was used. That material caused almost 100% spider mortality in the doses recommended for field spraying. Azadirachtin significantly reduced the rate at which O. lineatipes captured prey, while there was no significant difference in capture rates among spiders exposed to a control treatment and the E. ribes treatment. Considering its absence of unfavourable impacts on O. lineatipes in terms of mortality and predatory activity, the plant extract from E. ribes shows promise as a new biopesticide material. In contrast, azadirachtin, which has been considered as safe for non‐target organisms, exhibited slight lethal effect only in higher concentrations and strong sublethal effect by reducing spiders’ predation rate.     

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

Summary A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.     

Differential thresholds of azadirachtin for feeding deterrence and toxicity in locusts and an aphid

Conclusions The importance of using azadirachtin in the field at levels causing primary antifeedancy may have been overemphasised in the past. Currently, it is recommended that commercial preparations of neem be applied at 50–100 ppm (a.i.). While this would undoubtedly have an antifeedant effect in many phytophagous insects it may also harm beneficial species (Schmutterer, 1990). By lowering the concentration of azadirachtin applied to the crop, food intake by insect pests with low chemoreceptor sensitivity to azadirachtin, e.g. aphids (Nisbet et al., 1993; 1994), would not initially be affected. However, secondary antifeedant effects, IGR and sterilant effects could rapidly manifest themselves and bring about crop protection by reducing insect pest populations without harming natural predator or parasitoid populations.     

Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

Herbivory and novel weapons: no evidence for enhanced competitive ability or allelopathy induction of <Emphasis Type="Italic">Centaurea diffusa</Emphasis> by biological controls

Biological control of weeds by arthropod herbivores is thought to work by reducing the competitive ability of the weed relative to the surrounding vegetation. However, the assumption that herbivory reduces plant competitive ability has not been tested in most biological control systems, and counter to expectation, recent research on the impact of biological control agents on invasive Centaurea species suggests that this genus may respond to herbivory by increased competitive ability through enhanced plant re-growth and/or by inducing increased production of phytotoxic allelochemicals. We examined the impact of two biological control agents of the invasive plant diffuse knapweed (C. diffusa) to see if feeding by either of these insects would enhance the plant’s competitive ability or allelochemical output. Sub-lethal herbivory by either of the biological control agents significantly reduced knapweed performance when the plant was grown in competition with either of two native species. Competition with knapweed significantly reduced the performance of both native species (Artemisia frigida and Bouteloua gracilis), and herbivory by one of the biocontrol agents resulted in a small but significant increase in both native species’ performance. Diffuse knapweed’s putative allelochemical 8-hydroxyquinoline was not detected in experimental or field collected soils from knapweed-infested sites. In contrast to other studies on the impacts of biological control on other Centaurea species, these data support the premise that biological control agents may reduce invading plant competitive ability. We find no evidence for diffuse knapweed allelopathy mediated by 8-hydroxyquinoline or enhanced allelopathy in response to herbivory by biological control agents.     

Inhibition of growth,and effects on nutrient uptake of arctic graminoids by leaf extracts — allelopathy or resource competition between plants and microbes?

Previous research has shown that plant extracts, e.g. from boreal dwarf shrubs and trees, can cause reduced growth of neighbouring plants: an effect known as allelopathy. To examine whether arctic and subarctic plants could also be affected by leaching of phytochemicals, we added extracts from the commonly occurring arctic dwarf shrubs Cassiope tetragona and Empetrum hermaphroditum, and from mountain birch, Betula pubescens ssp. tortuosa to three graminoid species, Carex bigelowii, Festuca vivipara and Luzula arcuata, grown in previously sterilized or non-sterilized arctic soils. The graminoids in non-sterilized soil grew more slowly than those in sterilized soil. Excised roots of the plants in non-sterilized soil had higher uptake rate of labelled P than those in sterilized soil, demonstrating larger nutrient deficiency. The difference in growth rate was probably caused by higher nutrient availability for plants in soils in which the microbial biomass was killed after soil sterilization. The dwarf shrub extracts contained low amounts of inorganic N and P and medium high amounts of carbohydrates. Betula extracts contained somewhat higher levels of N and much higher levels of P and carbohydrates. Addition of leaf extracts to the strongly nutrient limited graminoids in non-sterilized soil tended to reduce growth, whereas in the less nutrient limited sterilized soil it caused strong growth decline. Furthermore, the N and P uptake by excised roots of plants grown in both types of soil was high if extracts from the dwarf shrubs (with low P and N concentrations) had been added, whereas the P uptake declined but the N uptake increased after addition of the P-rich Betula extract. In contrast to the adverse extract effects on plants, soil microbial respiration and soil fungal biomass (ergosterol) was generally stimulated, most strongly after addition of the Betula extract. Although we cannot exclude the possibility that the reduced plant growth and the concomitant stimulation of microbial activity were caused by phytochemicals, we believe that this was more likely due to labile carbon in the extracts which stimulated microbial biomass and activity. As a result microbial uptake increased, thereby depleting the plant available pool of N and P, or, for the P-rich Betula extract, depleting soil inorganic N alone, to the extent of reducing plant growth. This chain of events is supported by the negative correlation between plant growth and sugar content in the three added extracts, and the positive correlation between microbial activity, fungal biomass production and sugar content, and are known reactions when labile carbon is added to nutrient deficient soils.     

A temporal profile of the endocrine control of trypsin synthesis in the yellow fever mosquito,Aedes aegypti

Trypsin synthesis and secretion is induced after the female mosquito takes a blood meal. Its peak activity has been shown to be proportional to the amount and quality of food uptake. Further regulatory elements, hormones from the brain and the ovary, increase the synthethic rate of trypsin in the midgut by a factor of two. We investigated the temporal effect of removing the humoral factors by decapitation and ovariectomy. Trypsin synthesis was reduced to less than half its normal output when the operations were performed prior, or immediately after the blood meal. Postponing decapitation resulted in an increased activity. However, the dependence on hormones extended up to 14-16hrs after a meal, when maximal synthethic rates are assumed. Similarly, ovariectomy had a prolonged effect on trypsin synthesis. Finally, the lack of hormones reduced the synthetic capacity of the midgut even when small blood meals were given. We conclude that for continued efficient trypsin synthesis, humoral stimulation is necessary but is not part of the feedback mechanism that links the presence of food with the amount of trypsin secreted.     

Competition in foraging flocks of migrating semipalmated sandpipers

I examined the effect of competitor density on foraging success in staging semipalmated sandpipers (Calidris pusilla) foraging on a burrowing amphipod (Corophium volutator) in each of two study years. Little is known about the effect of competitor density when predation attempts disturb prey, causing a temporary decrease in food availability. Controlling for Corophium density and other potentially confounding factors such as temperature, pecking rate and capture rate increased linearly with sandpiper density. Success rate, the ratio of captures to pecks, was not influenced by sandpiper density. The effect of sandpiper density was similar in each of the two study years and was documented early and late in the low tide period. The increase in foraging rate is argued to be a response to increased competition for rapidly depleting prey at the temporal scale of exploitation by a flock. Potential fitness costs associated with higher foraging costs may include decreased ability to distinguish between the profitability of different prey and reduced vigilance against predators.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.