Chitin Biosynthesis Inhibitors Research Articles

Buprofezin delayed the molting of Pardosa pseudoannulata, a predatory enemy for insect pests, by suppressing chitin synthase 1 expression

Spiders, the major predatory enemies of insect pests in fields, are vulnerable to insecticides. In this study, we observed that the recommended dose of buprofezin delayed the molting of the pond wolf spider Pardosa pseudoannulata, although it had no lethal effect on the spiders. Since buprofezin is an insect chitin biosynthesis inhibitor, we identified two chitin synthase genes (PpCHS1 and PpCHS2) in P. pseudoannulata. Tissue-specific expression profiling showed that PpCHS1 was most highly expressed in cuticle. In contrast, PpCHS2 showed highest mRNA levels in the midgut and fat body. RNAi knockdown of PpCHS1 significantly delayed the molting of 12-days old spiderlings, whereas no significant effect on the molting was observed in the PpCHS2-silencing spiderlings. The expression of PpCHS1 was significantly suppressed in the spiderlings treated with buprofezin, but rescued by exogenous ecdysteroid ponasterone A (PA). Consistent with this result, the molting delay caused by buprofezin was also rescued by PA. The results revealed that buprofezin delayed the molting of spiders by suppressing PpCHS1 expression, which will benefit the protection of P. pseudoannulate and related spider species.

Insect Growth Disruptors Cause Mouthpart Malformations, Inhibition of Feeding, and Mortality in the Neotropical Brown Stink Bug Euschistus heros (Hemiptera: Pentatomidae)

The Neotropical brown stink bug, Euschistus heros, is the most important sucking pest of soybean crops, quantitatively and qualitatively affecting grain and seed. This study aimed to determine mouthpart deformities, feeding inhibition, and E. heros nymphs’ mortality when in contact with insect growth disruptors (IGDs). The effects of two insecticides of the chitin biosynthesis inhibitor group (CBIs), lufenuron and diflubenzuron, and the juvenile hormone analog pyriproxyfen were tested in the laboratory. CBI application promoted mouthpart deformities, feeding inhibition, alteration of metamorphosis, and mortality of E. heros nymphs. Although pyriproxyfen did not cause mouthpart deformation, it caused certain mortality for nymphs, possibly due to physiological disorders. Lufenuron caused feed inhibition and mortality in less time when compared with diflubenzuron and pyriproxyfen. It was also more toxic to 4th instar nymphs. The insecticides, when used in higher concentrations, increased nymph’s mortality in less time. Nymphs with deformed mouthparts were unable to feed and died. All IGDs in the tested concentrations caused 100% mortality and prevented adult formation.

Influência da adição de adjuvantes à calda de pulverização de teflubenzuron em linhagens resistentes e suscetíveis da lagarta falsa-medideira Chrysodeixis includens em soja (Lepidoptera: Noctuidae)

ABSTRACT: The soybean looper (SBL), Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), is a soybean and cotton pest in South America countries. Field-evolved resistance of SBL to inhibitors of chitin biosynthesis has been reported in Brazil; however, this mode of action is still widely used against SBL. On this basis, we conducted laboratory bioassays to investigate if adjuvants (Nimbus®, TA 35®, Break-Thru® S 240, and Rizospray Extremo®) added to the teflubenzuron spray increase the mortality of SBL strains (resistant, heterozygous, and susceptible to chitin biosynthesis inhibitors). Using chromatography analysis, we also evaluated the amount of teflubenzuron on soybean leaves when applied alone or in combination with adjuvants. In laboratory bioassays, the biological activity of teflubenzuron increased against the susceptible SBL strain when adjuvants were added. In contrast, no relevant effects of adjuvants added to the teflubenzuron spray against heterozygous and resistant SBL larvae were detected. In leaf bioassays, even leaves from the upper third part of the plants containing a significantly higher amount of teflubenzuron (3.4 mg/kg vs 1.7 and 0.6 mg/kg); the mortality of SBL strains was similar when teflubenzuron was applied alone or in mixture with adjuvants. Our findings indicated that adjuvants added to teflubenzuron spray do not provide a substantial increase in the mortality of SBL strains resistant to chitin biosynthesis inhibitors. Therefore, it is necessary to reduce the use of this mode-of-action insecticide against SBL and to give preference to other insecticides or control tactic.

Functional characterization of chitin synthesis pathway genes, HaAGM and HaUAP, reveal their crucial roles in ecdysis and survival of Helicoverpa armigera (Hübner)

The chitin metabolic pathway is one of the most lucrative targets for designing pest management regimes. Inhibition of the chitin synthesis pathway causes detrimental effects on the normal growth and development of insects. Phospho-N-acetylglucosamine mutase (AGM) and UDP-N-acetylglucosamine pyrophosphorylase (UAP) are two key chitin biosynthesis enzymes in insects including Helicoverpa armigera, a pest of global significance. In the present study, we have identified, cloned and recombinantly expressed AGM and UAP from H. armigera (HaAGM and HaUAP). Biochemical characterization of recombinant HaAGM and HaUAP exhibited high affinities for their natural substrates N-acetyl glucosamine-6-phosphate (Km 38.72 ± 2.41) and N-acetyl glucosamine-1-phosphate (Km 3.66 ± 0.13), respectively. In the coupled enzyme-catalytic assay, HaAGM and HaUAP yielded the end-products, inorganic pyrophosphate and UDP-GlcNAc, confirming their active participation in the chitin synthesis pathway of H. armigera. Gene expression profiling revealed that HaAGM and HaUAP genes were expressed in all developmental stages and key tissues. These genes also showed substantial responses towards the moulting hormone 20-hydroxyecdysone and chitin biosynthesis inhibitor, novaluron. Remarkably, the RNAi-mediated knockdown of either HaAGM or HaUAP led to severe developmental deformities and significant mortality ranging from 65.61 to 72.54%. Overall findings suggest that HaAGM and HaUAP play crucial roles in the ecdysis and survival of H. armigera. Further, these genes could serve as potential targets for designing pest management strategies for H. armigera.

Toxicological, biochemical and ultrastructural alterations induced by chlorfluazuron and Bacillus thuringiensis (diple d.f.) on Pectinophora gossypiella (saunders) larvae

Effects of the lethal concentrations of Chlorfluazuron and Bacillus thuringiensis (Diple D.f.) against Pectinophora gossypiella (Saund.) larvae treated as neonate were studied recently. The obtained results indicated that, Chlorfluazuron treatment showed great toxicity compared to B. thuringiensis. The biochemical response of 10-days old larvae was studied by the evaluation of the total soluble protein, total lipids, and transaminases activities: Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST), and the carbohydrate hydrolyzing enzymes (trehalas, invertase and amylase). The total protein content decreased to 43.26mg/dl and 26.24mg/dl for chlorfluazuron and Bacillus thuringiensis treatments, respectively compared to untreated control (100.1 mg/dl). In contrast, the total lipid levels were increased for the same compounds, recording values: 132.73 and 87.87 mg/dl, respectively, compared to control (36.51 mg/dl). Moreover, a high increase in the activity of all Carbohydrate hydrolyzing enzymes induced by both treatments compared to control. Activity of AST enzyme was declined to 5.44 and 12.66 mg glucose released/gm body weight/min for Chlorfluazuron and B. thuringiensis, respectively, compared to control (14.62 mg glucose released/gm body weight/min). In contrary, a great increase in the ALT activity was recorded with values: 129.49 and 159.61 mg/dl for the same compounds, respectively compared to untreated check (85.36 mg/dl). Newly hatched larvae of P. gossypiella treated with Chlorfluazuron as one of chitin biosynthesis inhibitors showed some histopathological alternations of the cuticle layers. The exo-cuticle became thicker with variable staining affinity, while the endo-cuticle became thin and faintly stains. The epidermal layer was formed mostly with irregularly distributed cubical cells varied in height and staining affinity. Furthermore, histopathological alternations, of midgut were obtained for Bt treated larvae of P. gossypiella representing extensive damage of the epithelial columnar cells with increased cytoplasm vacuolation at the Bt toxins. In addition, ultrastructure studies conjugated with observations for both Chlorfluazuron and Bt treated and untreated PBW larvae were massive supported by transmission electron microscopy (TEM).

The Myosin Motor Domain-Containing Chitin Synthases Are Involved in Cell Wall Integrity and Sensitivity to Antifungal Proteins in Penicillium digitatum.

Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new antifungal molecules. Among these are the antifungal peptides and proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the antifungal proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to antifungal proteins in P. digitatum.

Sublethal Effects of Insect Growth Regulators on Boll Weevil (Coleoptera: Curculionidae).

Immature stages of the boll weevil, Anthonomus grandis grandis Boh. (Coleoptera: Curculionidae), develop protected inside cotton fruiting structures. Therefore, the adult beetles have become the main target of insecticide applications. The use of insect growth regulators (IGRs) is recommended against immatures, even though they may also affect the survival and reproductive traits of adult insects. The present study evaluated the impact of a juvenile hormone analog (pyriproxyfen), an ecdysteroid agonist (methoxyfenozide), and a chitin biosynthesis inhibitor (lufenuron) on adult cotton boll weevils, a key cotton pest. Mated and virgin beetles were treated by feeding them contaminated squares and cotton leaf discs that were previously immersed into pyriproxyfen, methoxyfenozide, and lufenuron solutions at field-rate concentrations. After exposure, treated couples were caged onto cotton plants, and survival, fecundity, and egg viability were evaluated. The IGRs neither affected the survival nor fecundity of adult boll weevils. On the other hand, egg viability was significantly reduced by lufenuron, regardless of whether the females were treated premating or postmating or whether their pairs were either treated or untreated. However, egg viability increased as the females aged since the initial exposure date to lufenuron, indicating a potential transovarial effect of this insecticide. Our results indicate that pyriproxyfen and methoxyfenozide do not affect adult boll weevils, whereas lufenuron temporarily reduces the egg viability of this key cotton pest.

Cell Wall-Modifying Antifungal Drugs.

Antifungal therapy is a critical component of patient management for invasive fungal diseases. Yet, therapeutic choices are limited as only a few drug classes are available to treat systemic disease, and some infecting strains are resistant to one or more drug classes. The ideal antifungal inhibits a fungal-specific essential target not present in human cells to avoid off-target toxicities. The fungal cell wall is an ideal drug target because its integrity is critical to cell survival and a majority of biosynthetic enzymes and wall components is unique to fungi. Among currently approved antifungal agents and those in clinical development, drugs targeting biosynthetic enzymes of the cell wall show safe and efficacious antifungal properties, which validates the cell wall as a target. The echinocandins, which inhibit β-1,3-glucan synthase, are recommended as first-line therapy for Candida infections. Newer cell wall-active drugs in clinical development encompass next-generation glucan synthase inhibitors including a novel echinocandin and an enfumafungin, an inhibitor of Gwt1, a key component of GPI anchor protein biosynthesis, and a classic inhibitor of chitin biosynthesis. As the cell wall is rich in potential drug discovery targets, it is primed to help deliver the next generation of antifungal drugs.

Chitin Biosynthesis Inhibitors in Euschistus heros Fabr. (Hemiptera: Pentatomidae): Morphometric Alterations in Testes and Nuclei of Testicular Accessory Cells of Adults

Euschistus heros is an important pest in Brazilian agriculture growing importance in the general Neotropical realm. Its reproductive potential is the key factor for its characterization as a pest in major crops such as soybean and cotton. The aims of this study were to characterize morphometric parameters of testicles and testicular accessory cells-TACs nuclei of adults E. heros treated with chitin biosynthesis inhibitors (CBIs). The insecticides lufenuron (Match® 50 EC) and buprofezin (Applaud® 250 WP) were applied individually in 4th instar nymphs, remained in controlled conditions until the emergence of adult males. The testicles were identified and removed 72 h after emergency, fixed, photographed for anatomic analysis, and processed for morphometric analyses of the TACs nuclei. It was possible to observe that lufenuron and buprofezin decreased the testicular area. Buprofezin decreased the mean nuclear area analyzed in the TACs, and nuclear hypertrophy can indicate an activity on support and nutrition of germ line cells, presenting a possible effect on protein synthesis. The intense reaction for Fast green in control compared to buprofezin treatment may indicate that total protein (histones and non-histones) has been altered. The tested insecticides, with special focus on buprofezin may affect the final stages of reproductive development of E. heros, with potential to be used in field to the control of this pest.

Performance of reproductive system of Dichelops melacanthus (Hemiptera: Pentatomidae) subjected to buprofezin and pyriproxyfen: morphological analysis of ovarioles and testes

The use of insecticides known as insect growth regulators, which are considered more selective to natural enemies, may be an alternative to integrated pest management of stink bugs of the main crops in Brazil. The objective of this study was to evaluate histological changes in the reproductive system of Dichelops melacanthus as well as female fecundity and egg fertility after use of growth-regulating insecticides. The insecticides used were buprofezin (a chitin biosynthesis inhibitor) at a sublethal concentration (LC30 of 2.99 g L-1) and pyriproxyfen (a juvenile hormone analog) at a sublethal concentration (LC30 of 8.35 mL L-1). A completely randomized experimental design was used, with 10 repetitions and 10 insects per experimental unit. Two bioassays were performed: in the first bioassay, fecundity (eggs/female) and fertility (nymph eclosion) of the insects that survived the insecticides were evaluated; in the second bioassay, morphological alterations of the ovarioles of adult females and of the testes of adult male insects that survived the insecticides were evaluated. Buprofezin and pyriproxyfen did not affect the adult sex ratio or female fecundity. Pyriproxyfen reduced the percentage of ecloded nymphs (71.6%) compared with that of the control and buprofezin (96.4 and 90.6%, respectively) treatments and had an ovicidal effect, with direct and indirect action on embryogenesis. Morphological changes were observed in both treatments with buprofezin and pyriproxyfen. The alterations observed in female and male reproductive systems may occur by the action of buprofezin and pyriproxyfen on the morphology of both ovarioles and testes.

Performance of reproductive system of Dichelops melacanthus (Hemiptera: Pentatomidae) subjected to buprofezin and pyriproxyfen: morphological analysis of ovarioles and testes

The use of insecticides known as insect growth regulators, which are considered more selective to natural enemies, may be an alternative to integrated pest management of stink bugs of the main crops in Brazil. The objective of this study was to evaluate histological changes in the reproductive system of Dichelops melacanthus as well as female fecundity and egg fertility after use of growth-regulating insecticides. The insecticides used were buprofezin (a chitin biosynthesis inhibitor) at a sublethal concentration (LC30 of 2.99 g L-1) and pyriproxyfen (a juvenile hormone analog) at a sublethal concentration (LC30 of 8.35 mL L-1). A completely randomized experimental design was used, with 10 repetitions and 10 insects per experimental unit. Two bioassays were performed: in the first bioassay, fecundity (eggs/female) and fertility (nymph eclosion) of the insects that survived the insecticides were evaluated; in the second bioassay, morphological alterations of the ovarioles of adult females and of the testes of adult male insects that survived the insecticides were evaluated. Buprofezin and pyriproxyfen did not affect the adult sex ratio or female fecundity. Pyriproxyfen reduced the percentage of ecloded nymphs (71.6%) compared with that of the control and buprofezin (96.4 and 90.6%, respectively) treatments and had an ovicidal effect, with direct and indirect action on embryogenesis. Morphological changes were observed in both treatments with buprofezin and pyriproxyfen. The alterations observed in female and male reproductive systems may occur by the action of buprofezin and pyriproxyfen on the morphology of both ovarioles and testes.

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis

Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.

Comparative transcriptome analysis of lufenuron-resistant and susceptible strains of Spodoptera frugiperda (Lepidoptera: Noctuidae).

BackgroundThe evolution of insecticide resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) has resulted in large economic losses and disturbances to the environment and agroecosystems. Resistance to lufenuron, a chitin biosynthesis inhibitor insecticide, was recently documented in Brazilian populations of S. frugiperda. Thus, we utilized large-scale cDNA sequencing (RNA-Seq analysis) to compare the pattern of gene expression between lufenuron-resistant (LUF-R) and susceptible (LUF-S) S. larvae in an attempt to identify the molecular basis behind the resistance mechanism(s) of S. frugiperda to this insecticide.ResultsA transcriptome was assembled using approximately 19.6 million 100 bp-long single-end reads, which generated 18,506 transcripts with a N50 of 996 bp. A search against the NCBI non-redundant database generated 51.1 % (9,457) functionally annotated transcripts. A large portion of the alignments were homologous to insects, with the majority (45 %) being similar to sequences of Bombyx mori (Lepidoptera: Bombycidae). Moreover, 10 % of the alignments were similar to sequences of various species of Spodoptera (Lepidoptera: Noctuidae), with 3 % of them being similar to sequences of S. frugiperda. A comparative analysis of the gene expression between LUF-R and LUF-S S. frugiperda larvae identified 940 differentially expressed transcripts (p ≤ 0.05, t-test; fold change ≥ 4). Six of them were associated with cuticle metabolism. Of those, four were overexpressed in LUF-R larvae. The machinery involved with the detoxification process was represented by 35 differentially expressed transcripts; 24 of them belonging to P450 monooxygenases, four to glutathione-S-transferases, six to carboxylases and one to sulfotransferases. RNA-Seq analysis was validated for a number of selected candidate transcripts by using quantitative real time PCR (qPCR).ConclusionsThe gene expression profile of LUF-R larvae of S. frugiperda differs from LUF-S larvae. In general, gene expression is much higher in resistant larvae when compared to the susceptible ones, particularly for those genes involved with pathways for xenobiotic detoxification, mainly represented by P450 monooxygenases transcripts. Our data indicate that enzymes involved with the detoxification process, and mostly the P450, are one of the resistance mechanisms employed by the LUF-R S. frugiperda larvae against lufenuron.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2183-z) contains supplementary material, which is available to authorized users.

Insecticide Rotations for the Management of Lepidopteran Pests in Cabbage and Collards

Insecticide rotations for the management of the diamondback moth, Plutella xylostella (L), and other lepidopteran pests of cabbage and collard crops were evaluated in 2010, 2011, and 2012 at Tifton, GA, with the intent of identifying mode of action rotations equal in efficacy to single insecticide mode of action treatments. This was to demonstrate how insecticide rotations, which are purported to aid in the prevention of insecticide resistance, can be an economically-viable insect control option in Brassica crops. The insecticides and mode of actions tested included: chlorantraniliprole and flubendiamide (ryanodine receptor modulators), spinetoram (nicotinic acetylcholine receptor allosteric activator), indoxacarb (voltage-dependent sodium channel blocker), Bacillus thuringiensis subsp. aizawai (microbial disrupter of insect midgut membranes), novaluron (inhibitor of chitin biosynthesis) and zeta-cypermethrin (sodium channel modulator). The results demonstrated that all rotations were equally effective in the control lepidoteran pests in Brassica crops to the use of single insecticide treatments and that at least one rotation treatment provided the highest marketable yield each year.

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

Effects of the Insect Growth Regulators Novaluron and Diflubenzuron on the Brown Marmorated Stink Bug

The brown marmorated stink bug (BMSB), Halyomorpha halys (Stål), is an invasive pest from China that causes economic damage to field crops, vegetables, and tree fruit. Due to its destructive potential, applications of broad-spectrum insecticides have escalated. Researchers are trying to identify options for controlling BMSB that have less of a negative impact on non-target species. Chitin biosynthesis inhibitors are more selective than the commonly used pyrethroids and organophosphates. They are active on the larval stage of the insect and are reported as having sublethal effects such as reducing adult fecundity. In our studies, bioassays were completed with chitin biosynthesis inhibitors novaluron and diflubenzuron to evaluate the effectiveness of these insecticides on adult mortality, nymphal growth, adult fecundity, and egg hatch. Our data indicate that treatments of novaluron at 362.2 g ai/ha or diflubenzuron at 280.2 g ai/ha effectively controlled BMSB nymphs. However, the insecticides were not effective at reducing egg hatch, adult fecundity, or adult life span. If novaluron or diflubenzuron are used in agriculture for BMSB control, then the nymphal stage should be targeted. Accepted for publication 9 November 2012. Published 12 December 2012.

Functional analysis of a Hansenula polymorpha MNN2-2 homologue encoding a putative UDP-N-acetylglucosamine transporter localized in the endoplasmic reticulum

The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.

Synthesis and Biological Properties of Dihydro-Oxadiazine-Based Heterocyclic Derivatives

Dihydro-oxadiazine and its derivatives have been demonstrated to be important heterocyclic scaffold platform with bioactive diversity, which present wide activities such as cardiovascular, antitumor, antibacterial, antimicrobial, acricidal, insecticidal, plant-growth regulating, chitin biosynthesis inhibitors and monoamine oxidase inhibition. Versatile features of dihydro-oxadiazine heterocycles have emerged, so the aim of the present paper was to review the recent advances of dihydro-oxadiazine-based heterocyclic derivatives mainly including synthesis and biological activities.

Diacylhydrazine derivatives as novel potential chitin biosynthesis inhibitors: Design, synthesis, and structure–activity relationship

A series of diacylhydrazine derivatives containing hydrophobic alkyl chains have been designed and synthesized. The target molecules have been identified on the basis of analytical spectral (IR, 1H NMR, 13C NMR, and HRMS) data. All synthesized compounds have been screened for their potential inhibition in vitro against chitin synthesis using yeast cell extracts. The preliminary assays indicate that some of the compounds display moderate to good inhibitory activity. Structure–activity relationship (SAR) is also discussed based on the experimental data, and the further analysis of the quantitative structure–activity relationship (QSAR) indicates that the electronic parameter is the main factor to affect inhibition activities.

Mode of action of etoxazole

The mode of action of the 2,4-diphenyl-1,3-oxazoline acaricide/insecticide etoxazole has been argued to be moulting inhibition, but experimental results supporting this hypothesis are lacking. This study investigated the effect of etoxazole on chitin biosynthesis in the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Etoxazole induced moulting defects in fall armyworm larvae similar, if not identical, to those caused by benzoylphenylureas, a well-known class of insecticidal chitin biosynthesis inhibitors. Furthermore, in contrast to untreated larvae, the chitin content in the integuments of larvae several days after treatment did not differ from that in freshly ecdysed individuals, thus suggesting strong chitin biosynthesis inhibition in vivo. A more detailed investigation of the inhibitory potential by incubating cultured integument pieces from larvae of S. frugiperda with [14C]N-acetyl-D-glucosamine, a radiolabelled chitin precursor, revealed I50 values of 2.95 and 0.071 microM for etoxazole and triflumuron respectively. The incorporation of radiolabel into potassium hydroxide-resistant material was inhibited by etoxazole in a dose-dependent manner. Based on these results, it is concluded that the acaricidal and insecticidal mode of action of etoxazole is chitin biosynthesis inhibition.