Action of juvenoids on the metamorphosis of cyclorrhaphous Diptera

Administrations of high doses of juvenoids to the last instar larvae of cyclorrhaphous flies cause occasionally lethal defects in puparium formation but mostly affect only the pupal-adult transformation. In pupae, juvenoids impede proliferation and differentiation of the imaginal disks and of abdominal histoblasts: at low doses they cause incomplete rotation of male genitalia and deformations of the ovipositor, at higher doses their effects gradually spread from the tip of abdomen towards the middle of the body. The highest amounts influence the entire abdomen, size and pigmentation of the eyes, and development of hairs and sclerotization of the integument on the head and thorax. Various species slightly differ in the pattern of morphological effects produced, in the ability of affected insects to leave the puparium, and in the sensitivity to juvenoids of different types. A uniform scale for classification of morphological effects in the species examined is described in this paper. The most potent juvenoids are effective at doses around one nanogramme per specimen. Out of 29 selected compounds tested, isopropyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate is the most active juvenoid for Drosophila, Musca, and Sarcophaga; methyl 10,11-epoxy-3,7,11-trimethyl-2,6-tridecadienoate is the most active juvenoid for Ceratilis; and isopropyl 11-chloro-3,7,11-trimethyl-2-dodecenoate possesses the highest activity for Calliphora.     

Steroid conjugates: Synthesis and preliminary biological testing of pro-juvenoids

A series of 10 new pro-juvenoids (juvenogens, insect hormonogenic compounds, pro-drug-like agents) was synthesized using isomeric synthetic juvenoids (insect juvenile hormone analogs) and steroid molecules as patterns modifying parts of the complex hormonogenic molecules. In addition, several new synthons were prepared, which were required by the designed synthetic protocol to achieve the target molecules. These pro-juvenoids were subjected to the topical screening tests and to the drinking assays on the red firebug (Pyrrhocoris apterus), a convenient model laboratory phytophagous insect. Simple and efficient synthetic procedures for the preparation of the target pro-juvenoids and their synthons are presented. Furthermore, the biological activity of the pro-juvenoids in comparison with the activity of their parent juvenoids and that of several commercially available agents is demonstrated. Juvenoids and pro-juvenoids may replace toxic insecticides persistent in the insect pest control because they have no adverse effects on non-target organisms and/or human.     

Functional interpretation of APN receptor from M.sexta using a molecular model

Insect pests are the major cause of damage to commercially important agricultural crops. The continuous application of synthetic pesticides resulted in severe insect resistance by plants. This causes irreversible damage to the environment. Bacillus thuringiensis (Bt) emerged as a valuable biological alternative in pest control. However, insect resistance against Bt has been reported in many cases. Insects develop resistance to insecticides through mechanisms that reduce the binding of toxins to gut receptors. Nonetheless, the molecular mechanism of insect resistance is not fully understood. Therefore, it is important to study the mechanism of toxin resistance by analyzing amino‐peptidase‐N (APN) receptor of the insect M. sexta. A homology model of APN was constructed using Insight II molecular modeling software and the model was further evaluated using the PROCHECK program. Oligosaccharides participating in post translational modification were constructed and docked onto specific APN functional sites. Post analyses of the APN model provide insights on the functional properties of APN towards the understanding of receptor and toxin interactions. We also discuss the predicted binding sites for ligands, metals and Bt toxins in M. sexta APN receptor. These data help in the development of a roadmap for the design and synthesis of novel insect resistant Cry toxins.     

Cross communication between signaling pathways: juvenoid hormones modulate ecdysteroid activity in a crustacean

Methyl farnesoate is a juvenoid hormone that regulates a variety of processes in crustaceans including male sex determination among daphnids (Branchiopoda, Cladocera). The synthetic juvenoids pyriproxyfen and fenoxycarb mimic the action of methyl farnesoate in daphnids. In the present study we tested the hypothesis that juvenoids also can regulate ecdysteroid activity in a crustacean (Daphnia magna). Methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. Exposure of ecdysteroid-responsive cells to 20-hydroxyecdysone reduced cell proliferation and increased mRNA levels of the ecdysone receptor and its partner protein ultraspiracle. Co-treatment of cells with the juvenoid pyriproxyfen attenuated all of these ecdysteroid mediated responses. While juvenoids functioned as anti-ecdysteroids in both intact embryos and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. The combined effects of pyroproxyfen with the ecdysteroid synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone were evaluated in an effort to discern whether the action of the juvenoids were additive with those of know anti-ecdysteroids. The anti-ecdysteroid effects of pyriproxyfen were non-additive with those of either anti-ecdysteroid. Rather, joint effects conformed to a model of synergy. These results demonstrated that juvenoids elicit anti-ecdysteroidal activity in a crustacean through a unique mechanism of action. A model involving receptor partner deprivation is proposed that explains the synergistic interactions observed.     

Competitive antagonism of insect GABA receptors by 4-substituted 5-(4-piperidyl)-3-isothiazolols

γ-Aminobutyric acid (GABA) receptors are important targets of parasiticides/insecticides. Several 4-substituted analogs of the partial GABA_A receptor agonist 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) were synthesized and examined for their antagonism of insect GABA receptors expressed in Drosophila S2 cells or Xenopus oocytes. Thio-4-PIOL showed weak antagonism of three insect GABA receptors. The antagonistic activity of Thio-4-PIOL was enhanced by introducing bicyclic aromatic substituents into the 4-position of the isothiazole ring. The 2-naphthyl and the 3-biphenylyl analogs displayed antagonist potencies with half maximal inhibitory concentrations in the low micromolar range. The 2-naphthyl analog induced a parallel rightward shift of the GABA concentration–response curve, suggesting competitive antagonism by these analogs. Both compounds exhibited weak insecticidal activities against houseflies. Thus, the orthosteric site of insect GABA receptors might be a potential target site of insecticides.     

Effects of the anti hormone-hormone mimic ETB on the induction of insect juvenile hormone esterase in Trichoplusia ni.

Juvenile hormone (JH) esterases can be artificially induced to appear in the hemolymph of last instar larvae of the lepidopterous insect $\text{Trichoplusia}$$\text{ni}$ (Noctuidae) by topical treatment with JH I, JH II, or dihomo branched juvenoids. ETB (ethyl-4-[2-(t-butylcarbonyloxy) butoxy] benzoate; ZR-2646) at high doses is a weak inducer of JH esterase (JHE). However, at doses of ETB that induce only low levels of JHE activity, ETB will block the JHE induction caused by the dihomo juvenoid epofenonane and at higher doses will reduce the induction caused by JH I or JH II. ETB is not a JHE inhibitor; rather, it appears to be acting as a JH agonist/antagonist in normal larvae and in isolated abdomens. These effects of ETB on JHE induction may illustrate a new mode of action of anti-JH's.     

In vivo evaluation of the toxic activity and genotoxicity of the Hymenaea courbaril L.’s resin in Drosophila melanogaster

Due to the negative consequences carried by the usage of synthetic insecticides, a global interest into finding substitutes for these chemical compounds through natural products has arisen. When yielded to external attacks, plants generally produce metabolites to defend themselves. The physicochemical characteristics of this kind of compounds have allowed their usage as potential bioinsecticides. The Hymenaea courbaril L. (algarrobo) has proven to be a plant rich in metabolites with outstanding biological activity, in such a way that some of its extracts have been tested as insecticides. The goal of this study was to know the phytochemical composition of Hymenaea courbaril L.’s resin and perform evaluations in vivo of its toxic and genotoxic effects in the biological model Drosophila melanogaster. For this, two resin extracts were prepared and both a phytochemical analysis were carried out on them, having found in the ethanolic total extract the presence of terpenes, flavonoids and coumarins, while in the partial ethanolic extract only presence of terpenes and flavonoids was found. Drosophila larvae were submitted to different concentrations of the extracts and both the survival and the sexual ratio were evaluated, finding that larvae are more sensitive to the partial ethanolic extract. Subsequently, the induction of somatic mutation and mitotic recombination (SMART) was evaluated in the flies’ eyes. The most significant affectations at a genotoxic level were found when larvae were tested with the partial extract, indicating that possibly the coumarins absence makes this insect more susceptible to damages at a genetic material level.     

Action of ecdysoids,juvenoids, and non-hormonal agents on termination of pupal diapause in the flesh fly

Pupal diapause in Sarcophaga crassipalpis can be immediately terminated with high doses of ecdysterone or 5,β-hydroxyecdysterone. Within a range of lower doses diapause is not immediately terminated, but the duration of diapause is shortened. Injection of small doses at 3-day intervals is more effective than a single application of the same total amount. 5,β-hydroxy-ecdysterone is more effective than ecdysterone. The juvenoid isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate does not cause immediate termination of diapause but diapause is shortened. Simultaneous application of ecdysterone and the juvenoid synergistically produce a shorter duration of diapause than either hormone applied separately. Topical application of acetone and several other organic compounds can cause an immediate break of diapause. Neckligated pupae, however, do not respond to acetone treatment. Temperature shocks caused by brief exposure to high or low temperatures are ineffective.Although high doses of ecdysoids cause immediate termination of diapause, subsequent adult morphogenesis is protracted in direct proportion to the dose of ecdysoid. With the highest doses many flies fail to complete adult morphogenesis. Flies receiving high doses of ecdysoid are characterized by developmental abnormalities such as underdeveloped antennae, compound eyes, and mouthparts. By contrast, adultoids produced with juvenoids are characterized by abdominal abnormalities.     

Insect growth regulatory and larvicidal activity of chalcones against Aedes albopictus

Widespread use of chemical insecticides has resulted in development of insect resistance and natural products with biological activities could become an attractive alternative to control insect pests. In order to find more effective insecticides for controlling mosquito, various mosquitocidal compounds are studied. Recently, juvenile hormone antagonists (JHANs) have been found to be to safe and effective insecticides for control of mosquito. In order to identify novel insecticidal compounds with JHAN activity, several chalcones were surveyed on their JHAN activities and larvicidal activities against Aedes albopictus larvae. Among them, 2′‐hydroxychalcone and cardamonin showed high levels of JHAN and mosquito larvicidal activities. These results suggested that chalcones with JHAN activity could be useful for control of mosquito larvae.     

Isolation, primary culture and morphological characterization of oenocytes from Aedes aegypti pupae

Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.     

Insect growth inhibition by tocotrienols and hydroquinones from Roldana barba-johannis

The methanol extract from the aerial parts of Roldana barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and their corresponding acetylated and methylated derivatives showed insecticidal and insect growth regulatory activities against the Fall Armyworm [Spodoptera frugiperda J.E. Smith, (Lepidoptera: Noctuidae)], an important insect pest of corn. The most active compounds were sargachromenol and its acetylated derivative; sargahydroquinoic acid and its acetylated derivative; and a mixture of sargachromenol, sargahydroquinoic acid, and sargaquinoic acid (6:3:1) and the acetylated form of this mixture. All these compounds and mixtures had significant inhibitory effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. These substances may be useful as natural insecticidal agents.     

Glycosidic juvenogens: derivatives bearing alpha,beta-unsaturated ester functionalities

A series of the protected alkyl glycosides 5a/5b-12a/12b was synthesized from the parent isomeric alcohols (insect juvenile hormone bioanalogs; juvenoids), 4-[4'-(2'-hydroxycyclohexyl)methylphenoxy]-3-methyl-but-2-enoic acid ethyl ester (1a/1b-4a/4b; racemic structures) and (1a-4a; enantiopure structures). Cadmium carbonate was used as a promoter of this Koenigs-Knorr reaction, and the products were obtained in 82-92% yields. Deprotection of the carbohydrate functionality of 5a/5b-12a/12b was carefully performed using ethanolysis in the presence of zinc acetate, due to the presence of another ester functionality in the aglycone part of the molecule of protected alkyl glycosides. Resulting alkyl glycosides 13a/13b-20a/20b (diastereoisomeric mixtures) and 13a-20a (enantiopure compounds), biochemically activated hormonogenic compounds (juvenogens), were obtained in 82-93% yields. Finally, chiral HPLC separation of the diastereoisomeric mixtures of alkyl glycosides was applied to get sufficient quantities of the respective enantiomers 13b-20b of the alkyl glycosides for their structure elucidation and (13)C chemical shift assignment by (1)H and (13)C NMR spectroscopy. Partial introductory entomological screening tests of the target alkyl glycosides 13a/13b-20a/20b were performed on the red firebug (Pyrrhocoris apterus). The results of this biological testing clearly demonstrated the time-extended effect of several juvenogens on P. apterus due to their biochemical activation, i.e., hydrolysis of the juvenogen molecule, which results in liberation of the biologically active juvenoid in the insect organism.     

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis,biological activity,and molecular docking studies

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC₅₀ values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC₅₀ values of 2.0 and 0.9?μM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100?μg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.     

Discovery,synthesis, and evaluation of N-substituted amino-2(5H)-oxazolones as novel insecticides activating nicotinic acetylcholine receptors

N-Substituted amino-2(5H)-oxazolones A are a novel class of insecticides acting as nicotinic acetylcholine receptor (nAChR) agonists and show potent activity against hemipteran insect species. Here we report the discovery and preparation of this class of chemistry. Our efforts in SAR elucidation, biological activity evaluation, as well as mode-of-action studies are also presented.     

Enzymatic properties of α-amylase in the midgut and the salivary glands of mulberry moth, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

The pyralid moth, Glyphode pyloalis Walker, is an important pest of the mulberry. Amylases are the hydrolytic enzymes that catalyze the hydrolysis of the α-D-(1,4)-glucan linkage in glycogen and other related carbohydrates. Laboratory-reared fifth stadium larvae were randomly selected; the midgut (MG) and the salivary glands (SG) were removed by dissection under a dissecting microscope and α-amylase activity was assayed using the dinitrosalicylic acid procedure. The activity of α-amylase in the MG and the SG were 0.011 and 0.0018 μmol/min, respectively. The optimal pH and temperature for α-amylase were 9 for MG at 37–40 °C and 10 for SG at 37 °C respectively. Various concentrations of compounds (NaCl, KCl, MgCl₂, Urea, EDTA, SDS and CaCl₂) had differential effects on the enzyme activity. Plant amylase inhibitors may play an important role against insect pests. Hence, the characterization of digestive enzymes and the examination of their inhibitors may be a useful tool in future management of this important mulberry pest.     

昆虫碱性磷酸酶的研究进展

碱性磷酸酶存在于昆虫的头、唾液腺（唾液）、肠道、马氏管、表皮、血淋巴、脂肪体、生殖系统、附肢等部位,广泛参与了昆虫的发育、神经传导、激素合成、物质代谢、滞育、社会型昆虫亚种形成等过程。同时碱性磷酸酶与昆虫抗性有关,特别涉及到对Bt制剂的阻滞作用,其本身也是某些农药的靶标酶,某些生物源化合物及病毒、真菌也可以影响其活性。昆虫碱性磷酸酶的研究,将有助于提高对昆虫生化机制及代谢过程的认识,并为害虫治理和资源昆虫饲养提供新的思路。本文综述了国内外对昆虫碱性磷酸酶的研究状况,并描述了昆虫碱性磷酸酶的生化性质及其与生理功能的关系。     

Cyanogenic glucosides in the biological warfare between plants and insects: the Burnet moth-Birdsfoot trefoil model system

Cyanogenic glucosides are important components of plant defense against generalist herbivores due to their bitter taste and the release of toxic hydrogen cyanide upon tissue disruption. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their own predator defense. Burnet moths (Zygaena) sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) and, in parallel, are able to carry out de novo synthesis of the very same compounds. The ratio and content of cyanogenic glucosides is tightly regulated in the different stages of the Zygaena filipendulae lifecycle and the compounds play several important roles in addition to defense. The transfer of a nuptial gift of cyanogenic glucosides during mating of Zygaena has been demonstrated as well as the possible involvement of hydrogen cyanide in male assessment and nitrogen metabolism. As the capacity to de novo synthesize cyanogenic glucosides was developed independently in plants and insects, the great similarities of the pathways between the two kingdoms indicate that cyanogenic glucosides are produced according to a universal route providing recruitment of the enzymes required. Pyrosequencing of Z. filipendulae larvae de novo synthesizing cyanogenic glucosides served to provide a set of good candidate genes, and demonstrated that the genes encoding the pathway in plants and Z. filipendulae are not closely related phylogenetically. Identification of insect genes involved in the biosynthesis and turn-over of cyanogenic glucosides will provide new insights into biological warfare as a determinant of co-evolution between plants and insects.     

Synthesis of Novel (9S)‐Acyloxy Derivatives of Quinidine and Dihydroquinidine as Insecticidal Agents

Endeavor to discover biorational natural products‐based insecticides, two series (30) of novel (9S)‐acyloxy derivatives of quinidine and dihydroquinidine were prepared and assessed for their insecticidal activity against Mythimna separata in vivo by the leaf‐dipping method at 1 mg/mL. Among all the compounds, especially four derivatives exhibited the best insecticidal activity with final mortality rates of 71.4 %, 75.0 %, 71.4 %, and 75.0 %, respectively. Relatively speaking, 9‐hydroxy group is well tolerated, and the results showed that after modification of the hydroxy group with an acyloxy group, the insecticidal activity was significantly increased; the configuration at C8/9 position is important for insecticidal activity, and the (9S)‐configuration is optimal; modification of the out‐ring double bond is acceptable, and hydrogenation of the double bond enhances insecticidal activity. These preliminary results will pave the way for further modification of quinidine in the development of potential new insecticides.     

Screening of insecticidal activity of plant essential oils and extract-based formulations against four agricultural insect pests and their risk assessment

Frankliniella occidentalis (Pergande), Myzus persicae (Sulzer), Plutella xylostella (L), and Spodoptera exigua (Hübner) are notorious agricultural insect pests worldwide. Due to the rapid development of resistance to the conventional insecticides, the number of available insecticides might be reduced in a near future, and botanical insecticides can be considered as one of the potential candidates to solve this problem. In the present study, test formulations were prepared using plant essential oils and extracts as main active ingredients, and their insecticidal activity and phytotoxicity were examined. Among the combinations tested, two oil-in-water formulations of NRS-13 containing thyme white oil and NRS-24 containing basil and rosemary oils were selected based on their notable insecticidal activity and low phytotoxicity. Chemical analyses on the active essential oils, the quantitative analysis of the major components in the formulations, insecticidal activities under pot and field conditions, their acute toxicity on mammals, and ecotoxicity on fish were also examined. The NRS-13 (BaechooSafer^TM) and NRS-24 (JindiOut^TM) formulations showed species-specific insecticidal activity against P. xylostella and M. persicae, respectively. In addition, both formulations produced low acute oral and dermal toxicity, skin and eye irritation, and fish toxicities, which suggests those formulations based on essential oils could be considered as insecticides for pest control in small-scale environments in organic farming.