Key Event Title
|Level of Biological Organization|
|cuticle secreting cell|
Key Event Components
|chitin synthase activity||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|SAM depletion leading to population decline (1)||MolecularInitiatingEvent|
|CHS-1 inhibition leading to mortality||MolecularInitiatingEvent|
|Anopheles gambiae||Anopheles gambiae||High||NCBI|
|Tribolium castaneum||Tribolium castaneum||High||NCBI|
|Trichoplusia ni||Trichoplusia ni||High||NCBI|
|Hyalophora cecropia||Hyalophora cecropia||High||NCBI|
|Bradysia hygida||Bradysia hygida||Moderate||NCBI|
|Mamestra brassicae||Mamestra brassicae||Moderate||NCBI|
|Chilo suppressalis||Chilo suppressalis||Moderate||NCBI|
|Locusta migratoria||Locusta migratoria||Moderate||NCBI|
|Nilaparvata lugens||Nilaparvata lugens||Moderate||NCBI|
|Aphis glycines||Aphis glycines||Moderate||NCBI|
|Lepeophtheirus salmonis||Lepeophtheirus salmonis||Moderate||NCBI|
|Panonychus citri||Panonychus citri||Moderate||NCBI|
|Grapholita molesta||Grapholita molesta||Moderate||NCBI|
|Ectropis obliqua||Ectropis obliqua||Moderate||NCBI|
|Tigriopus japonicus||Tigriopus japonicus||Moderate||NCBI|
Key Event Description
Chitin synthases are essential enzymes for all organisms synthesizing chitin, for example arthropods and fungi (Latgé 2007; Merzendorfer 2011). Chitin synthases polymerize chitin and subsequently translocate chitin through the cell membrane (Merzendorfer 2006; Merzendorfer 2011). In arthropods, two isoforms of the chitin synthase are known, CHS1, which is responsible for the synthesis of cuticular chitin, and chitin synthase isoform 2, which synthesizes chitin in the midgut (Arakane et al. 2005). In this MIE, inhibition of CHS-1 is characterized. The biological state being measured is the activity of the enzyme. CHS-1 has an essential role in the cuticle biology, as it constitutes the last and most critical step in the chitin biosynthetic pathway by catalyzing the polymerization of UDP-GlcNAc to chitin (Merzendorfer and Zimoch 2003; Merzendorfer 2006).
How It Is Measured or Detected
Since the purification or even recombinant production of CHS1 has not been achieved yet, the most common way is to use crude enzyme preparations for CHS1 activity assays. It is noteworthy that in crude enzyme preparations of whole organisms both CHS isoforms, CHS1 and CHS2, are present. However, the expression of CHS1 was shown to be much higher than CHS2 in Anopheles gambiae (Zhang et al. 2012), therefore the effect of CHS2 may be regarded as negligible. Alternatively, the digestive tract of the respective organism could be removed before producing the enzyme preparation. Different ways exist to detect the activity of the enzyme. One can incubate the enzyme preparation with radioactively labelled chitin precursors (e.g. 14C-UDP-GlcNAc) and measure radioactivity in the formed chitin chains by scintillation counting (Cohen 1982; Cohen and Casida 1990). Another approach for the detection of CHS1 activity involves the binding of formed chitin chains to wheat germ agglutinin (WGA) which possesses specific chitin binding properties (Lucero et al. 2002; Zhang and Yan Zhu 2013). The assay builds on the principle of a sandwich-ELISA, where chitin binds to a layer of WGA. A second layer of WGA which is conjugated to horseradish peroxidase (HRP) is then added and subsequently incubated with a HRP substrate. The cleavage of the HRP substrate leads to color formation and the amount of chitin synthesized can be determined colorimetrically.
Domain of Applicability
Taxonomic: Effect data for the occurrence of CHS1 inhibition exist from Dipteran, Lepidopteran and Coleopteran insect species. Sequence alignment of CHS1 protein sequences using the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS, https://seqapass.epa.gov/seqapass) tool, yielded susceptibility predictions for various insect species, arachnids and crustacean taxa such as branchiopods, hexanauplia, malocostraca and merostomata. However, most of the protein sequences were not identified as CHS1. The alignment of amino acid residues believed to be critical for ligand binding were therefore carried out with sequences identified as CHS1. Evidence was rated as high for species with a susceptibility prediction and/or effect data. Evidence was rated as moderate when only alignment data were available. Although most of the sequences are not annotated as CHS1, all arthropods rely on the synthesis of cuticular chitin therefore it is extremely likely that the AOP is applicable to the whole phylum of arthropods.
Life stage: This MIE is applicable for organisms undergoing continuous molt cycles. Namely larval stages of insects and all life stages of crustaceans and arachnids.
Sex: The MIE is applicable to all sexes.
Chemical: Substances known to trigger inhibit CHS-1 are of the family of pyrimidine nucleosides (e.g. polyoxin D, polyoxin B and nikkomycin Z) (Cohen and Casida 1982; Kuwano and Cohen 1984; Cohen and Casida 1990; Zhang and Yan Zhu 2013; Osada 2019). There also exists evidence for the phthalimide captan to inhibit CHS-1 activity in vitro (Cohen and Casida 1982). However, as phthalimides are known to covalently bind to thiol groups in proteins (Lukens and Sisler 1958), it is not clear if the inhibition is due to specific CHS-1 inhibition or due to unspecific protein binding.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Stressors known to competitively inhibit CHS1 are polyoxin B, polyoxin D and Nikkomycin Z (Cohen and Casida 1982; Cohen and Casida 1990; Zhang and Yan Zhu 2013). There may also be stressors that inhibit CHS-1 in a non-competitive manner which may become apparent in further characterization efforts of this MIE. There is also a study that reports inhibition of CHS-1 by the phthalimide fungicide captan (Cohen and Casida 1982). However, it remains elusive if the observed inhibition is due to specific interaction with the enzyme or due to unspecific protein binding which is the predominant mode of action of phthalimides (Lukens and Sisler 1958).
Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost M, Beeman RW. 2005. The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix. Insect Mol Biol. 14(5):453–463. doi:10.1111/j.1365-2583.2005.00576.x.
Cohen E. 1982. In vitro chitin synthesis in an insect: formation and structure of microfibrils. Eur J Cell Biol. 26(2):289–294.
Cohen E, Casida JE. 1982. Properties and inhibition of insect integumental chitin synthetase. Pestic Biochem Physiol. 17(3):301–306. doi:10.1016/0048-3575(82)90141-9.
Cohen E, Casida JE. 1990. Insect and Fungal Chitin Synthetase Activity: Specificity of Lectins as Enhancers and Nucleoside Peptides as Inhibitors. Pestic Biochem Physiol. 37(3):249–253. doi:10.1016/0048-3575(90)90131-K.
Kuwano E, Cohen E. 1984. The use of a Tribolium chitin synthetase assay in studying the effects of benzimidazoles with a terpene moiety and related compounds. Agric Biol Chem. 48(6):1617–1620. doi:10.1080/00021369.1984.10866362.
Latgé JP. 2007. The cell wall: A carbohydrate armour for the fungal cell. Mol Microbiol. 66(2):279–290. doi:10.1111/j.1365-2958.2007.05872.x.
Lucero HA, Kuranda MJ, Bulik DA. 2002. A nonradioactive, high throughput assay for chitin synthase activity. Anal Biochem. 305(1):97–105. doi:10.1006/abio.2002.5594.
Lukens RJ, Sisler HD. 1958. 2-Thiazolidinethione-4-carboxylic acid from the reaction of captan with cysteine. Science (80- ). 127(3299):650. doi:10.1126/science.127.3299.650.
Merzendorfer H. 2006. Insect chitin synthases: A review. J Comp Physiol B Biochem Syst Environ Physiol. doi:10.1007/s00360-005-0005-3.
Merzendorfer H. 2011. The cellular basis of chitin synthesis in fungi and insects: Common principles and differences. Eur J Cell Biol. 90(9):759–769. doi:10.1016/j.ejcb.2011.04.014. http://dx.doi.org/10.1016/j.ejcb.2011.04.014.
Merzendorfer H, Zimoch L. 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol. 206(24):4393 LP – 4412. doi:10.1242/jeb.00709. http://jeb.biologists.org/content/206/24/4393.abstract.
Osada H. 2019. Discovery and applications of nucleoside antibiotics beyond polyoxin. J Antibiot (Tokyo). 72(12):855–864. doi:10.1038/s41429-019-0237-1. http://dx.doi.org/10.1038/s41429-019-0237-1.
Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae. Insect Sci. 20(2):158–166. doi:10.1111/j.1744-7917.2012.01568.x.
Zhang X, Zhang J, Park Y, Zhu KY. 2012. Identification and characterization of two chitin synthase genes in African malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol. 42(9):674–682. doi:10.1016/j.ibmb.2012.05.005. http://dx.doi.org/10.1016/j.ibmb.2012.05.005.