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Neonicotinoid insecticides (NNI) are agonists of insect nicotinic acetylcholine receptors (nAChR) that induce non-elucidate mechanisms of abnormal behavior in insects. In this work, we investigated the effects of sublethal doses of the neonicotinoid thiamethoxam (TMX) on neurochemical and physiological parameters in cockroaches. Sublethal doses of TMX (0.01–10 ng.g−1 body mass) caused significant alterations in most of the neurophysiological parameters evaluated. TMX reduced sustained locomotor activity by 19.9–25.8 %, depending on the dose. Leg grooming activity increased by 124.5 ± 3.4 %, 158.7 ± 3.5 %, 168.3 ± 3.4 %, and 160.4 ± 3.4 % (mean ± SEM) with TMX doses of 0.01, 0.1, 1, and 10 ng.g−1, respectively. Exploratory activity was significantly reduced only at the lowest TMX dose (0.01 ng.g−1) – the time spent immobile increased from 30 % to ∼45 %, whereas none of the doses affected the walking speed. Treatment with TMX (0.01 ng.g−1) markedly reduced the olfactory sensitivity of the cockroaches and also reduced the mechanosensory action potential amplitude, rise time and decay time by 61.2 ± 19 %, 50 ± 4 %, and 76.8 ± 9.5 %, respectively. In semi-isolated heart preparations, TMX caused positive chronotropism (increases of 34.7 ± 15.9 %, 26.8 ± 7.8 %, 43.0 ± 16.5 %, and 19.0 ± 13.7 % for 0.01, 0.1, 1, and 10 ng of TMX, respectively). TMX attenuated the activity of glutathione-S-transferase by 35.1 ± 6.4 % at the highest dose tested (10 ng.g−1). TMX caused alterations in the metal ion content of cockroach brains that varied with the dose tested and the ion examined. These findings indicate that sublethal doses of TMX can interfere with normal neurological function in cockroaches and disrupt brain metal ion homeostasis. [Display omitted] • Thiamethoxam is an agonist of insect nicotinic acetylcholine receptors (nAChR). • Thiamethoxam disrupt the somatosensorial integration in insects. • Thiamethoxam induces functional alterations of insect brain metal ion homeostasis. • These actions adversely affect neuronal pathways involved in insect behavior. [ABSTRACT FROM AUTHOR]

Beauvericin is a cyclic hexadepsipeptide produced as secondary metabolites of an entomopathogenic fungus, Beauveria bassiana that shown nematocidal, insecticidal and cytotoxic activities. Glyphodes pyloalis Walker is a destructive pest of mulberry trees that extensive feeding of larvae on the leaves, pile of excreta and transmission of viral diseases to silkworm are of its constraints on regional sericulture. Since sustainable and environmental friendly control of G. pyloalis is of importance, the current study aimed to produce beauvericin by culturing a native isolate of B. bassiana to Czapek medium and extract it by high performance liquid chromatography using RPC18 column. The extracted beauvericin was exposed to the 4th instar larvae of G. pyloalis through leaf dipping method to determine mortality and lethal concentrations. Results showed the LC 50 of 0.918 μg/ml of beauvericin against the larvae. The LC 30 concentration of beauvericin was nano-formulated and treated orally against G. pyloalis larvae to determine its possible effects on nutritional indices, digestive and antioxidative enzymes. Results showed the decreased level of nutritional indices and imposing the metabolic cost of the treated larvae compared to control. Digestive enzymes were also negatively influenced following larval feeding on the leaves treated by beauvericin except for α-glucosidase and lipase. Antioxidative enzymes were also affected in the treated larvae by beauvericin in that their activity significantly increased except for gluxcose-6-phosphate dehydrogenase. Results of the current study revealed the nano-formulation of beauvericin affected physiological performance of the larvae in a sublethal concentration. These results imply on potential of nano-formulated beauvericin to be used against G. pyloalis as a nature-based insecticide. [Display omitted] • Beauvericin was toxic against Glyphodes pyloalis larvae by LC 50 of 0.918 μg/ml. • Sub-lethal concentration of beauvericin decreased the level of nutritional indices. • Digestive enzymes were negatively influenced in the beauvericin-treated larvae. • The activity of antioxidative enzymes significantly incraesed after beauvericin treatment. [ABSTRACT FROM AUTHOR]

Plant based insecticides are considered among the most economic and ecofriendly chemicals for the protection of plants and stored grains. The cowpea weevil (Callosbruchus maculatus) causes more than 90% damage to sored grains in three to six months. The current study investigates insecticidal potentials of five selected botanicals: Melia azedarach, Nicotiana rustica, Azadirachta indica, Nicotiana tabacum and Thuja orientalis. They are explored at six different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) against C. maculatus and compared to effects of distilled water which is used as a control. Toxicities of 3%(V/V) extracts of N. tabacum, N. rustica, A. indica and T. orientalis against C. maculatus were 100%, 86.11%, 80.56% and 72.22%, respectively. Maximum mortality was caused by N. tabacum and N. rustica (100%), followed by A. indica (82%), whereas minimum mortality was observed in T. orientalis (64%) at 2.5%. Several phytochemicals, alkaloids, saponins, diterphenes, phytosterol, flavonoids and phenols were identified in N. tabacum and N. rustica, while few were present in A. indica. Phytosterol was present in greatest abundance. Saponins were only detected in aqueous extracts of N. rustica and N. tabacum. Taken together, these results indicate the utility of N. tabacum, N. rustica and A. indica as potential botanicals to control pest beetle and cowpea weevil.

The aim of this study was to produce Bacillus thuringiensis-based biopesticide using starch-producing industry wastewater (SIW) fortified with soybean medium and optimize the formulated product using different adjuvants. This study was necessary as low endotoxin concentration is obtained in formulated biopesticide when SIW alone is used as fermentation medium. The fermentation runs were conducted using SIW alone and SIW fortified with 25% soybean (w/v) medium in 2000 L and 150 L bioreactor, respectively. SIW supplemented with soybean medium showed an increase in cell count (from 1.95 × 108 to 1.65 × 109 CFU mL–1), spore synthesis (from 1.5 × 108 to 1.35 × 109 CFU mL–1) and endotoxin concentration (from 436 to 1170 μg mL–1) when compared to SIW medium alone. The fermented broth was concentrated using continuous centrifugation and adjuvants were added for biopesticide formulation in order to enhance its resistance against UV rays and rainfastness. Entomotoxicity of the formulation produced using fermented broth of SIW fortified with soybean (38,000 IU μL–1) was higher than that obtained by SIW medium alone (21,000 IU μL–1), commercial biopesticide Foray 76B (20,000 IU μL–1) and Btk sander's (12,500 IU μL–1). [ABSTRACT FROM AUTHOR]

Ureases are metalloenzymes that hydrolyze urea to ammonia and carbamate. The main urease isoforms present in the seeds of Canavalia ensiformis (jack bean urease - JBU and canatoxin) exert a variety of biological activities. The insecticidal activity of JBU is mediated, at least in part, by jaburetox (Jbtx), a recombinant peptide derived from the JBU amino acid sequence. In this article, we review the neurotoxicity of Jbtx in insects. The insecticidal activity of Jbtx has been investigated in a variety of insect orders and species, including Blattodea (the cockroaches Blatella germânica, Nauphoeta cinerea, Periplaneta americana e Phoetalia pallida), Bruchidae (Callosobruchus maculatus - cowpea weevil), Diptera (Aedes aegypti - mosquito), Hemiptera (Dysdercus peruvianus - cotton stainer bug; Oncopeltus fasciatus - large milkweed bug, and the kissing bugs Rhodnius prolixus and Triatoma infestans), Lepidoptera (Spodoptera frugiperda - fall army worm) and Orthoptera (Locusta migratoria - locust). In N. cinerea, the injection of Jbtx induces marked alteration of locomotor and grooming behavior, whereas in T. infestans Jbtx causes leg paralysis, an extension of the proboscis and abnormal antennal movements. Electromyographical analysis showed that Jbtx causes complete neuromuscular blockade in P. pallida. The same treatment in N. cinerea and L. migratoria causes a decrease in the action potential firing rate. Jbtx forms membrane pore-channels compatible with cations in bilipid membranes. A study using B. germanica voltage-gated sodium (Nav1.1) channels that were heterologously expressed in Xenopus laevis oocytes correlated the entomotoxicity of Jbtx with the activation of these channels. Taken together, these findings demonstrate the potential of this peptide as a natural pesticide. [ABSTRACT FROM AUTHOR]

Abstract Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea , Jbtx (32 μg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ± 85 cm and 2573 ± 89 s, p <.05, n = 40). Jbtx (8 to 32 μg/g body weight, respectively) also increased the leg and antennae grooming activities (p <.05, n = 40, respectively). Jbtx (8 to 16 μg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p <.05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10−7 and 2.5 × 10−3 μg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p <.05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels. Graphical abstract Unlabelled Image Highlights • The entomotoxic effect of Jaburetox (Jbtx) is complex and alters insect behavior. • Jbtx activates voltage-gated sodium channels to modulate insect behavior. • Further toxicological studies will unveil the risk assessment of Jaburetox. [ABSTRACT FROM AUTHOR]

The use of natural bioactive compounds mainly proteinaceous secondary metabolites of fungi is one of the promising pest control methods because of their lethal effects on insects in low concentration, limited persistence in environment and easily decomposition into environmentally safe compounds. The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is a destructive pest of olive fruits around the world. In the current study, the proteinaceous compounds were extracted from the two isolates (MASA and MAAI) of Metarhizium anisopliae and their effects were evaluated on toxicity, feeding performance and antioxidant system of the adult's olive flies. Both extracts from MASA and MAAI showed entomotoxicity against the adults by 2.47 and 2.38 mg/mL as LC 50 concentrations. Also, LT 50 values were recorded 1.15 and 1.31 days for MASA and MAAI, respectively. No statistical differences were recorded in the consumption rate of the adults on control and secondary metabolite contained protein hydrolysate. In contrast, the adults fed on LC 30 and LC 50 concentrations of MASA and MAAI demonstrated significant reduction in the activities of digestive alpha-amylase, glucosidases, lipase, trypsin, chymotrypsin, elastase, amino- and carboxypeptidases. Activity of antioxidant enzymes changed in the adults of B. oleae fed on the fungal secondary metabolites. Catalase, Peroxidase and Superoxide dismutase elevated in the treated adults with the highest amounts of MAAI. Similar results were found in activity of ascorbate peroxidase and glucose-6-phosphate dehydrogenase except for malondialdehyde amount in which no statistical differences were recorded between treatments and control. Relative gene expression of caspase enzymes revealed the higher expression in the treated B. oleae compared to control with the highest level of caspase 8 for MASA and caspases 1 and 8 for MAAI. Results of our study showed that the secondary metabolites extracted from the two isolates of M. anisopliae caused mortality, interrupted digestion and induced oxidative stress in the adults of B. oleae. [Display omitted] • MASA and MAAI extracts showed entomotoxicity by 2.47 and 2.38 mg/mL as LC 50. • No statistical differences were recorded in solution consumption of the adults on control and treatment. • MASA and MAAI demonstrated significant differences in the activities of digestive enzymes. • Activity of antioxidant enzymes elevated in the adults of B. oleae fed on the fungal secondary metabolites. • Relative gene expression of caspase enzymes revealed the higher expression in the treated B. oleae compared to control. [ABSTRACT FROM AUTHOR]

Colocasia esculenta tuber agglutinin (CEA), a mannose binding lectin, exhibits insecticidal efficacy against different hemipteran pests. Dysdercus cingulatus, red cotton bug (RCB), has also shown significant susceptibility to CEA intoxication. However, the molecular basis behind such entomotoxicity of CEA has not been addressed adequately. The present study elucidates the mechanism of insecticidal efficacy of CEA against RCB. Confocal and scanning electron microscopic analyses documented CEA binding to insect midgut tissue, resulting in an alteration of perimicrovillar membrane (PMM) morphology. Internalization of CEA into insect haemolymph and ovary was documented by western blotting analyses. Ligand blot followed by mass spectrometric identification revealed the cognate binding partners of CEA as actin, ATPase and cytochrome P450. Deglycosylation and mannose inhibition assays indicated the interaction to probably be mannose mediated. Bioinformatic identification of putative glycosylation or mannosylation sites in the binding partners further supports the sugar mediated interaction. Correlating entomotoxicity of CEA with immune histological and binding assays to the insect gut contributes to a better understanding of the insecticidal potential of CEA and endorses its future biotechnological application.

Rhinella icterica is a poisonous toad whose toxic secretion has never been studied against entomotoxic potential. Sublethal doses of Rhinella icterica toxic secretion (RITS) were assayed in Nauphoeta cinerea cockroaches, in order to understand the physiological and behavioral parameters, over the insect central and peripheral nervous system. RITS (10 μg/g) injections, induced behavioral impairment as evidenced by a significant decrease (38 ± 14%) in the distance traveled ( p  < .05), followed by an increase (90 ± 6%) of immobile episodes ( p  < .001, n  = 28, respectively). In cockroaches semi-isolated heart preparations, RITS (16 μg/200 μl) induced a significant irreversible dose-dependent negative chronotropism, reaching ~40% decrease in heart rate in 20 min incubation. In in vivo cockroach neuromuscular preparations, RITS (20, 50 and 100 μg/g of animal weight) induced a time-dependent inhibition of twitch tension that was complete for 20 μg/g, in 120 min recordings. RITS (10 μg/g) also induced a significant increase in the insect leg grooming activity (128 ± 10%, n  = 29, p  < .01), but not in the antennae counterparts. The RITS increase in leg grooming activity was prevented in 90% by the pretreatment of cockroaches with phentolamine (0.1 μg/g). The electrophysiological recordings of spontaneous neural compound action potentials showed that RITS (20 μg/g) induced a significant increase in the number of events, as well as in the rise time and duration of the potentials. In conclusion, RITS showed to be entomotoxic, being the neuromuscular failure and cardiotoxic activity considered the main deleterious effects. The disturbance of the cockroaches' behavior together with the electrophysiological alterations, may unveil the presence of some toxic components present in the poison with inherent biotechnological potentials. [ABSTRACT FROM AUTHOR]

The use of higher dosage and repeated applications of conventional pesticides have led to the rapid development of insect resistance to pesticide and adverse effects on human health and environment. Accordingly, researchers are prompted to identify an alternative entomotoxic agent for crop protection. Nanocides are being considered as alternatives to conventional insecticides because they are expected to lessen the application rate and reduce the chances of resistance development in pests. In this study, we evaluated the entomotoxic effects of nanosilica on larvae of Plutella xylostella, in a laboratory by using dust spray, larva dipping, leaf dipping, and solution spray methods. Dust treatment showed a more highly significant effect than the other three treatments. The mortality percentage increased up to 58% and 85% at 24 and 72 h after treatment, respectively, when nanosilica was applied at a rate of 1 mg cm−2. In all four bioassays, mortality rate increased with both increased time after nanosilica exposure and increased concentration. Light microscopy and scanning electron microscopy images showed that larval death was due to desiccation, body wall abrasion, and spiracle blockage. These results suggested that nanosilica can be an alternative to conventional pesticides if dust formulation would be properly used. [ABSTRACT FROM AUTHOR]

In this paper we present observations of individuals of the bee species Trigona recursa feeding on the fruits of Crotalaria micans. This plant, which contains pyrrolizidine alkaloids, is known to be toxic to humans, mammals and poultry. Over the course of three days, we observed a large number of bees feeding on many individual Crotalaria micans plants in an urban fragment of Brazilian Savanna. The bees preferred greener fruits, which are the softest and most toxic. Consumption of the plant had no immediately apparent fatal effect on the bees, since we did not find any dead individuals near the observation site. Some insect species are known to use pyrrolizidine and alkaloids for defense by incorporating them into their body or using them as precursors to pheromones. Trigona recursa and other bee species have not been previously recorded consuming Crotalaria micans and it is unclear what their motivation may be. We present these observations as a novel finding of the feeding behavior of Trigona recursa.

Fipronil (Fpl), an insecticide belonging to the class of phenylpyrazoles, is associated with the widespread mortality of pollinator insects worldwide. Based on studies carried out on residual concentrations of Fpl commonly found in the environment, in this study, we evaluated the sublethal effects of Fpl on behavior and other neurophysiological parameters using the cockroach Nauphoeta cinerea as a biological model. Sublethal doses of Fpl (0.1-0.001 μg g-1) increased the time spent grooming and caused dose-dependent inhibition of exploratory activity, partial neuromuscular blockade in vivo and irreversible negative cardiac chronotropism. Fpl also disrupted learning and olfactory memory formation at all doses tested. These results provide the first evidence that short-term exposure to sublethal concentrations of Fpl can significantly disrupt insect behavior and physiology, including olfactory memory. These findings have implications for current pesticide risk assessment and could be potentially useful in establishing a correlation with pesticide effects in other insects, such as honey bees., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Kitchen garbage is usually discharged from restaurants, public catering rooms and school canteens, and it is reported that 2000 t kitchen garbage would be produced per day in Beijing, China. In other big cities similar phenomena can be observed, and it is reported that half of the municipal waste is composed of kitchen garbage. Kitchen garbage is characterized by high moisture content, easily deteriorating and abundant in nutrition, but it should be treated as a kind of resource rather than simple waste because it is mainly composed of starch, protein, cellulose, fat, and trace element, such as potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), and iron (Fe); kitchen garbage can be regarded as a suitable substrate for microbial growth. In this research, kitchen garbage was used as the raw material to produce Bacillus thuringiensis (Bt) bio-pesticide which is one of the most commonly used and extensively studied bio-pesticides because of its various field applications, such as in agriculture, forestry, and public health sectors. However, the costly raw materials, high equipment investment, and complicated operation procedures in traditional Bt production process have seriously constrained its use at the industrial production scale. In this work we conducted to determine the potential and superiority of using kitchen garbage for Bt bio-pesticide production through semi-solid fermentation to reduce the production cost, and the effects of several parameters on the stability of the product were also studied. In this work, the semi-solid state sample with 75% water content presented the highest δ-endotoxin efficiency of 833 μg/mL, which increased by 54% compared to that of the solid-state sample (water content of 50%) and 162% compared to that of the submerged sample (water content of 99%), and proved that using kitchen garbage as the raw material for Bt production through semi-solid state fermentation has obvious advantages compared with traditional techniques and some similar studies. Mainly because of its nutritious substrate and effective fermentation mode, this technology can not only solve the problem of civil solid waste pollution but also bring a cost-effective resource for Bt bio-pesticide production. Among the main affecting factors during Bt bio-pesticide manufacturing and application, salinity, pH value and temperature had the minimal impact on the stability of the parasporal crystal; when these 3 factors were controlled at 3%, 11 and 60 ℃, respectively, the entomotoxicity of parasporal crystal could still remain above 70% after the fermentation of less for 48 h, and therefore common fermentation process would not inactivate the goal product sharply. By contrast, ultraviolet irradiation had more significant impact on parasporal crystal’s stability, and the entomotoxicity of parasporal crystal declined by 50% when exposed to the ultraviolet (36 W, 60 μW/cm2, 40 cm) for about 3 h. On account of this, polylactic acid was used as the carrier to produce Bt slow-release formulation to mitigate the effects of ultraviolet. Experiments showed that the activity of Bt slow-release formulation remained over 75% of its initial value after 72 h ultraviolet irradiation, which was 10 times the untreated parasporal crystal. And the release rates of parasporal crystal in formulation were generally metastable in a 30-day period, and the compact structure of polylactic acid was proven to effectively protect δ-endotoxin from ultraviolet damage. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis ( Bt ) bio-pesticides is a kind of selective and efficient pesticides, but the high cost of raw materials always limits its development, food waste contains superior nutritional elements which could sustain the growth of most microorganisms. So we choose food waste as the material, explored its feasibility of producing bio-pesticides and optimized the influence parameters during the fermentation process. In this work, food waste with 75% water content presented δ-endotoxin efficiency of 862 μg/mL, spore count of 2.23×10 11 CFU/mL and entomotoxicity of 56560 IU/mg. The influence factors during fermentation process were optimized by asingle-factor indextest, and the result showed that initial pHhad a max effect on the fermentation performance. The optimum fermentation conditions were0.2% inoculum size, 2% salt content, 5% or less oil content, and the initial pH adjusted to 7.0, inoptimized conditions, the δ-endotoxin outputwas2310 μg/mL and the entomotoxicity was 56875IU/mgafter fermentation. It seemed to be a promising way to make use of food waste. [ABSTRACT FROM AUTHOR]

Colocasia esculenta tuber agglutinin (CEA), a mannose binding lectin, exhibits insecticidal efficacy against different hemipteran pests. Dysdercus cingulatus, red cotton bug (RCB), has also shown significant susceptibility to CEA intoxication. However, the molecular basis behind such entomotoxicity of CEA has not been addressed adequately. The present study elucidates the mechanism of insecticidal efficacy of CEA against RCB. Confocal and scanning electron microscopic analyses documented CEA binding to insect midgut tissue, resulting in an alteration of perimicrovillar membrane (PMM) morphology. Internalization of CEA into insect haemolymph and ovary was documented by western blotting analyses. Ligand blot followed by mass spectrometric identification revealed the cognate binding partners of CEA as actin, ATPase and cytochrome P450. Deglycosylation and mannose inhibition assays indicated the interaction to probably be mannose mediated. Bioinformatic identification of putative glycosylation or mannosylation sites in the binding partners further supports the sugar mediated interaction. Correlating entomotoxicity of CEA with immune histological and binding assays to the insect gut contributes to a better understanding of the insecticidal potential of CEA and endorses its future biotechnological application. [ABSTRACT FROM AUTHOR]

Biopesticides are usually sprayed on forests by using planes made up of aluminum alloy. Bioval derived from starch industry wastewater (SIW) in suspension form was developed as stable anticorrosive biopesticide formulation. In this context, various anticorrosion agents such as activated charcoal, glycerin, ethylene glycol, phytic acid, castor oil and potassium silicate were tested as anticorrosive agents. There was no corrosion found in Bioval formulation where potassium silicate (0.5% w/v) was added and compared with Foray 76 B, as an industrial standard, when stored over 6 months. In relation to other parameters, the anticorrosion formulation of Bioval+buffer+KSi reported excellent zeta potential (−33.19 ± 4 mV) and the viscosity (319.13 ± 32 mPa.s) proving it's stability over 6 months, compared to the standard biopesticide Foray 76 B (−36.62 ± 4 mV potential zeta, pH 4.14 ± 0.1 and 206 ± 21 mPa.s viscosity). Metal analysis of the different biopesticides showed that Bioval+buffer+KSi has no corrosion (5.11 ± 0.5 mg kg−1of Al and 13.53 ± 1.5 mg kg−1of Fe) on the aluminum alloy due to the contribution of sodium acetate buffer at pH 5. The bioassays reported excellent results for Bioval+Buffer+KSi (2.95 ± 0.3 × 109CFU mL−1spores and 26.6 ± 2.7 × 109IU L−1Tx) compared with initial Bioval (2.46 ± 0.3 × 109CFU mL−1spores and 23.09 ± 3 × 109IU L−1Tx) and Foray 76 B (2.3 ± 0.2 × 109CFU mL−1spores and 19.950 ± 2.1 UI L−1Tx) which was due to the break-up of the external chitinous membrane due to abrasive action of potassium silicate after ingestion by insects. The contribution of sodium acetate buffer and potassium silicate (0.5% and at pH = 5) as anticorrosion agent in the Bioval allowed production of an efficient biopesticide with a reduced viscosity and favorable pH as compared to Foray 76 B which enhanced the entomotoxic potential against spruce budworm (SB) larvae (Lepidoptera: Choristoneura fumiferana). [ABSTRACT FROM AUTHOR]

Swainsonine, a secondary metabolite from Metarhizium anisopliae has been extensively studied in the complementary areas of therapeutics and toxicology. This work aims to develop a simple UV-HPLC method of analyses for swainsonine in Metarhizium fermentation broth and to explore its in situ entomotoxic activities. The partially purified broth was quantitatively analyzed using middle UV (205 nm)-reverse phase HPLC method with different mobile phases and gradient programmes. Swainsonine was eluted as single peak at (te) 6.0-6.9 min with average concentration of 4.04 ± 0.52 μg/mL using optimal mobile phase (0.1% trifluoroacetic acid in water and acetonitrile). The mass spectrometry analysis further indicated the characteristic MS1 species for swainsonine, [M+H]+ 174.30 in corresponding HPLC peaks. The antiproliferative effects of swainsonine on lepidopteran, Sf-21 cells were determined through 3-(4, 5-dimethylthia-zol-2-yl)−2, 5-diphenyl tetrazolium bromide (IC50 standard = 3.90 μM and IC50 purified = 5.27 μM) and trypan blue dye exclusion (IC50 standard = 6.91 μM and IC50 purified = 8.67 μM) assays. The fluorescence activated cell sorting evaluation of Sf-21 cells showed nearly 35% and 42% of population in various apoptotic stages at 36 h, when treated with standard and purified swainsonine, respectively. The morphodimensional field emission scanning electron and atomic force microscopic analyses further confirmed the characteristic apoptotic features like membrane blebbings, ruptures and volume shrinkage in the lepidopteran cells after 24-36 h of post-treatment incubation. The study describes the potential entomotoxic activities of swainsonine and its role in the virulence of Metarhizium spp. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1196-1205, 2014 [ABSTRACT FROM AUTHOR]

Plant based insecticides are considered among the most economic and ecofriendly chemicals for the protection of plants and stored grains. The cowpea weevil ( Callosbruchus maculatus ) causes more than 90% damage to sored grains in three to six months. The current study investigates insecticidal potentials of five selected botanicals: Melia azedarach, Nicotiana rustica, Azadirachta indica , Nicotiana tabacum and Thuja orientalis. They are explored at six different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) against C. maculatus and compared to effects of distilled water which is used as a control. Toxicities of 3%(V/V) extracts of N. tabacum, N. rustica, A. indica and T. orientalis against C. maculatus were 100%, 86.11%, 80.56% and 72.22%, respectively. Maximum mortality was caused by N. tabacum and N. rustica (100%), followed by A. indica (82%), whereas minimum mortality was observed in T. orientalis (64%) at 2.5%. Several phytochemicals, alkaloids, saponins, diterphenes, phytosterol, flavonoids and phenols were identified in N. tabacum and N. rustica, while few were present in A. indica. Phytosterol was present in greatest abundance. Saponins were only detected in aqueous extracts of N. rustica and N. tabacum. Taken together, these results indicate the utility of N. tabacum , N. rustica and A. indica as potential botanicals to control pest beetle and cowpea weevil.

Abstract: Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leafworm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3days with 9μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC–MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA. [Copyright &y& Elsevier]

Abstract: Mathematic relationships between spore concentrations, delta-endotoxin concentrations and entomotoxicity (Tx) of Bacillus thuringiensis var. kurstaki HD-1 (Btk HD-1) preparations produced in six different media were analysed. The relationship between delta-endotoxin and spore concentration and SpTx-spore (specific Tx per 1000 spore) and spore concentration produced in the different media (starch industry wastewater (SIW) with total solids (TS) concentration of 15g/L, SIW with TS of 30g/L, SIW supplemented with 0.2% (w/v) colloidal chitin, SIW supplemented with 1.25% (w/v) cornstarch and 0.2% (v/v) Tween 80, secondary sludge, and semi-synthetic medium) strictly followed the Power law. Tx and delta endotoxin concentration followed the exponential relation whereas a definite relation between Tx and spore concentration could not be established. Spore and delta-endotoxin produced at the early time (12h) during fermentation might be more toxic than those produced during latter period of fermentation irrespective of media used. Tx and delta-endotoxin concentration exhibited a semi-log linear relationship. Based on these findings, delta-endotoxin concentration can be determined rapidly to monitor the progress of the biopesticide production process. [Copyright &y& Elsevier]

BACKGROUND: In this study, the effects of three saponins and one sapogenin with a triterpenoid or steroid structure in two lepidopteran insect cell lines, ovarian Bm5 and midgut CF-203 cells, were analysed with regard to cell viability, cell membrane permeation, EcR responsiveness and DNA fragmentation. In addition, the entomotoxic action of Q. saponaria saponin with primary midgut cell cultures and larval stages of the cotton leafworm Spodoptera littoralis was tested. RESULTS: Both lepidopteran cell lines show a high sensitivity to all four sapo(ge)nins, with a concentration-dependent viability loss and EC50 values of 25-100 µ M in MTT bioassays. A trypan blue assay with Q. saponaria saponin confirmed rapid cell membrane permeation to be a cause of cytotoxicity. Saponins caused no EcR activation in Bm5 cells, but a loss of ecdysteroid signalling was observed with IC50 values of 5-10 µ M. Lower saponin concentrations induced DNA fragmentation, confirming their potential to induce apoptosis. Finally, Q. saponaria saponin caused cytotoxicity in primary midgut cell cultures of S. littoralis (EC50 = 4.7 µ M) and killed 70-84% of S. littoralis larvae at pupation at 30-70 mg g−1, while lower concentrations retarded larval weight gain and development. CONCLUSIONS: The data obtained provide evidence that saponins exert a strong activity on lepidopteran cells, presumably based on a cytotoxic action due to permeation of the cell membrane. Primary midgut cell cultures and larvae of S. littoralis showed high sensitivity to Q. saponaria saponin, indicating the insect midgut as a primary target for entomotoxicity and the potential use of saponins in the control of pest Lepidoptera. Copyright © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Pest management researchers currently reappraise the use of inert dust-based insecticides because of the growing problem of environmental pollution and increasing insect resistance associated with conventional insecticides. Diatomaceous earth, which is amorphous micron-sized silica derived from fossilized phytoplankton, has become popular as an alternative insecticidal agent in European countries. In this investigation the insecticidal efficacy of amorphous lipophilic silica nanoparticle was examined on red flour beetle (Tribolium castaneum), a stored grain insect pest. The biosafety of this silica nanoparticle formulation was studied in MRC-5 cell line with water-soluble tetrazolium and lactate dehydrogenase activity assays. Acute oral toxicity of these nanocides was studied in mice model following OECD guidelines for testing of chemicals as well as the effects of particle exposure on mouse blood parameters, serum biochemical levels, and histopathological changes in various organs. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis var. kurstaki biopesticide was produced in batch and fed-batch fermentation modes using starch industry wastewater as sole substrate. Fed-batch fermentation with two intermittent feeds (at 10 and 20 h) during the fermentation of 72 h gave the maximum delta-endotoxin concentration (1,672.6 mg/L) and entomotoxicity (Tx) (18.5 × 106 SBU/mL) in fermented broth which were significantly higher than maximum delta-endotoxin concentration (511.0 mg/L) and Tx (15.8 × 106 SBU/mL) obtained in batch process. However, fed-batch fermentation with three intermittent feeds (at 10, 20 and 34 h) of the fermentation resulted in the formation of asporogenous variant (Spo−) from 36 h to the end of fermentation (72 h) which resulted in a significant decrease in spore and delta-endotoxin concentration and finally the Tx value. Tx of suspended pellets (27.4 × 106 SBU/mL) obtained in fed-batch fermentation with two feeds was the highest value as compared to other cases. [ABSTRACT FROM AUTHOR]

Abstract: Photoprotection (against UV-A and UV-B radiations) of the active components of Bacillus thuringiensis var. kurstaki obtained from the fermentation of various culture media was investigated. The culture media comprised: starch industry wastewater; secondary wastewater sludge (non-hydrolyzed and hydrolyzed) and soya (used as a reference). Photoprotection was carried out by using various UV-protection additives, namely, para-aminobenzoic acid, lignosulfonic acid and molasses at different concentrations (0.1%, 0.15% and 0.2%, w/w). In the absence of UV-protection agents, secondary sludge demonstrated natural UV protection with half-lives ranging from 3.25 to 3.4 d. The half-life for soya and starch industry wastewater was 1.9 and 1.8 d, respectively. Para-amino-benzoic acid as a UV-protection agent at 0.20% (w/w) gave excellent UV-protection for soya and starch industry wastewater with half-lives being 5.9 and 7 d, respectively. Likewise, lignosulfonic acid at 0.20% (w/w) was an effective photostabilizer for hydrolyzed and non-hydrolyzed secondary sludge with half-lives of 7.25 and 8 d, respectively. Hence, when similar concentration of the UV-protection additives was used, photoprotection was higher for the alternative media than the conventional soya medium, validating the technical feasibility of using three additives. [Copyright &y& Elsevier]

Total solids (TS) concentrations ranging from 15 to 66 g L-1 of starch industry wastewater (SIW) were tested as raw material for the production of Bacillus thuringiensis var. kurstaki HD-1 (Btk) biopesticide in shake flasks and a 15 L bench-scale fermenter. Shake flask studies revealed a higher delta-endotoxin concentration of Btk at 30 g L-1 TS concentration and 2.5% (v v-1) volume of pre-culture. The fermenter experiments conducted using SIW at 30 g L-1 TS concentration under controlled conditions of temperature, pH and dissolved oxygen showed higher spore count, enzyme production (protease and amylase) and delta-endotoxin concentration as compared with those of SIW at 15 g L-1 TS concentration. The entomotoxicity, at the end of fermentation, with SIW at 30 g L-1 solids concentration (17.8 × 109 SBU L-1, measured against spruce budworm) was considerably higher as compared with entomotoxicity at 15 g L-1 solids concentration (15.3 × 109 SBU L-1) and semi-synthetic medium (11.7 × 109 SBU L-1). The pellet, comprising spores and delta-endotoxin complex obtained after centrifugation and followed by resuspension (in supernatant) in one-tenth of the original volume, of SIW at 30 g L-1 solids concentration media registered the highest potential for application (to protect forests against spruce budworm) than other media in term of entomotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: Ribosome-inactivating proteins (RIPs) from plants inhibit protein synthesis by inactivating ribosomes. Some two-chain (type 2) RIPs are highly toxic and may play a role in plant defense. The lower toxicity of single-chain (type 1) RIPs reflects the lack of a protein domain able to bind to, and translocate the toxin across cell membranes. We studied the effect of single-chain RIPs, lychnin, momordin, gelonin, PAP-S and saporin S-6, in larvae of Anticarsia gemmatalis and Spodoptera frugiperda. After ingesting a total dose of 20 or 40μg of the toxins, weight gain, survival rate, lesions in DNA and oxidative status (catalase and superoxide dismutase activities and lipidic peroxidation) of RIP-treated insects were assayed. Momordin was the less toxic in the biossays. S. frugiperda had a more pronounced weight loss on the 4th day of treatment and A. gemmatalis on the 10th day. RIP-induced mortality reached 57.13% for A. gemmatalis and 29.45% for S. frugiperda. RIP-treated insects showed a 2–3-fold increase in DNA lesions as assessed by the comet assay, but there were no correlations between stress markers and DNA damage. We conclude that single-chain RIPs are entomotoxic to lepidopteran insects causing extensive DNA lesions. [Copyright &y& Elsevier]

Abstract: Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94×107 CFUmL−1 at 96h) was obtained at 30gL−1 suspended solids. The maximum lytic enzyme activities were achieved around 12–30h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96h of fermentation at 30gL−1 suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290SBUμL−1 at 30gL−1 suspended solids concentration at the end of fermentation (96h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35gL−1 compared to 30 and 25gL−1. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k L a at 35gL−1 did not increase in comparison to 30gL−1 due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent. [Copyright &y& Elsevier]

Abstract: The study investigates the recovery of active components (crystal proteins, spores and other factors of virulence) of Bacillus thuringiensis (Bt) based biopesticides from centrifuged supernatant, by ultrafiltration. The centrifuged fermented broths comprised, starch industry wastewater, non-hydrolyzed and hydrolyzed wastewater sludge and semi-synthetic soya medium (as control). The ultrafiltration membrane of 5kDa gave highest recovery of the active components and increased the entomotoxicity in the retentates by 7.9%, 10.5%, 9.0%, 5.7%, for semi-synthetic soya medium, starch industry wastewater, non-hydrolyzed and hydrolyzed wastewater sludge, respectively. However, the retention of suspended solids on the membrane (measured via mass balance) varied with the type of fermented broths and was very high for hydrolyzed sludge (soya-15%; starch industry wastewater-12%; non-hydrolyzed sludge-7% and hydrolyzed sludge-68%). This reflected the deposit on the membrane. In the given context, scale-up of the ultrafiltration process will give better efficacy for non-hydrolyzed sludge and starch industry wastewater in comparison to soya and hydrolyzed sludge medium. [Copyright &y& Elsevier]

Abstract: Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia (≈3.02 and 4.2×1010 CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6×108 in control to 3.0×107 CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO3 were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/μL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone. [Copyright &y& Elsevier]

Abstract: Starch industry wastewater (SWW), slaughterhouse wastewater (SHWW) and secondary sludges from three different wastewater treatment plants (Jonquière—JQS, Communauté Urbaine de Québec—CUQS and Black lake—BLS) were used as raw materials for the production of Bacillus thuringiensis (Bt) based biopesticides in a pilot scale fermentor (100L working volume). The slaughterhouse wastewater exhibited the lowest Bt growth and entomotoxcity (Tx) potential (measured against spruce budworm) due to low availability of carbon, nitrogen and other nutrients. Performance variation (growth, sporulation, proteolytic activity and Tx potential) within the three types of sludges was directly related to the availability of nitrogen and carbohydrates, which could change with sludge origin and methods employed for its generation. The Tx potential of Bt obtained in different secondary sludges (JQS: 12×109 SBU/L; CUQS: 13×109 SBU/L and BLS: 16×109 SBU/L) and SWW (18×109 SBU/L) was higher than the soybean based synthetic medium (10×109 SBU/L). The maximum protease activity was obtained in CUQ secondary sludge (4.1IU/mL) due to its high complex protein concentration. Nevertheless, high carbohydrate concentration in SWW repressed enzyme production. The secondary sludges and SWW were found to be suitable raw materials for high potency Bt biopesticide production. [Copyright &y& Elsevier]

Abstract: Bacillus thuringiensis (Bt) production using wastewater sludge as a raw material was studied in bench scale fermentor at different sludge solids concentration. The highest cell and spore concentrations and entomotoxicity were observed at a suspended solids concentration of 20–25g/l. Maximal protease activity also increased with solids concentration, however, it decreased after attaining a peak at about 36h. Maximal protease activity (2.3–2.8IU/ml) obtained at 25 and 20g/l sludge suspended solids concentration was 1.75 and 1.5 times that of the maximal activity obtained at 15g/l. Irrespective of the sludge suspended solids concentration, protease activity (until its peak) showed an exponential relationship with total cell count and a linear relationship with entomoxicity potency. An exponential correlation was also established between spore concentration and entomotoxicity. The correlations tested using a regression model were highly significant. [Copyright &y& Elsevier]

Abstract: Studies were conducted on harvesting of Bacillus thuringiensis (Bt)-based biopesticides from fermented broths of starch industry wastewater (SIW), wastewater sludge (raw and hydrolyzed—NH and TH, respectively) and semi-synthetic soyameal to enhance entomotoxicity (Tx) by centrifugation. Pertinent factors influencing Tx, solids concentration, pH, temperature and centrifugal force were investigated. The centrifugate solids concentration beyond 100g/l did not enhance Tx, instead caused pellet formation. Centrifugation efficiency (Tx recovery) was higher at pH 4, and temperature 20°C for starch wastewater (98%), wastewater sludge (98% and 97.8% for non-hydrolyzed and hydrolyzed, respectively) and soya broth (83%). For maximum Tx recovery (SIW—95%; NH—90%; TH—98% and soya—78%), the centrifugal force and time required was 48000g and 30min, respectively. Losses in recovery efficiency were lower for SIW and wastewater sludge in comparison to soya on adopting commercially recommended centrifugal force of 9000g. The settling velocity computations for different fermented broths enabled calculation of Σ factor for continuous commercial centrifuge of a given capacity and hence simulation of power requirements. It was established that power requirements for a given Tx recovery efficiency were highest for conventional medium (soya) in comparison to other waste-based fermented broths. [Copyright &y& Elsevier]

Abstract: Feasibility of production of antagonistic Trichoderma sp. conidial spores using wastewater sludge as a raw material employing different suspended solids concentration (10–50g/l) was investigated in shake flasks. Maximum conidial spore count obtained for raw sludge was 1.98×104 CFU/ml, which was enhanced by sludge pre-treatments (alkaline and thermal alkaline). Conidial spore count ranging from 1.3×106 to 2.8×107 CFU/ml was observed for alkaline and thermal alkaline treated sludges. Optimal suspended solids concentration was 30g/l (107 CFU/ml) whereas, lower (&#60;20g/l) and higher (>30g/l) solids concentration were less efficient. Thermal alkaline pre-treated sludge showed diauxic growth due to multiplicity of sludge biodegradability. A simple, modified CFU filtration technique was also developed for fungal spore assessment in sludge. Bioassay of fermented sludge against spruce budworm larvae showed entomotoxicity (15036SBU/μl), on par with Bacillus thuringiensis biopesticides. This study successfully demonstrated potential of wastewater sludge as a raw material for production of value added product, aiding in sludge management and proliferation of eco-friendly and economical biocontrol agents. [Copyright &y& Elsevier]

Studies were conducted on the production ofBacillus thuringiensis(Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38-55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3-5.5 e+08 cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity. [ABSTRACT FROM AUTHOR]

This study focuses on development of aqueous flowable (suspension) formulations for Bacillus thuringiensis (Bt) based biopesticides from wastewater sludge. Different inerts like sorbitol, sodium monophosphate, sodium metabisulphite, sorbic acid, propionic acid, Tween-80, Triton X-100 and glycerol were tested for formulations. Five different formulations for non-hydrolyzed (NH) secondary sludges were tried and the best combination selected on the basis of various physical parameters like viscosity, particle size, suspendibility, entomotoxicity, and microbiological purity tests. F5 formulations (for secondary sludge) comprising sorbitol, sodium monophosphate and sodium metabisulphite gave better physical and biological characteristics with a small effect on entomotoxicity and spore concentration after 120 days at pH 6, 6.5 and temperatures 40 and 50°C and viscosity change at 40 and 50°C. The formulations were more stable at pH 4.0 to 5.0 and temperatures 4 to 30°C whereas at pH 6.0 and 6.5 and temperatures 40 and 50°C, there was degeneration of the product. Lower proteolytic activity and physical factors like ionic strength and surface group changes at pH 6 and 6.5 were responsible for the instability of the formulation. [ABSTRACT FROM AUTHOR]

• Toad (R. icterica) parotoid secretion were tested in locust isolated muscle. • LC-DAD-MS/MS analysis detected at least 28 compounds in the secretion. • The secretion caused negative chronotropism in semi-isolated heart. • The secretion enhanced the amplitude of contraction and tonus of oviduct muscle. Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 μg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001–100 μg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 μg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A 2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides. [ABSTRACT FROM AUTHOR]

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1 , is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development. IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides., (Copyright © 2019 American Society for Microbiology.)