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Created at: 2020-11-11 13:00

AOP ID and Title:

AOP 360: Chitin synthase 1 inhibition leading to mortality
Short Title: CHS-1 inhibition leading to mortality

Graphical Representation

Authors

Simon Schmid 1,2, You Song 1, and Knut Erik Tollefsen 1,2

1 Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349, Oslo, Norway

2 Faculty of Environmental Science and Resource Management, Department of Environmental Sciences (IMV), Norwegian University of Life Sciences (NMBU), N-1432, Ås, Norway

Contact: Simon.Schmid@niva.no


Status

Author status OECD status OECD project SAAOP status
Open for comment. Do not cite

Abstract

Arthropods heavily rely on chitin synthesis as chitin is one of the main constituents of the cuticle. Successful molting, and therefore a successful development necessitates stability and integrity of the cuticle. The cuticular chitin synthase (CHS1) is the key enzyme in the biosynthetic pathway and arthropods are therefore especially dependent on its proper function.
The present AOP describes the effects of chemical inhibition of the cuticular chitin synthase (CHS1) on the molting process leading to increased mortality in arthropods. Inhibition of CHS1 is the molecular initiating event and leads to a decreased chitin content in the arthropod cuticle which leaves the organism immature at the stage for ecdysis. This phenomenon can be described as premature molting. The organism eventually dies due to being stuck in the old cuticle or due to the consequences of a weak exoskeleton after ecdysis.
The AOP is considered to be very consistent. Essentiality of key events was rated as high for every key event and the biological plausibility was rated as high for the whole AOP. However, there does not exist very much empirical evidence that allows to draw a representative conclusion on dose and time concordance along the AOP. Therefore, empirical evidence and also the quantitative understanding was considered to be low. The overall confidence in the AOP was valued as moderate.
The present AOP will guide assay development for further experimental studies by revealing data and knowledge gaps. One of its primary applications will also be providing guidance in screening strategies in order to broaden its chemical applicability domain.


Background

Arthropods need to shed their exoskeleton in order to grow and reproduce. This process, also called molting or ecdysis, is mediated by behavioural mechanisms which involve the skeletal muscles (Ayali 2009; Song et al. 2017a). In order to properly shed its cuticle, the organism needs to possess a newly synthesized cuticle that possesses a certain integrity to support this process. Since chitin is a major constituent of the cuticle, it contributes substantially to its integrity (Cohen 2001; Vincent and Wegst 2004). Chitin is synthesized from uridine diphosphate-N-Acetylglucosamine (UDP-GlcNAc) in a polymerization reaction by the transmembrane enzyme chitin synthase isoform 1 (CHS-1). CHS-1 is localized on the apical side in the cuticular epithelium.
Since chitin and the process of chitin synthesis does not occur in vertebrates, it can and has been exploited for the design of pest controlling agents. Inhibitors of chitin synthesis may not only be of use for the control of unwanted arthropods and fungi, they may also pose a risk for beneficial arthropods such as insects and crustaceans. Disruption of chitin synthesis or the endocrine mechanisms controlling molting generally lead to a disruption of ecdysis (Merzendorfer et al. 2012; Song et al. 2017a; Song et al. 2017b). If the amount of chitin in the cuticle decreases, the affected organism may not be able to molt properly and will most probably die of starvation or suffocation (Camp et al. 2014; Song et al. 2017a). Alternatively, if molting is completed despite an immature cuticle, the organism may be deformed and die as a consequence of a weak cuticle.
Therefore, the present AOP should build the basis of a mechanistic approach for the systematic evaluation and the risk assessment of chemicals interfering with chitin synthesis by directly inhibiting CHS-1.


Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
1 MIE 1522 Increase, Chitin synthase 1 inhibition Increase, CHS-1 inhibition
2 KE 1523 Decrease, Cuticular chitin content Decrease, Cuticular chitin content
3 KE 1524 Increase, Premature molting Increase, Premature molting
4 AO 350 Increase, Mortality Increase, Mortality

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Increase, Chitin synthase 1 inhibition adjacent Decrease, Cuticular chitin content Moderate Low
Decrease, Cuticular chitin content adjacent Increase, Premature molting Moderate Low
Increase, Premature molting adjacent Increase, Mortality Moderate Low

Stressors


Name Evidence
Polyoxin B High
Polyoxin D High
Nikkomycins High
Captan Moderate
Captafol Moderate
Folpet Moderate

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability
Life Stage Evidence
larvae High
Juvenile High
Adult Moderate
Taxonomic Applicability
Term Scientific Term Evidence Links
Pieris brassicae Pieris brassicae High NCBI
Anopheles gambiae Anopheles gambiae High NCBI
Lucilia cuprina Lucilia cuprina High NCBI
Tribolium castaneum Tribolium castaneum High NCBI
Bombyx mori Bombyx mori High NCBI
Anopheles quadrimaculatus Anopheles quadrimaculatus High NCBI
Trichoplusia ni Trichoplusia ni High NCBI
Artemia salina Artemia salina High NCBI
Daphnia magna Daphnia magna High NCBI
Hyalophora cecropia Hyalophora cecropia High NCBI
Ostrinia nubilalis Ostrinia nubilalis 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
Sex Applicability
Sex Evidence
Unspecific Moderate

Taxonomic: Effect data along the AOP exist from Dipteran, Lepidopteran and Coleopteran insect species as well as from Branchiopods and Anostracans of the crustacea . 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 CHS-1. 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 CHS-1, all arthropods rely on the synthesis of cuticular chitin therefore it is extremely likely that the AOP is applicable to all arthropods.

Life stage: The AOP is applicable for organisms undergoing continuous molt cycles. As insects do not molt in their adulthood, the AOP is only applicable for larval and pupal stages of insects. Crustaceans and arachnids grow and molt throughout their lifetime (Passano 1961; Uhl et al. 2015), which makes the AOP applicable to all life stages, where juvenile life stages might be more susceptible to chemical perturbations due to higher growth rate and therefore more frequent molting .

Sex: The AOP is applicable to all sexes.

Chemical: Substances known to trigger the MIE and leading to the AO are of the family of pyrimidine nucleosides (e.g. polyoxin D, polyoxin B and nikkomycin Z) (Osada 2019). There also exists evidence for phthalimides (captan, captafol and folpet) to inhibit CHS-1 activity and to decrease the cuticular chitin content in vitro (Cohen and Casida 1982; Gelman and Borkovec 1986). However, as these substances 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.

Essentiality of the Key Events

The essentiality of all key events was considered as high. Essentiality evaluations were mainly based on specifically designed studies demonstrating the expected effect pattern predicted by the AOP to occur after knockdown of CHS-1.

Inhibition, Cuticular chitin synthase (High): Knockdown of the cuticular chitin synthase leads to the expected pattern of effects described in this AOP. It decreases the cuticular chitin content and leads to premature molting associated mortality in insects (Arakane et al. 2005; X. Zhang et al. 2010; Li et al. 2017; Zhai et al. 2017). If the cuticular chitin content was not directly measured as endpoint, knockdown of the CHS-1 led directly to the occurrence of premature molting associated increase of mortality (Chen et al. 2008; X. Zhang et al. 2010; Wang et al. 2012; Yang et al. 2013; Shang et al. 2016; Mohammed et al. 2017; Wang et al. 2019; Ye et al. 2019; Ullah et al. 2020)

Decrease, Cuticular chitin content (High): Abolishment of the cuticular chitin synthesis through knockdown of CHS-1 leads to premature molting associated mortality (Arakane et al. 2005; X. Zhang et al. 2010; Li et al. 2017; Zhai et al. 2017). By knocking down the UDP-GlcNAc pyrophosphorylase (UAP), which catalyzes the last sugar conversion before the polymerization to chitin, it was shown that reduced chitin synthesis leads to the same outcome as the knockdown of CHS-1. Namely premature molting and increased mortality (Arakane et al. 2011; Liu et al. 2013). Knockdown of trehalase genes, which constitutes the start of the chitin synthetic pathway and convert trehalose to glucose, leads to a similar pattern of effects, namely premature molting associated mortality (Chen et al. 2010; Shi et al. 2016).

Increase, Premature molting (High): Several studies show that premature molting is a direct consequence of decreased chitin synthesis and leads to increased mortality. The KE is consistently listed as cause for mortality when CHS-1 is knocked down throughout a number of studies (Arakane et al. 2005; Chen et al. 2008; J. Zhang et al. 2010; X. Zhang et al. 2010; Wang et al. 2012; Yang et al. 2013; Shang et al. 2016; Li et al. 2017; Mohammed et al. 2017; Zhai et al. 2017; Wang et al. 2019; Ye et al. 2019; Ullah et al. 2020).

Increase, Mortality (High): Increased mortality was observed in all of the abovementioned studies.

Weight of Evidence Summary

Biological Plausibility: The biosynthesis of chitin is well characterized and is conserved among arthropods. Although the exact mode of action of chitin synthases remains elusive, it is widely accepted and well established that the chitin synthase is the key enzyme in the pathway, polymerizing chitin using UDP-N-Acetylglucosamine as substrate (Merzendorfer and Zimoch 2003).
Arthropod cuticles mostly consist of chitin embedded into a matrix of cuticular proteins. It is therefore widely accepted that chitin contributes crucially to the quality and function of the cuticle (Reynolds 1987; Muthukrishnan et al. 2012). The molting process requires the new cuticle to be strong enough to withstand the stresses of ecdysis.
During ecdysis, arthropods pause food intake and growth. If ecdysis is initiated before the new cuticle is strong enough, the organism likely dies of starvation or growth arrest (Song, Villeneuve, et al. 2017). It was also reported that certain arthropods pause respiration during ecdysis, which may lead to suffocation (Camp et al. 2014).
Based on the well-established biological knowledge on the processes this AOP bases on, the biological plausibility for all KER was rated as high.

Empirical Evidence: Empirical evidence assessment was conducted on the basis of in vitro and in vivo experiments performed with stressors affecting key events throughout the AOP. Studies showed that the key events are affected by model stressors such as Polyoxin D and Nikkomycin Z, which are able to competitively inhibit CHS1 (Endo et al. 1970). Several studies provide evidence that polyoxin B, polyoxin D and nikkomycin Z trigger the MIE (Cohen 1982; Turnbull and Howells 1982; Kuwano and Cohen 1984; Cohen and Casida 1990; Zhang and Yan Zhu 2013). Also the cuticular chitin content was shown to be decreased by polyoxin D and nikkomycin Z (Gijswijt et al. 1979; Calcott and Fatig 1984; Gelman and Borkovec 1986; Zhuo et al. 2014). The AO is supported by in vivo studies with polyoxin D and nikkomycin Z (Tellam et al. 2000; Tellam and Eisemann 2000; Zhu et al. 2007; Zhang and Yan Zhu 2013; New Zealand Environmental Protection Authority 2015).
A major data gap constitutes the absence of data covering the KE “Increase, premature molting”. This KE is mentioned in some studies but never assessed as an individual endpoint (Gijswijt et al. 1979; Tellam et al. 2000).
Another major data gap is the lacking quantitative data connecting KE by KERs. As endpoints were only measured as individual endpoints and not in sequence, it makes it nearly impossible to evaluate the dose and temporal concordance for the KEs and KERs.
Based on the major data gaps and therefore the lacking information on dose and temporal concordance of the KER empirical evidence was evaluated to be low for the whole AOP.

Overall confidence in the AOP: Both, essentiality of KEs and the biological plausibility of the whole AOP were considered to be high. However, due to lack of quantitative data, empirical evidence was judged to be low. Therefore the overall confidence in the AOP was evaluated as moderate.

Quantitative Consideration

Quantitative data are limited for all KER and therefore the whole AOP. Therefore, predictions on the occurrence of downstream KE and the AO on the basis of the occurrence of upstream KEs is not readily feasible. Quantitative understanding of the AOP was therefore considered to be low.

Considerations for Potential Applications of the AOP (optional)

Arthropods are responsible for many functions in terrestrial as well as aquatic ecosystems and are therefore jointly responsible for ecosystem health (Seastedt and Crossley 1984; Losey and Vaughan 2006; LeBlanc 2007). Therefore, it is important to develop AOPs which enhance the mechanistic knowledge on chemicals, such as chitin synthesis inhibitors, which may pose a risk to non-target arthropods. Those AOPs will contribute to the systematic use of mechanistic data to preserve beneficial arthropod populations and ecosystem health.
The present AOP will help to guide future experimental studies by identifying data gaps and missing links. This will lead to the identification and development suitable bioassays in order to populate the AOP with (quantitative) experimental data which may allow for predictions of regulatory relevant endpoints on the basis of the occurrence of the MIE.
The present AOP may also guide screening strategies in order to broaden its chemical applicability domain. The identified substances may then be prioritized and undergo a thorough hazard assessment.
As there already exist approaches to assess mixture toxicity using the AOP framework (Altenburger et al. 2012; Beyer et al. 2014), the present AOP could be employed for the effect assessment  of mixtures of chemicals that share the same KEs (e.g. AOP #361, aopwiki.org/aops/361, AOP #358, aopwiki.org/aops/358, and AOP #359, aopwiki.org/aops/359).

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Appendix 1

List of MIEs in this AOP

Event: 1522: Increase, Chitin synthase 1 inhibition

Short Name: Increase, CHS-1 inhibition

Key Event Component

Process Object Action
chitin synthase activity decreased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:342 - S-adenosylmethionine depletion leading to population decline (1) MolecularInitiatingEvent
Aop:360 - Chitin synthase 1 inhibition leading to mortality MolecularInitiatingEvent

Stressors

Name
Polyoxin B
Polyoxin D
Nikkomycins
Captan

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
cuticle secreting cell

Organ term

Organ term
epithelium

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).



Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
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
Life Stage Applicability
Life Stage Evidence
larvae High
Juvenile High
Adult Moderate
Sex Applicability
Sex Evidence
Unspecific Moderate

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.


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.


References

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.


List of Key Events in the AOP

Event: 1523: Decrease, Cuticular chitin content

Short Name: Decrease, Cuticular chitin content

Key Event Component

Process Object Action
cuticle development cuticle decreased

Stressors

Name
Polyoxin D
Nikkomycins
Captan
Captafol
Folpet

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
cuticle

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Pieris brassicae Pieris brassicae High NCBI
Lucilia cuprina Lucilia cuprina High NCBI
Bombyx mori Bombyx mori High NCBI
Artemia salina Artemia salina High NCBI
Ostrinia nubilalis Ostrinia nubilalis High NCBI
Life Stage Applicability
Life Stage Evidence
larvae High
Juvenile High
Adult Moderate
Sex Applicability
Sex Evidence
Unspecific Moderate

Taxonomic: Effect data for the occurrence of this KE exist from Pieris brassicae, Lucilia cuprina, Bombyx mori, Artemia salina and Ostrinia nubilalis, defining its taxonomic applicability. Most likely, this KE is applicable to the whole phylum of arthropods, as they all rely on chitin as part of their exoskeleton.

Life stage: This KE is applicable for organisms synthesizing chitin in order to grow and develop, namely larval stages of insects and all life stages of crustaceans and arachnids.

Sex: This KE is applicable to all sexes.

Chemical: Substances known decrease the cuticular chitin content are of the family of pyrimidine nucleosides (e.g. polyoxin D and nikkomycin Z) (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Zhuo et al. 2014; Osada 2019). There also exists evidence for phthalimides (captan, captafol and folpet) to to decrease the cuticular chitin content in vitro (Gelman and Borkovec 1986). However, as these substances 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.


Key Event Description

This key event describes the decrease in cuticular chitin content. Chitin is a major part of the arthropod cuticle and therefore also responsible for its integrity (Reynolds 1987; Muthukrishnan et al. 2012). The cuticle is the exoskeleton of arthropods and has manifold functions, it protects organisms from predators, loss of water, acts as a physical barrier against microbial pathogens and provides support for muscular function (Vincent and Wegst 2004). Hence, cuticular chitin is also indispensable for the development of arthropods, as an immaculate cuticle is required for proper molting and therefore also for the growth of an organism.
During molting, the newly secreted cuticle is subject to mechanical stress associated and therefore needs to possess enough structural and functional integrity. The ecdysis motor program, which constitutes the behavioral part of the cuticle shedding requires the newly secreted cuticle to possess a certain strength to support for muscular force in order to shed the old cuticle (Ewer 2005). Cuticular integrity is also important after ecdysis, as insects and crustaceans expand their new cuticle by increasing internal pressure by swallowing air and water, respectively. This happens in order to expand and provide stability to the new cuticle until it is hardened (tanned) (Clarke 1957; Lee 1961; Dall et al. 1978; deFur et al. 1985).


How it is Measured or Detected

Several ways to determine cuticular chitin are described in the literature. Some of them are based on the determination of amino sugars after digestion or hydrolysis of chitin. For example, after the digestion of chitin by a bacterial chitinase, the GlcNAc amount can be determined colorimetrically by a modified Morgan-Elson assay (Reissig et al. 1955; Arakane et al. 2005). Alternatively, one can also quantify glucosamine colorimetrically after deacetylation and hydrolysis of chitin (Lehmann and White 1975; Zhang and Zhu 2006).
There also exists an approach based on the detection of fluorescence after staining with calcofluor white. In this assay, no treatment of the samples is necessary, the detection is carried out in homogenates of the respective organisms as calcofluor white directly binds to chitin (Henriques et al. 2020).
Chitin can also be quantified using radioactively labelled precursors (e.g. 14C-UDP-GlcNAc) which are incorporated into in vitro cultured integument pieces or into the cuticle of whole organisms (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986).


References

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.

Calcott PH, Fatig RO. 1984. Inhibition of Chitin metabolism by Avermectin in susceptible Organisms. J Antibiot (Tokyo). 37(3):253–259. doi:10.7164/antibiotics.37.253.

Clarke KU. 1957. On the Increase in Linear Size During Growth in Locusta Migratoria L. Proc R Entomol Soc London Ser A, Gen Entomol. 32(1–3):35–39. doi:10.1111/j.1365-3032.1957.tb00361.x.

Dall W, Smith DM, Press B. 1978. Water uptake at ecdysis in the western rock lobster. J Exp Mar Bio Ecol. 35(1960). doi:10.1016/0022-0981(78)90074-6.

deFur PL, Mangum CP, McMahon BR. 1985. Cardiovascular and Ventilatory Changes During Ecdysis in the Blue Crab Callinectes Sapidus Rathbun. J Crustac Biol. 5(2):207–215. doi:10.2307/1547867.

Ewer J. 2005. How the ecdysozoan changed its coat. PLoS Biol. 3(10):1696–1699. doi:10.1371/journal.pbio.0030349.

Gelman DB, Borkovec AB. 1986. The pharate adult clasper as a tool for measuring chitin synthesis and for identifying new chitin synthesis inhibitors. Comp Biochem Physiol Part C, Comp. 85(1):193–197. doi:10.1016/0742-8413(86)90073-3.

Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in Pieris brassicae (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.

Henriques BS, Garcia ES, Azambuja P, Genta FA. 2020. Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining. Front Physiol. 11(February):1–10. doi:10.3389/fphys.2020.00117.

Lee RM. 1961. The variation of blood volume with age in the desert locust (Schistocerca gregaria Forsk.). J Insect Physiol. 6(1):36–51. doi:10.1016/0022-1910(61)90090-7.

Lehmann PF, White LO. 1975. Chitin Assay Used to Demonstrate Renal Localization and Cortisone-Enhanced Growth of Aspergillus fumigatus Mycelium in Mice. Infect Immun. 12(5):987–992.

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.

Muthukrishnan S, Merzendorfer H, Arakane Y, Kramer KJ. 2012. Chitin Metabolism in Insects. Elsevier B.V. http://dx.doi.org/10.1016/B978-0-12-384747-8.10007-8.

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.

Reissig JL, Strominger JL, Leloir LF. 1955. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem.:959–966.

Reynolds SE. 1987. The cuticle, growth and moulting in insects: The essential background to the action of acylurea insecticides. Pestic Sci. 20(2):131–146. doi:10.1002/ps.2780200207.

Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly Lucilia cuprina. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.

Vincent JFV, Wegst UGK. 2004. Design and mechanical properties of insect cuticle. Arthropod Struct Dev. 33(3):187–199. doi:10.1016/j.asd.2004.05.006.

Zhang J, Zhu KY. 2006. Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron. Insect Biochem Mol Biol. 36(9):712–725. doi:10.1016/j.ibmb.2006.06.002.

Zhuo W, Fang Y, Kong L, Li X, Sima Y, Xu S. 2014. Chitin synthase A: A novel epidermal development regulation gene in the larvae of Bombyx mori. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.


Event: 1524: Increase, Premature molting

Short Name: Increase, Premature molting

Key Event Component

Process Object Action
ecdysis, chitin-based cuticle decreased

Stressors

Name
Polyoxin D
Nikkomycins

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Pieris brassicae Pieris brassicae High NCBI
Lucilia cuprina Lucilia cuprina High NCBI
Life Stage Applicability
Life Stage Evidence
larvae High
Juvenile High
Adult Moderate
Sex Applicability
Sex Evidence
Unspecific Moderate

Taxonomic: Effect data for the occurrence of this KE exist from Pieris brassicae and Lucilia cuprina, defining its taxonomic applicability. However, all arthropods undergo molting, so it is highly likely that this KE is applicable to the whole phylum of arthropods.

Life stage: This KE is applicable for organisms that undergo molting in order to grow and develop, namely larval stages of insects and all life stages of crustaceans and arachnids.

Sex: This KE is applicable to all sexes.

Chemical: Substances known to induce premature molting are of the family of pyrimidine nucleosides (e.g. polyoxin D and nikkomycin Z) (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008).


Key Event Description

This key event is measured on the level of the individual. In order to grow and develop, arthropods need to shed their exoskeleton periodically (Heming 2018). If they are not able to molt properly, the organism will eventually die. Premature molting summarizes a variety of effects related to molting disruption. It describes the unsuccessful molting where the organism is not able to shed the old cuticle, but also other effects related to molting in an immature stage where the new cuticle is not mature enough for the molt, such as rupture of the new cuticle and associated desiccation, deformities, higher susceptibility to pathogens or impaired locomotion.


How it is Measured or Detected

Premature molting can be determined by observation. For example, during an OECD 202 Daphnia sp. Acute immobilization test (OECD 2004), the cumulative number of molts can be assessed as an additional endpoint. One could even prolong the test to 96h to get a clearer result of this endpoint. Additionally, one could apply histopathological methods to monitor the maturity of the newly synthesized cuticle (e.g. thickness of procuticle).


References

Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of Bombyx mori (Lepidoptera: Bombycidae), Mamestra brassicae, Mythimna separata, and Spodoptera litura (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.

Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in Pieris brassicae (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.

Heming BS. 2018. Insect development and evolution. Ithaca: Cornell University Press.

OECD. 2004. Test No. 202: Daphnia sp. Acute Immobilisation Test. OECD Guidel Test og Chem Sect 2.(April):1–12. doi:10.1787/9789264069947-en. [accessed 2020 Jun 5]. https://www.oecd-ilibrary.org/environment/test-no-202-daphnia-sp-acute-immobilisation-test_9789264069947-en.

Tellam RL, Vuocolo T, Johnson SE, Jarmey J, Pearson RD. 2000. Insect chitin synthase. cDNA sequence, gene organization and expression. Eur J Biochem. 267(19):6025–6043. doi:10.1046/j.1432-1327.2000.01679.x.


List of Adverse Outcomes in this AOP

Event: 350: Increase, Mortality

Short Name: Increase, Mortality

Key Event Component

Process Object Action
mortality increased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:4 - Ecdysone receptor agonism leading to incomplete ecdysis associated mortality KeyEvent
Aop:286 - Mitochondrial complex III inhibition leading to growth inhibition (1) AdverseOutcome
Aop:287 - Mitochondrial complex III inhibition leading to growth inhibition (2) AdverseOutcome
Aop:331 - Formation of DNA photoproducts leading to growth inhibition (1) AdverseOutcome
Aop:327 - Excessive reactive oxygen species production leading to mortality (1) AdverseOutcome
Aop:328 - Excessive reactive oxygen species production leading to mortality (2) AdverseOutcome
Aop:329 - Excessive reactive oxygen species production leading to mortality (3) AdverseOutcome
Aop:330 - Excessive reactive oxygen species production leading to mortality (4) AdverseOutcome
Aop:343 - S-adenosylmethionine depletion leading to population decline (2) AdverseOutcome
Aop:342 - S-adenosylmethionine depletion leading to population decline (1) AdverseOutcome
Aop:358 - Chitinase inhibition leading to mortality AdverseOutcome
Aop:359 - Chitobiase inhibition leading to mortality AdverseOutcome
Aop:360 - Chitin synthase 1 inhibition leading to mortality AdverseOutcome
Aop:361 - Sulfonylureareceptor binding leading to mortality AdverseOutcome

Stressors

Name
Polyoxin D
Nikkomycins

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Lucilia cuprina Lucilia cuprina High NCBI
Daphnia magna Daphnia magna High NCBI
Life Stage Applicability
Life Stage Evidence
All life stages High
Sex Applicability
Sex Evidence
Unspecific High

Taxonomic: This AO is applicable to all living organisms.

Life stage: This AO is applicable to all life stages.

Sex: This AO is applicable to all sexes.

Chemical: Substances known to increase mortality in arthropods are of the family of pyrimidine nucleosides (e.g. polyoxin D and nikkomycin Z) (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008).


Key Event Description

This key event is observed at the biological level of the individual and describes the increase of mortality of individuals upon exposure to a stressor.


How it is Measured or Detected

The AO can be detected by observation, for example by immobilization of the respective organisms. There exist guidelines for the characterization of this AO in arthropods. For example, the OECD 202 Daphnia sp. Acute immobilization test (OECD 2004) which can also be modified depending on the effect one expects.


Regulatory Significance of the AO

The Adverse Outcome is highly significant from a regulatory point of view. It is employed as regulatory endpoint in most studies assessing the toxicity of stressors.


References

Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of Bombyx mori (Lepidoptera: Bombycidae), Mamestra brassicae, Mythimna separata, and Spodoptera litura (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.

Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in Pieris brassicae (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.

OECD. 2004. Test No. 202: Daphnia sp. Acute Immobilisation Test. OECD OECD Guidelines for the Testing of Chemicals, Section 2. [accessed 2020 Mar 3]. https://www.oecd-ilibrary.org/environment/test-no-202-daphnia-sp-acute-immobilisation-test_9789264069947-en.

Tellam RL, Vuocolo T, Johnson SE, Jarmey J, Pearson RD. 2000. Insect chitin synthase. cDNA sequence, gene organization and expression. Eur J Biochem. 267(19):6025–6043. doi:10.1046/j.1432-1327.2000.01679.x.


Appendix 2

List of Key Event Relationships in the AOP