Enhanced activity of an insecticidal protein, trypsin modulating oostatic factor (TMOF), through conjugation with aliphatic polyethylene glycol

BACKGROUND: Trypsin modulating oostatic factor (TMOF), a decapeptide (Tyr-Asp-Pro-Ala-Pro₆) isolated from the ovaries of the adult yellow fever mosquito, Aedes aegypti, regulates trypsin biosynthesis. TMOF per os is insecticidal to larval mosquitoes and a good model for the development of technologies to enhance protein insecticide activity by reduced catabolism and/or enhanced delivery to the target. RESULTS: TFA-TMOF-K (TFA = trifluoro acetyl) allowed the specific conjugation of monodispersed, aliphatic polyethylene glycol (PEG) to the amino group of lysine-producing TMOF-K-methyl(ethyleneglycol)₇-O-propionyl (TMOF-K-PEG₇P). The addition of lysine to TMOF reduced its per os larval mosquitocidal activity relative to the parent TMOF, but conjugation of TMOF-K with methyl(ethyleneglycol)₇-O-propionyl increased its toxicity 5.8- and 10.1-fold above that of TMOF and TMOF-K for Ae. aegypti. Enhanced insecticidal activity was also found for larval Ae. albopictus and for neonates of Heliothis virescens and Heliocoverpa zea. Only TMOF-K was found by MS/MS in the hemolymph for H. virescens fed on TMOF-K-PEG₇P. No TMOF, TMOF-K or PEGylated TMOF-K was detected in the hemolymph after topical applications. CONCLUSIONS: This research suggests that aliphatic PEG polymers can be used as a new method for increasing the activity of insecticidal proteins. Copyright © 2011 Society of Chemical Industry     

Non-target toxicology of a new mosquito larvicide, trypsin modulating oostatic factor

Trypsin modulating oostatic factor (TMOF), a peptide hormone originally isolated from the ovaries of adult Aedes aegypti, is currently under commercial development as a new pesticide chemistry with a novel mode of action for the control of larval mosquitoes. The objective of the current research is to evaluate potential risks of the use of TMOF as an insecticide on non-target organisms. TMOF (YDPAP₆) was degraded in vitro (as determined by HPLC and LC/MS) to DPAP₆, PAP₆, and then AP₆ by leucine aminopeptidase, a pancreatic enzyme found in the digestive system of vertebrates. The rate of degradation of TMOF and PAP₆ was significantly greater than that of DPAP₆, while no metabolism of AP₆ was found. TMOF technical insecticide was produced on a commercial scale by recombinant yeast (heat-killed before application). The technical TMOF when administered in a single dose by gavage to male and female mice at 2000 mg dry weight/kg body weight produced no negative effects as compared to controls up to 12 days after treatment. When male and female mallard ducks were treated by gavage with 1250 mg dry weight of technical TMOF/kg body weight each day for 5 days, again no toxic effects were noted through 35 days after the last treatment. TMOF technical insecticide was also applied to the shaved skin of male and female rabbits at the rate of 2000 mg/kg for 1-2 days, with no effect. The end point observations in these in vivo experiments were mortality; changes in growth rate, behavior, body structure, and color; and possible lesions observed during necropsy. Finally, Daphnia incubated with technical TMOF in rearing water at the level of 1.0 × 10⁶ yeast cells/ml (10 mg/ml) also demonstrated no negative effects on mortality, growth, molting, time to first brood, and production of viable neonates. It appears from these studies that TMOF can be degraded by vertebrate digestive proteases and technical TMOF is not toxic to the non-target organisms examined.     

Toxicity of novel aromatic and aliphatic organic acid and ester analogs of trypsin modulating oostatic factor to larvae of the northern house mosquito, Culex pipiens complex, and the tobacco hornworm, Manduca sexta

Eight non-peptidic chemical analogs of trypsin modulating oostatic factor (TMOF, NH₂-YDPAP₆), an insect hormone inhibiting trypsin biosynthesis in mosquitoes, were synthesized based on the structure of the native peptide. The median lethal concentration (LC₅₀) for the chemical analogs, TMOF and FDPAP (a peptidic analog of TMOF) was estimated for larvae of the northern house mosquito, the Culex pipiens complex, using a static 5-day bioassay. Four of these compounds demonstrated the same larvicidal activity as TMOF, while three of these compounds were 1.2-2.5-fold more active than TMOF. The compounds introduced by injection were toxic to fourth instars of the tobacco hornworm, Manduca sexta, except for TMOF, FDPAP, and PPHEN. Injection of TMOF and FDPAP into fourth stadium and TMOF into second stadium M. sexta had no effect on trypsin activity, growth, or mortality. Apparently the mosquito hormone is inactive in the tobacco hornworm at the developmental stages examined. Three TMOF analogs (CHEA, PHEA, and PHA) demonstrating the highest activity by injection in M. sexta were also found to be toxic by injection in fourth instars of the tobacco budworm, Heliothis virescens, and the cotton bollworm, Helicoverpa zea, as well as adult male German cockroaches, Blattela germanica. A two-choice feeding bioassay with H. virescens indicated that at least one of the TMOF analogs, PHEA, has anti-feeding properties.     

Inhibitory effect of the antifeeding compound AC-24,055 [4′-(3,3-dimethyl-1-triazeno) acetanilide] on digestive enzymes of Spodoptera littoralis larvae

The effect of the antifeeding compound AC-24,055 on the main digestive enzymes of Spodoptera littoralis was determined. The inhibitory effect of AC-24,055 on the amylolytic and proteolytic activity increased with concentration and was well correlated with the antifeedant activity. After 3-day feeding on sugar beet leaves treated with 0.1% AC-24,055, more than 90% suppression of both amylase and protease activity was obtained, whereas at some lower concentrations a slight increase in invertase activity, especially after prolonged feeding, was observed. At 0.1% AC-24,055 the inhibition of the proteolytic activity was considerably stronger than it was in starved larvae. These results suggest that the inhibitory effect on the digestive enzymes is due not only to starvation but also to the effect of the antifeedant. Protease and amylase inhibition was obtained also by injecting AC-24,055 into the larval hemolymph. Maximum inhibition of both amylase and protease, about 50% and 25%, respectively, was reached 2 days after an injection of 5 μg AC-24,055; it then decreased gradually and was abolished 4 days after injection.Adding massive doses of AC-24,055 to the enzyme reaction mixture did not affect the in vitro activity of protease, amylase, or invertase. This indicates that AC-24,055 affects the digestive enzymes indirectly, probably by inhibiting a physiological system affecting the production of these enzymes.     

The effect of insecticides on osmotic and lonic balance in the locust Schistocerca gregaria

Schistocerca poisoned with insecticides may lose more than 30% of their body weight and 60% of their hemolymph volume within 24 hr. In spite of this and the impairment of the nervous system, poisoned Schistocerca show a remarkable ability to control the osmotic concentration and ionic composition of the remaining hemolymph. Much of the water loss from poisoned locusts can be attributed to evaporation through the spiracles, due to the progressive failure of the animal to close the spiracular valves. Another significant factor is a massive transport of fluid across the gut wall into the fore and midgut. This fluid may be regurgitated causing a further loss in weight.     

Phenyltin compounds inhibit digestive enzymes of Tribolium confusum larvae

The potency of dietary phenyltin compounds in inhibiting the growth of first and fourth instar Tribolium confusum L. larvae and the gut proteolytic activity of fourth instar larvae decreases in the order of triphenyltin chloride (Ph₃SnCl) ? diphenyltin dichloride (Ph₂SnCl₂) ? phenyltin trichloride or tetraphenyltin. The growth retardation, which prolongs the larval stage without affecting pupation or emergence, may result from an antifeeding effect involving gut protease inhibition by Ph₃Sn⁺ and Ph₂Sn²⁺. Gut amylase and invertase activities are less sensitive than the protease activity to in vivo inhibition. Under in vitro conditions, relatively high concentrations of Ph₃SnCl and Ph₂SnCl₂ are required for inhibition, the order of enzyme sensitivity is protease > amylase > invertase, and Ph₂SnCl₂ is more potent than Ph₃SnCl. Proteins such as casein, albumin and hemoglobin, but not carbohydrates such as starch and sucrose bind Ph₃Sn⁺ so it is inaccessible for inhibition of digestive enzymes. The level of Ph₃Sn⁺ inhibiting gut protease in vivo is far below that necessary for in vitro inhibition of this enzyme activity. It is speculated that the in vivo inhibitory effects of Ph₃Sn⁺ and Ph₂Sn²⁺ on digestive enzymes may result from binding to the enzyme protein, its zymogen or to other proteins involved in production of the digestive enzymes.     

Dieldrin transport in the insect: an examination of Gerolt's hypothesis

[¹⁴C]-Dieldrin was detected in the hemolymph of adult male Periplaneta americana and P. brunnea 2.5 hr after an acetone solution of radiodieldrin (RD) was applied to the pronotum. Sephadex thin-layer gel filtration (TLG) of cell-free hemolymph (HL) from cockroaches to which RD was applied, and TLG of HL to which RD was added directly, indicated that dieldrin binds to a protein (s) of ca. 18,900 mol wt and two groups of proteins of mol wt ≥ 160,000. The capacity of Periplaneta HL for RD at 22°C was to at least 57 μM, but the highest concentration of RD detected in HL after topical application was 29.9 μM. The hemocytes contained about 37% of the dieldrin in whole cockroach hemolymph. When isolated cockroach legs were perfused with HL containing protein-bound RD, autoradiography of the dissected legs revealed ¹⁴C counts in the tissues. These findings are consistent with the translocation of topical dieldrin by hemolymph, but do not eliminate the possibility of translocation via the tracheal system.     

DDT-lipoprotein complex in the American cockroach hemolymph: A possible way of insecticide transport

The binding in vitro of ¹⁴C-DDT to proteins from the hemolymph of the American cockroach was shown by means of electrophoresis and isoelectric focusing on polyacrylamide gels. The study of ¹⁴C-DDT penetration into the insect implies the hemolymph as one of the possible manners by which this insecticide could penetrate the central nervous system, and other target organs. The possibility of DDT being transported by the hemolymph bound to lipoproteins is discussed.     

斜纹夜蛾四龄幼虫暴食相关基因的鉴定和通路分析

为绿色高效防治斜纹夜蛾Spodoptera litura,采用高通量测序技术对斜纹夜蛾暴食前期（3龄期）和暴食初始期（4龄期）进行转录组测序,筛选暴食前期和暴食初始期斜纹夜蛾差异基因,并对其高表达基因的代谢通路进行KEGG富集分析,随机选择缬氨酸、亮氨酸和异亮氨酸降解通路的5个关键基因对其表达量进行测定,以验证转录组测序结果的正确性。结果显示,从转录组数据中共拼接了17 045条unigenes,其中有876条是新发现的unigenes,16 625条unigenes有注释信息;5个基因的实时荧光定量PCR结果与转录组测序分析结果一致,表明转录组分析结果可信;在暴食初始期高表达的基因主要为血淋巴脂多糖结合蛋白基因,暴食初始期高表达基因富集度最大的通路主要为支链氨基酸代谢通路,其次是脂肪酸降解通路,表明暴食初始期斜纹夜蛾在增强其消化系统功能和营养吸收能力的同时,也加强了其能量代谢,为幼虫获取更多的食物提供动力。     

Tracing a 14C-labeled carbamate molluscicide through the digestive system of deroceras reticulatum (müller)

The passage of a ¹⁴C-labeled carbamate, 2-(2-chloro-1-methoxy-' ethoxy)phenyl N-methylcarbamate, or its labeled metabolites through the alimentary system of the grey garden slug Deroceras reticulatum was examined by autoradiographic studies and scintillation counting. It was demonstrated that, in a first step, the molluscicide penetrated the cells of the oesophagus and the crop. It was quickly transported by the hemolymph to the periphery of the body and re-entered the cells of the digestive tract and the mid-gut gland in a second step from the hemolymph side. The crypt cells of the mid-gut gland are discussed as cells involved in detoxification, and connective tissue cells as the major storage sites of the labeled material. Excretion in feces and secretion in mucus are thought to be the routes of ¹⁴C elimination.     

Mode of action of N-ethylmaleimide as a parathion synergist in Triatoma infestans

N-Ethylmaleimide (NEM) injected in sublethal doses enhanced the triatomicide effect of parathion. The study of the possible causes responsible for this synergistic effect in Triatoma infestans showed (1) a depletion of SH groups in the hemolymph and the central nervous system (CNS) caused by NEM; (2) protein and lipid covalent binding of [¹⁴C]NEM in the CNS; (3) partial inhibition by NEM of the glutathione S-transferase detoxifying pathway; (4) enhanced penetration of parathion and paraoxon into the insect CNS pretreated with NEM; (5) significant change in the electrophoretic and histochemical pattern of acetylcholinesterase inhibition in NEM-pretreated insects. The results obtained suggest that NEM synergizes parathion in adult T. infestans by inhibiting glutathione-dependent detoxification and enhancing penetration into the CNS.     

Toxicity nanoinsecticide based on clove essential oil against Tribolium castaneum (Herbst)

This study aimed to characterize nanoparticles loaded with clove (Syzygium aromaticum) essential oil-based polyethylene glycol (PEG) and to know their insecticidal activity against red flour beetle (Tribolium castaneum). The nanoparticles have irregular shapes in good dispersion. The nanoformulation could not enhance clove oil contact toxicity to T. castaneum, but could protect the oil from degradation and evaporation while simultaneously allowing sustained release, as indicated by the continued high toxicity for 16 weeks of storage.     

Physiological effects of a benzyl-1,3-benzodioxole chemosterilant on Sarcophaga bullata

Ovarian development of adult female Sarcophaga bullata was arrested by benzyl-1,3-benzodioxole (J2581) when fed in concentrations of 5–10 mg/g sugar. An accumulation of hemolymph protein, especially vitellogenin, was associated with the disruption of ovarian development, indicating that the benzyl-1,3-benzodioxole affects the sequestration of vitellogenin from the hemolymph into the ripening follicles. Exogenous supplementation with ecdysterone or juvenile hormone analog (ZR515) did not restore normal ovarian growth.     

Biological and biochemical responses of Biomphalaria alexandrina to some extracts of the plants Solanum siniacum and Artemisia judaica L.

The molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of Solanum siniacum and Artemisia judaica L. plants against Biomphalaria alexandrina snails was carried out. The tests revealed plant’s ethanol extract was more toxic to the snails than the other tested extracts. Therefore, it was tested against snails’ fecundity (M_x), reproduction rate (R₀) and their infection with Schistosoma mansoni miracidia. In addition, biochemical parameters and the activities of some enzymes in tissues of snails treated with the two tested plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of B. alexandrina snails to plant’s ethanol extract led to a significant reduction in their survival and snails’ fecundity, reproduction rate. In addition, it caused a considerable reduction in the infectivity of S. mansoni miracidia to the snails. Also, it caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results revealed that the glucose concentration in hemolymph and Lactate level in soft tissues of treated snails were increased (P < 0.001) while glycogen, total protein, the lipid content and the pyruvate level in snail’s tissues decreased (P < 0.001). The activities of lactic dehydrogenase (LDH), pyruvatekinase (PK) and cytochrome oxidase (CY) enzymes in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with the two tested plants while protease (PR) activity increased (P < 0.001). It is concluded that the application of LC₂₅ of ethanol extract of S. siniacum and A. judaica L. may be helpful in snail control as it interferes with the snails’ biology and physiology.     

Triphenyl derivatives of group IV elements as inhibitors of growth and digestive enzymes of Tribolium castaneum larvae

The potency of triphenyl derivatives of group IV elements in inhibiting the growth of first and fourth instar Tribolium castaneum larvae is much greater for Ph₃SnCl and Ph₃PbCl than for Ph₃GeCl, Ph₃SiOH, and Ph₃CCl. Pupated larvae emerge normally, showing that the pupal stage is not affected. The larval growth retardation may result from an antifeeding effect involving digestive enzyme inhibition. Ph₃SnCl and Ph₃PbCl at 500 μmol/kg of diet completely inhibit larval growth (fourth instar larvae) and provide a reduction of 56–59, 29–33, and 2–15% in the in vivo activity of invertase, amylase, and protease, respectively. Under these conditions Ph₃GeCl, Ph₃SiOH, and Ph₃CCl are essentially inactive. High concentrations (5 × 10^?4 and 2 × 10^?3M) of Ph₃SnCl and Ph₃GeCl acting in vitro strongly inhibit invertase, amylase, and protease activities, whereas Ph₃PbCl is moderately inhibitory and Ph₃SiOH and Ph₃CCl are inactive. When both in vivo and in vitro findings are considered, Ph₃SnCl is the most potent inhibitor of larval digestive enzymes.     

Pharmacokinetic behavior of enantiomeric pyrethroid esters in the cockroach,Periplaneta americana L

The potent pyrethroid insecticide NRDC 157 (I; 3-phenoxybenzyl (1R, cis)-3 - (2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate) and its insecticidally inactive 1S, cis enantiomer (II) have similar cuticular penetration rates and are detectable in the hemolymph, nerve cord, fat body, and midgut of the American cockroach, Periplaneta americana, within 2 hr of topical application at 0.17 μg/g, a just-lethal dose of I. At this dose, both enantiomers show similar distribution patterns in these tissues, but symptoms of intoxication are seen only with I. Steady state concentrations of both enantiomers in the hemolymph and nerve cord are between 1.2 × 10^?7 and 1.7 × 10^?7M. I and II are not detectably hydrolyzed by fat body, nerve cord, and hemolymph preparations and are not detectably oxidized by fat body preparations in vitro, but a very low oxidation rate is measured for II, but not I, in nerve cord preparations. These results do not demonstrate a pharmacokinetic basis for the difference in insecticidal activity between enantiomers and therefore it is likely that the site of pyrethroid action is stereospecific. The use of the inactive enantiomer II as a model to study the effects of dose on penetration and distribution in the absence of symptoms is explored.     

Triphenyl and tetraphenyl derivatives of group V elements as inhibitors of growth and digestive enzymes of Tribolium confusum and Tribolium castaneum larvae

Tetraphenylstibonium chloride (Ph₄SbCl) at a dietary level of 500–2000 μmol/kg inhibits larval growth, gut invertase activity, pupation, and emergence of Tribolium confusum and Tribolium castaneum. Tetraphenylphosphonium chloride (Ph₄PCl) in this concentration range inhibits growth and gut invertase activity without affecting pupation and emergence whereas tetraphenylarsonium chloride is essentially inactive in all respects. Triphenylstibine (Ph₃Sb) but not the other triphenyl derivatives (amine, phosphine, and arsine) inhibits both larval growth and gut digestive enzyme activities (invertase and protease) in vivo. Protease but not invertase is inhibited in vitro by Ph₄PCl and Ph₄SbCl at 2 × 10^?3M. Ph₄PCl appears to act as an antifeeding agent and Ph₄SbCl and Ph₃Sb as more general toxicants.     

Effects of pyriproxyfen, a juvenile hormone analog, on development of the mealworm, Tenebrio molitor

Pyriproxyfen, an analog of the juvenile hormone (JH) in insects, has been evaluated in vivo and in vitro on an important stored product pest, the mealworm Tenebrio molitor. On newly ecdysed pupae, this insect growth regulator (IGR) was applied topically (at 0.10 and 0.20 μg/insect). Enzyme immunoassay measurements showed a decrease of the ecdysteroid titer in the hemolymph of treated specimens. In addition, the treatment induced an increase of the protein concentration in the hemolymph, however, there was no significant effect on the protein band pattern. Using in vitro pupal integument explant cultures, pyriproxyfen (at 1 and 10 μM in the medium) caused a significant inhibition of ecdysteroid amounts and of the thickness of new cuticle secreted. These new results are discussed in relation to the physiology of this IGR with a JH action.     

Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH₄)₂SO₄ precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici (Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.     

Tissue analysis and hemolymph translocation of [14C]chlorpyrifos sorbed from treated surfaces by American cockroaches

Using radiotracer methodology and dissection techniques it was demonstrated that [¹⁴]chlorpyrifos and/or its ¹⁴C-labeled metabolite(s) concentrated mainly in the gut tissues and malpighian tubules of American cockroaches, Periplaneta americana (Linnaeus), following sorption from a treated surface. Significantly lower (P ≤ 0.10) amounts of ¹⁴C were present in testes samples and no radioactive material was detected in brain tissue. After 41.5–48 hr of exposure of adult male American cockroaches to sublethal concentrations of [¹⁴C]chlorpyrifos, radioactivity was detected in the hemolymph of all cockroaches tested. The hemolymph accounted for 30.83% of the total sorbed ¹⁴C. A parabiotic experiment confirmed translocation of chlorpyrifos and/or its ¹⁴C-labeled metabolite(s) in hemolymph.