Significance of the sulfonylurea receptor (SUR) as the target of diflubenzuron in chitin synthesis inhibition in Drosophila melanogaster and Blattella germanica

Diflubenzuron (DIMILIN) is a powerful insecticidal chemical which has been known for many years to inhibit chitin synthesis in vivo in insects and related arthropod species. However, its action mechanism has remained unresolved partly because of its inaction on any of the enzymes involved in chitin synthesis in vitro. Based on our previous work (Diflubenzuron affects gamma-thioGTP stimulated Ca2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L. Insect Biochem. Mol. Biol. 24 (1994) 1009) showing that diflubenzuron inhibits Ca2+ uptake by vesicles obtained from the integument of American cockroach, Periplaneta americana (L.), in vitro, we tested the hypothesis that the action site of diflubenzuron is an ABC (ATP binding cassette) transporter, probably a sulfonylurea-sensitive transporter. Glibenclamide, one of the most commonly used sulfonylureas for type II diabetes treatment, was the positive control. When given to immature insects, glibenclamide clearly caused toxicity, with symptoms indicating molting abnormality comparable to diflubenzuron. Its LD50 (0.472 microg/nymph) was approximately 2.8 times the value obtained for diflubenzuron (0.17 microg/nymph, topical) in German cockroach, Blattella germanica (L.). However, in terms of the inhibitory activities on chitin synthesis, in isolated integuments glibenclamide showed an identical potency to diflubenzuron in B. germanica nymphs. A competitive binding assay with [3H]-glibenclamide and unlabeled diflubenzuron clearly established that the latter is capable of competitively displacing the former radioligand. The KD values observed for vesicles prepared from fruit fly larvae, Drosophila melanogaster M., were 44.9 nM for glibenclamide and 65.0 nM for diflubenzuron, respectively. Furthermore, glibenclamide was found to affect Ca2+ uptake by isolated cuticular vesicles from B. germanica in a manner very similar to diflubenzuron. These results support our conclusion that the sulfonylurea receptor (SUR) is the target of diflubenzuron in inhibition of chitin synthesis in these two insect species.     

氟虫脲对东亚飞蝗和中华稻蝗几丁质合成酶基因表达的影响

为了探讨氟虫脲可能的作用靶标及毒性机制, 本研究以重要农业害虫东亚飞蝗Locusta migratoria manilensis (Meyen)和中华稻蝗Oxya chinensis (Thunberg)为材料, 采用简并引物扩增中华稻蝗几丁质合成酶1基因（OcCHS1）的部分cDNA序列; 以氟虫脲浸渍法处理2龄中期中华稻蝗及1, 2和3龄东亚飞蝗若虫为处理组, 丙酮处理为对照组, 使用RT-PCR和实时荧光定量PCR方法分析氟虫脲对蝗虫几丁质合成酶基因mRNA表达的影响。结果获得的OcCHS1部分cDNA序列, 其长度为312 bp, 编码104个氨基酸, GenBank登录号为HM214491, 与东亚飞蝗几丁质合成酶1基因（LmCHS1）在氨基酸水平上相似度达95％。RT-PCR结果显示, 处理组几丁质合成酶1扩增带均强于对照组。实时荧光定量PCR结果表明: 与对照组相比, 处理组中华稻蝗2龄中期若虫OcCHS1 mRNA表达提高了1.02倍, 东亚飞蝗1, 2, 3龄若虫LmCHS1 mRNA表达分别提高了34%, 82%和89%, 差异显著（P<0.05）。分析基因表达提高的原因是几丁质合成受阻后基因表达水平的一种代偿性增加, 由此推测几丁质合成酶可能是氟虫脲作用的靶标之一。     

Insect chitin synthase cDNA sequence, gene organization and expression.

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.     

Cloning and characterization of a chitin synthase cDNA from the mosquito Aedes aegypti

Characterization of the enzymes involved in the chitin biosynthetic pathway in mosquitoes is critical due to the importance of chitin in the formation of the peritrophic matrix [PM] and its potential impact on vector competence. Chitin is the homopolymer of the amino sugar N-acetyl-D glucosamine [GlcNAc]. The final step of incorporation of GlcNAc into the chitin polymer is catalyzed by the enzyme chitin synthase [CS]. CS is a membrane bound enzyme, but the mechanism of its action in the biosynthesis of the PM is not understood. We have isolated and sequenced a CS-encoding cDNA clone from the mosquito Aedes aegypti, compared its sequence with CS from other organisms and studied its RNA expression. The cDNA is 3.5 kb in length with an open reading frame of 2.6 kb that encodes a protein of 865 amino acids with a predicted molecular mass of 99.5 kDa. The putative translation product shares 90% similarity to two CS proteins from Caenorhabditis elegans and 50% similarity to Saccharomyces cerevisiae in the catalytic domain of CS enzymes. Data suggest that CS is a single copy gene. RT-PCR analysis shows CS message in whole non-blood-fed females, whole blood-fed females, non-blood-fed midguts and in midguts dissected at different time points post-blood-feeding. In situ hybridization studies of midgut samples revealed that CS mRNA increases following a bloodmeal and is localized to the periphery of the epithelial cells facing the midgut lumen.     

Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.     

Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.     

Two chitin synthases in Saccharomyces cerevisiae

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Regulation of chitin synthesis in the larval midgut of Manduca sexta

In insects, chitin is not only synthesized by ectodermal cells that form chitinous cuticles, but also by endodermal cells of the midgut that secrete a chitinous peritrophic matrix. Using anti-chitin synthase (CHS) antibodies, we previously demonstrated that in the midgut of Manduca sexta, CHS is expressed by two cell types, tracheal cells forming a basal tracheal network and columnar cells forming the apical brush border [Zimoch and Merzendorfer, 2002, Cell Tissue Res. 308, 287-297]. Now, we show that two different genes, MsCHS1 and MsCHS2, encode CHSs of midgut tracheae and columnar cells, respectively. To investigate MsCHS2 expression and activity in the course of the larval development, we monitored chitin synthesis, enzyme levels as well as mRNA amounts. All of the tested parameters were significantly reduced during molting and in the wandering stage when compared to the values obtained from intermolt feeding larvae. By contrast, MsCHS1 appeared to be inversely regulated because its mRNA was detectable only during the molt at the time when tracheal growth occurs at the basal site of the midgut. To further examine midgut chitin synthesis, we measured enzyme activity in crude midgut extracts and different membrane fractions. When we analysed trypsin-mediated proteolytic activation, a phenomenon previously reported for insect and fungal systems, we recognized that midgut chitin synthesis was only activated in crude extracts, but not in the 12,000 g membrane fraction. However, proteolytic activation by trypsin in the 12,000 g membrane fraction could be reconstituted by re-adding a soluble fraction, indicating that limited proteolysis affects an unknown soluble factor, a process that in turn activates chitin synthesis.     

The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei

White shrimp, Litopenaeus vannamei, which had been injected with chitin at 4, 6 and 8 microg g(-1) or chitosan at 2, 4 and 6 microg g(-1), were challenged with pathogen Vibrio alginolyticus at 2 x 10(6) colony-forming units (cfu) shrimp(-1) and then placed in seawater of 34 per thousand. The survival of shrimp that received chitin or chitosan at either dose was significantly higher than that of control shrimp after 1 day, and at the termination of the experiment (6 days after the challenge). In another experiment, the total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, and phagocytic activity to V. alginolyticus were measured when L. vannamei (10.4 +/- 0.7 g) were injected individually with chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1). L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased significantly its THC and respiratory burst after 2 days. L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) still maintained significantly higher phenoloxidase activity after 6 days. L. vannamei received chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased its phagocytic activity against V. alginolyticus after 1 day, respectively. It is therefore concluded that L. vannamei that received chitin at 6 microg g(-1) or chitosan at 4 microg g(-1) or less increased its immune ability and resistance to V. alginolyticus infection.     

Benzoylphenyl ureas inhibit chitin synthesis without interfering with amino sugar uptake in imaginal wing discs of Plodia interpunctella (Hübner)

We tested the hypothesis that the inhibition of chitin synthesis by benzoylphenyl ureas could be explained by their effect on the uptake of GlcNAc into chitin. Our test system consisted of organ cultures of wing imaginal discs from Plodia interpunctella. These wing discs synthesize chitin in response to 20-hydroxyecdysone or RH 5849, a non-steroidal ecdysteroid mimic. Two benzoylphenyl ureas, diflubenzuron and teflubenzuron, inhibited ecdysteroid-dependent chitin synthesis in the wing discs. However, although chitin synthesis was inhibited, there was no corresponding diminution of amino sugar uptake by the imaginal discs. In another experiment 20-hydroxyecdysone stimulated uptake of two sugars, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, which are not synthesized into chitin. Transport of these non-metabolized sugars was unaffected by the inhibitors. In an additional test of the effects on precursor transport, the action of ecdysone (alpha-ecdysone) was examined. Ecdysone stimulated amino sugar uptake, but not chitin synthesis. Neither diflubenzuron nor teflubenzuron inhibited ecdysone-dependent precursor transport. Finally, we examined ecdysteroid-induced uptake of amino sugars by an imaginal disc derived cell line IAL-PID2. In this case, also, GlcNAc transport was not inhibited significantly by either diflubenzuron or teflubenzuron. From these observations we conclude that inhibition of uptake of amino sugars does not account for the ability of teflubenzuron and diflubenzuron to inhibit chitin synthesis in P. interpunctella wing discs.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).