The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Arakane, Yasuyuki; Muthukrishnan, Subbaratnam; Kramer, Karl J.; Specht, Charles A.; Tomoyasu, Yoshinori; Lorenzen, Marcé D.; Kanost, Michael R.; Beeman, Richard W.

doi:10.1111/j.1365-2583.2005.00576.x

Cited by 291 publications

(323 citation statements)

References 26 publications

(35 reference statements)

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“…2 C and E). Molting defects in TcKnk dsRNA-treated beetles closely resembled those previously observed after dsRNA-mediated knockdown of T. castaneum chitin synthase A (TcChs-A), which is essential for cuticular chitin synthesis (12). Quantitative chemical analysis revealed that insects subjected to RNAi for TcKnk were severely deficient in total chitin, comparable to insects in which chitin synthesis was prevented by RNAi for TcChs-A (Fig.…”

Section: Resultssupporting

confidence: 57%

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Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Chaudhari

Arakane

Specht

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

110

127

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During each molting cycle of insect development, synthesis of new cuticle occurs concurrently with the partial degradation of the overlying old exoskeleton. Protection of the newly synthesized cuticle from molting fluid enzymes has long been attributed to the presence of an impermeable envelope layer that was thought to serve as a physical barrier, preventing molting fluid enzymes from accessing the new cuticle and thereby ensuring selective degradation of only the old one. In this study, using the red flour beetle, Tribolium castaneum, as a model insect species, we show that an entirely different and unexpected mechanism accounts for the selective action of chitinases and possibly other molting enzymes. The molting fluid enzyme chitinase, which degrades the matrix polysaccharide chitin, is not excluded from the newly synthesized cuticle as previously assumed. Instead, the new cuticle is protected from chitinase action by the T. castaneum Knickkopf (TcKnk) protein. TcKnk colocalizes with chitin in the new cuticle and organizes it into laminae. Down-regulation of TcKnk results in chitinase-dependent loss of chitin, severe molting defects, and lethality at all developmental stages. The conservation of Knickkopf across insect, crustacean, and nematode taxa suggests that its critical roles in the laminar ordering and protection of exoskeletal chitin may be common to all chitinous invertebrates.RNAi | nikkomycin | phylogenetic tree | transmission electron microscopy | chitin synthase D uring development, insects must undergo periodic molting to accommodate growth and to overcome the rigid constraints imposed by portions of their chitinous exoskeletons (1, 2). This process entails the complete replacement of the entire outer shell of the insect, including digestion; resorption and recycling of the inner, more pliable layers; and shedding of the outer, more highly sclerotized and waterproofed layers, which are either discarded or, in some cases, ingested for further recycling (1-4). The molting process is hormonally initiated by 20-hydroxyecdysone and begins with the epidermis secreting what will become the outer layers of the new cuticle that separate the epidermal layer from the overlying old cuticle. An "apolytic space" then forms, separating new (inner) from old (outer) cuticles (5). With the delicate epidermis now protected by the first layers of new cuticle, the molting fluid in the apolytic space can digest the inner layers of the outer (old) cuticle. According to long-held dogma, protection of the new cuticle from degradation by molting fluid enzymes is conferred by a thin, nonchitinous envelope (previously termed the "cuticulin" layer or the "outer epicuticle") deposited by the epidermal cells just before the secretion of new cuticular chitin underneath (3). This envelope was believed to form a protective barrier against proteolytic and chitinolytic enzymes of the molting fluid, thereby confining their actions to the proximal layers of the old (outer) exoskeleton while preventing digestion of the newly deposit...

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Section: Resultssupporting

confidence: 57%

“…dsRNA-treated pharate pupal and pharate adult insects were collected for chitin content analysis. Chitin content was measured with a modified Morgan-Elson method as described previously (12). Insects treated with dsRNA for T. castaneum Vermilion (TcVer) and TcChs-A were used as negative and positive experimental controls, respectively (n = 5).…”

Section: Methodsmentioning

confidence: 99%

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Chaudhari

Arakane

Specht

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

110

127

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“…Whereas tyrosinases, which are oxidases that contain two copper ions and catalyze the oxidation of ortho-diphenols, have been studied extensively for their roles in melanization, wound healing, and insect immunity (7,(9)(10)(11), less information is available about the importance in insect developmental physiology of laccases, which are oxidases that contain four copper ions and have ortho-and para-diphenol oxidizing activities (4,12). To establish the involvement of either or both of these phenoloxidase genes in insect cuticle tanning, RNA interference (RNAi) experiments were performed by using the red flour beetle Tribolium castaneum, an economically important agricultural pest species that is exquisitely sensitive to dsRNAmediated posttranscriptional gene silencing (13,14). The results presented here demonstrate that a specific laccase gene plays the major if not exclusive role in the tanning of larval, pupal, and adult cuticles of T. castaneum.…”

mentioning

confidence: 99%

Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning

Arakane

Muthukrishnan

Beeman

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

345

343

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Cuticle tanning (or sclerotization and pigmentation) in invertebrates involves the oxidative conjugation of proteins, which renders them insoluble and hardens and darkens the color of the exoskeleton. Two kinds of phenoloxidases, laccase and tyrosinase, have been proposed to participate in tanning, but proof of the true identity of the enzyme(s) responsible for this process has been elusive. We report the cloning of cDNAs for laccases and tyrosinases from the red flour beetle, Tribolium castaneum, as well as their developmental patterns of expression. To test for the involvement of these types of enzymes in cuticle tanning, we performed RNA interference experiments to decrease the levels of individual phenoloxidases. Normal phenotypes were obtained after dsRNA-mediated transcript depletion for all phenoloxidases tested, with the exception of laccase 2. Insects injected with dsRNA for the laccase 2 gene failed to tan, were soft-bodied and deformed, and subsequently died in a dsRNA dose-dependent fashion. The results presented here support the hypothesis that two isoforms of laccase 2 generated by alternative splicing catalyze larval, pupal, and adult cuticle tanning in Tribolium.exoskeleton ͉ sclerotization ͉ pigmentation ͉ Tribolium castaneum ͉ RNA interference

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“…10,11) To date, two CHS genes have been identified in the genome of the fruit fly Drosophila melanogaster, 12) the tobacco hornworm Manduca sexta, 13) and the red flour beetle Tribolium castaneum. 14) In T. castaneum, for example, CHSA (also called CHS1) is responsible for synthesizing epidermal cuticle, whereas CHSB (CHS2) is responsible for synthesis of the midgut peritrophic matrix. 14) Meanwhile, a mite growth inhibitor, etoxazole [4-(4-tertbutyl-2-ethoxyphenyl)-2-(2,6-difluorophenyl)-1,3-oxazoline], belonging to Group 10B in the IRAC MoA classification, 15) has been reported to induce molting defects, reduce chitin content, and inhibit the incorporation of radiolabeled GlcNAc into chitinous material in the fall armyworm Spodoptera frugiperda, as was also observed after benzoylurea treatment.…”

Section: Introductionmentioning

confidence: 99%

“…14) In T. castaneum, for example, CHSA (also called CHS1) is responsible for synthesizing epidermal cuticle, whereas CHSB (CHS2) is responsible for synthesis of the midgut peritrophic matrix. 14) Meanwhile, a mite growth inhibitor, etoxazole [4-(4-tertbutyl-2-ethoxyphenyl)-2-(2,6-difluorophenyl)-1,3-oxazoline], belonging to Group 10B in the IRAC MoA classification, 15) has been reported to induce molting defects, reduce chitin content, and inhibit the incorporation of radiolabeled GlcNAc into chitinous material in the fall armyworm Spodoptera frugiperda, as was also observed after benzoylurea treatment. 16) Although both benzoylureas and etoxazole are known to inhibit chitin synthesis, their molecular mode of action has been an enigma.…”

Section: Introductionmentioning

confidence: 99%

Benzoylurea resistance in western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): the presence of a point mutation in chitin synthase 1

Suzuki

Shiotsuki

Jouraku

et al. 2017

Journal of Pesticide Science

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We examined the susceptibility of field strains (BO-1, BO-2, TO-1, and YH-1) and one laboratory strain (H-1) of the western flower thrip, Frankliniella occidentalis, to benzoylureas. LC 50 values of novaluron were determined as 0.64 ppm against laboratory strain and 2.1-130 ppm against field strains. In the presence of piperonyl butoxide, a cytochrome P450 inhibitor, the insecticidal activity of novaluron tended to be enhanced. To examine whether point mutations in chitin synthase 1 (CHS1) discovered in an etoxazole-resistant strain of Tetranychus urticae and a benzoylurea-resistant strain of Plutella xylostella exist in F. occidentalis, the nucleotide sequence of CHS1 was analyzed. We found a nonsynonymous substitution that corresponded to the location of the mutations found in T. urticae and P. xylostella in the field strains of F. occidentalis but not in the laboratory strain, indicating that this point mutation might be associated with the benzoylurea resistance exhibited by the field strains.

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The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Cited by 291 publications

References 26 publications

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning

Benzoylurea resistance in western flower thrips <i>Frankliniella occidentalis</i> (Thysanoptera: Thripidae): the presence of a point mutation in <i>chitin synthase 1</i>

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