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<h2>Authors</h2>
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<p><span style="font-size:14px">Simon Schmid <sup>1,2</sup>, You Song <sup>1</sup>, and Knut Erik Tollefsen <sup>1,2</sup></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Simon Schmid <sup>1,2</sup>, You Song <sup>1</sup>, and Knut Erik Tollefsen <sup>1,2,3</sup></span></span></p>
<p><span style="font-size:14px"><sup>1</sup> Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349, Oslo, Norway</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><sup>1</sup> Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Økernveien 94, N-0579, Oslo, Norway</span></span></p>
<p><span style="font-size:14px"><sup>2</sup> Faculty of Environmental Science and Resource Management, Department of Environmental Sciences (IMV), Norwegian University of Life Sciences (NMBU), N-1432, Ås, Norway</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><sup>2</sup> </span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">Faculty of Environmental Science and Resource Management<span style="background-color:white"><span style="color:#212121"> (MINA)</span></span>, Norwegian University of Life Sciences (NMBU), N-1432, Ås, Norway</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><sup>3</sup> </span></span><span style="font-size:11.0pt"><span style="background-color:white"><span style="font-family:"Arial",sans-serif"><span style="color:#212121">Centre for Environmental Radioactivity (CERAD), Norwegian University of Life Sciences (NMBU), N-1432 Ås, Norway</span></span></span></span></p>
<span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><span style="background-color:white"><span style="color:black">This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 859891 and was supported by NIVA’s Computational Toxicology Program, NCTP (<a href="https://www.niva.no/en/projectweb/nctp" style="color:blue; text-decoration:underline">www.niva.no/nctp</a>).</span></span></span></span></p>
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<h2>Abstract</h2>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:#212529">In order to grow and develop, arthropods need to shed their exoskeleton (or cuticle) periodically and replace it with a new one in a process called molting. Successful molting, and therefore a successful development necessitates stability and integrity of the cuticle to support muscular contractions involved in the shedding of the old cuticle. The integrity of the cuticle is largely dependent on the <em>N</em>-acetylglucosamine (GlcNAc) polymer chitin. Therefore, arthropods heavily rely on chitin synthesis as chitin is one of the main constituents of the cuticle. The cuticular chitin synthase (CHS-1) 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 (CHS-1) on the molting process leading to increased mortality in arthropods. Inhibition of CHS-1 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 concordance along the AOP whereas time concordance can be supported by knockdown studies of CHS-1. Therefore, empirical evidence was considered to be moderate and 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 identify chemicals directly interacting with CHS-1.</span></span></span></span></span></p>
<h2>Abstract</h2>
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<p><span style="font-size:14px">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.<br />
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.<br />
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.<br />
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.</span></p>
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<h3>Background</h3>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Arthropods (including insects, crustaceans and arachnids) 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-<em>N</em>-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.<br />
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.</span><br />
<span style="font-size:14px">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.</span></span></p>
<h3>Background</h3>
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<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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-<em>N</em>-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.<br />
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.</span></span><br />
<span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
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<p><span style="font-size:14px"><strong><span style="background-color:#FFFFFF">Taxonomic: </span></strong><span style="background-color:#FFFFFF">E</span>ffect 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, <a href="https://seqapass.epa.gov/seqapass/info.xhtml">https://seqapass.epa.gov/seqapass</a>) 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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong><span style="background-color:#ffffff">Taxonomic: </span></strong><span style="background-color:#ffffff">Since the whole phylum of arthropods is dependent on the synthesis of chitin to molt successfully, it is extremely likely that the AOP is applicable to all arthropods.</span><strong><span style="background-color:#ffffff"> </span></strong><span style="background-color:#ffffff">E</span>ffect data along the AOP exist from Dipteran, Lepidopteran and Coleopteran insect species as well as from Branchiopods and Anostracans of the crustacea. Although data is limited, KEs seem to be well conserved across taxa, as shown in available studies with specific stressors known to inhibit CHS and in studies where CHS-1 was knocked down by RNA interference. However, due to limited data availability, it was not possible to cover whole taxa but rather single species in the assessment of KEs. Alignment of amino acid residues in the catalytic center of CHS-1 using the Sequence Alignment to Predict Across Species Susceptibility tool (SeqAPASS, <a href="https://seqapass.epa.gov/seqapass/info.xhtml">https://seqapass.epa.gov/seqapass</a>, </span></span><span style="font-size:14px"><span style="font-family:"Arial",sans-serif">LaLone et al. 2016</span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">), confirmed structural and functional conservation in various insect, arachnid and crustacean species, strenghtening the evidence for the applicability domain to be the whole phylum of arthropods. However, taxonomic applicability may not only be defined by structural conservation of the protein sequence. So the evidence for the taxonomic applicability for species with support only from sequence alignment was judged as moderate, whereas evidence for species with support from sequence alignment and effect data was judged as high.</span></span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Life stage: </strong>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.</span></span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>The AOP is applicable to all sexes.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Sex: </strong>The AOP is applicable to all sexes.</span></span></p>
<p><span style="font-size:14px"><strong>Chemical: </strong>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 <em>in vitro</em> (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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Chemical: </strong>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 <em>in vitro</em> (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.</span></span></p>
<h3>Essentiality of the Key Events</h3>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
<h3>Essentiality of the Key Events</h3>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px"><strong>Inhibition, Cuticular chitin synthase (High): </strong></span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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)<strong>. </strong>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)</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Inhibition, Chitin synthase 1 (High): </strong>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)<strong>. </strong>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)</span></span></p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px"><strong>Decrease, Cuticular chitin content (High): </strong></span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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).</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Decrease, Cuticular chitin content (High): </strong>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 content 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 decreased cuticular chitin content and premature molting associated mortality (Chen et al. 2010; Shi et al. 2016).</span></span></p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px"><strong>Increase, Premature molting (High): </strong></span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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).</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Increase, Premature molting (High): </strong>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).</span></span></p>
<p><strong><font face="arial, helvetica,sans-serif"><span style="font-size:14px">Increase, Mortality (High): </span></font></strong><font face="arial, helvetica, sans-serif"><span style="font-size:14px">Increased mortality was observed in all of the abovementioned studies.</span></font></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:14px">Increase, Mortality (High): </span></strong><span style="font-size:14px">Increased mortality was observed in all of the abovementioned studies.</span></span></p>
<h3>Weight of Evidence Summary</h3>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px"><strong>Biological Plausibility: </strong></span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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-<em>N</em>-Acetylglucosamine as substrate (Merzendorfer and Zimoch 2003).<br />
<h3>Weight of Evidence Summary</h3>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Biological Plausibility: </strong>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-<em>N</em>-Acetylglucosamine as substrate (Merzendorfer and Zimoch 2003).<br />
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.<br />
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).<br />
Based on the well-established biological knowledge on the processes this AOP bases on, the biological plausibility for all KER was rated as high.</span></span></p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px"><strong>Empirical Evidence:</strong>Empirical evidence assessment was conducted on the basis of<em>in vitro</em>and<em>in vivo</em>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).<br />
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).<br />
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.<br />
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.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><strong><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:#212529">Empirical Evidence: </span></span></span></strong><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:#212529">Empirical evidence assessment was conducted on the basis of <em>in vitro </em>and <em>in vivo</em> 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 in cell free systems of coleopteran, lepidopteran and dipteran insect species<span style="font-size:10.5pt"><span style="background-color:white"><span style="color:#212529"> (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 </span></span></span>in lepidopteran and dipteran species as well as in the crustacean <em>Artemia salina</em><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:#212529"> (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 in dipteran insects and <em>Daphnia magna</em> (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 for KERs. As endpoints were only measured as individual endpoints and not in sequence, it makes it nearly impossible to evaluate the dose for the KEs and KERs. However, data from studies where CHS-1 was knocked down are able to support temporal concordance for all KERs. Knockdown of CHS-1 led to decreased chitin content and subsequently to premature molting associated mortality (</span></span></span>Arakane et al., 2005; Li et al., 2017)<span style="font-size:10.5pt"><span style="background-color:white"><span style="color:#212529">. Based on the major data gaps and therefore the lacking information on dose concordance as well as the given time concordance, empirical evidence was evaluated to be moderate for the whole AOP.</span></span></span></span></span></span></span></span></p>
<p><strong><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">Overall confidence in the AOP: </span></span></strong><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
<h3>Quantitative Consideration</h3>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:14px">Overall confidence in the AOP: </span></strong><span style="font-size:14px">Both, essentiality of KEs and the biological plausibility of the whole AOP were considered to be high. However, due to missing quantitative data and the lack of evidence for dose concordance, empirical evidence was judged to be moderate. Therefore the overall confidence in the AOP was evaluated as moderate.</span></span></p>
<h3>Quantitative Consideration</h3>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
</div>
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<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<hr>
<p><span style="font-size:14px">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.<br />
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.<br />
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.<br />
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, <a href="https://aopwiki.org/aops/361">aopwiki.org/aops/361</a>, AOP #358, <a href="https://aopwiki.org/aops/358">aopwiki.org/aops/358</a>, and AOP #359, <a href="https://aopwiki.org/aops/359">aopwiki.org/aops/359</a>).</span></p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">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.<br />
The present AOP will help to guide future experimental studies by identifying data gaps. 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.<br />
The present AOP may also guide screening strategies in order to identify chemicals inhibiting CHS-1. The identified substances may then be prioritized and undergo a thorough hazard assessment.<br />
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, <a href="https://aopwiki.org/aops/361">aopwiki.org/aops/361</a>, AOP #358, <a href="https://aopwiki.org/aops/358">aopwiki.org/aops/358</a>, and AOP #359, <a href="https://aopwiki.org/aops/359">aopwiki.org/aops/359</a>).</span></span></p>
</div>
<!-- reference section, text as of right now but should be changed to be handled as table -->
<div id="references">
<h2>References</h2>
<hr>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Altenburger R, Scholz S, Schmitt-Jansen M, Busch W, Escher BI. 2012. Mixture toxicity revisited from a toxicogenomic perspective. Environ Sci Technol. 46(5):2508–2522. doi:10.1021/es2038036.</span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Shang F, Xiong Y, Xia WK, Wei DD, Wei D, Wang JJ. 2016. Identification, characterization and functional analysis of a chitin synthase gene in the brown citrus aphid, <em>Toxoptera citricida</em> (Hemiptera, Aphididae). Insect Mol Biol. 25(4):422–430. doi:10.1111/imb.12228.</span></span></p>
<p><span style="font-size:14px">Song Y, Evenseth LM, Iguchi T, Tollefsen KE. 2017b. Release of chitobiase as an indicator of potential molting disruption in juvenile <em>Daphnia magna</em> exposed to the ecdysone receptor agonist 20-hydroxyecdysone. J Toxicol Environ Heal - Part A Curr Issues. 80(16–18):954–962. doi:10.1080/15287394.2017.1352215. https://doi.org/10.1080/15287394.2017.1352215.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Shi JF, Xu QY, Sun QK, Meng QW, Mu LL, Guo WC, Li GQ. 2016. Physiological roles of trehalose in <em>Leptinotarsa</em> larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes. Insect Biochem Mol Biol. 77:52–68. doi:10.1016/j.ibmb.2016.07.012.</span></span></p>
<p><span style="font-size:14px">Song Y, Villeneuve DL, Toyota K, Iguchi T, Tollefsen KE. 2017a. Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development. Environ Sci Technol. 51(8):4142–4157. doi:10.1021/acs.est.7b00480.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Song Y, Evenseth LM, Iguchi T, Tollefsen KE. 2017b. Release of chitobiase as an indicator of potential molting disruption in juvenile <em>Daphnia magna</em> exposed to the ecdysone receptor agonist 20-hydroxyecdysone. J Toxicol Environ Heal - Part A Curr Issues. 80(16–18):954–962. doi:10.1080/15287394.2017.1352215. https://doi.org/10.1080/15287394.2017.1352215.</span></span></p>
<p><span style="font-size:14px">Tellam RL, Eisemann C. 2000. Chitin is only a minor component of the peritrophic matrix from larvae of <em>Lucilia cuprina</em>. Insect Biochem Mol Biol. 30(12):1189–1201. doi:10.1016/S0965-1748(00)00097-7.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Song Y, Villeneuve DL, Toyota K, Iguchi T, Tollefsen KE. 2017a. Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development. Environ Sci Technol. 51(8):4142–4157. doi:10.1021/acs.est.7b00480.</span></span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Tellam RL, Eisemann C. 2000. Chitin is only a minor component of the peritrophic matrix from larvae of <em>Lucilia cuprina</em>. Insect Biochem Mol Biol. 30(12):1189–1201. doi:10.1016/S0965-1748(00)00097-7.</span></span></p>
<p><span style="font-size:14px">Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly <em>Lucilia cuprina</em>. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
<p><span style="font-size:14px">Uhl G, Zimmer SM, Renner D, Schneider JM. 2015. Exploiting a moment of weakness: Male spiders escape sexual cannibalism by copulating with moulting females. Sci Rep. 5(July):1–7. doi:10.1038/srep16928.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly <em>Lucilia cuprina</em>. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.</span></span></p>
<p><span style="font-size:14px">Ullah F, Gul H, Wang X, Ding Q, Said F, Gao X, Desneux N, Song D. 2020. RNAi-mediated knockdown of chitin synthase 1 (CHS1) gene causes mortality and decreased longevity and fecundity in <em>Aphis gossypii</em>. Insects. 11(1):1–11. doi:10.3390/insects11010022.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Uhl G, Zimmer SM, Renner D, Schneider JM. 2015. Exploiting a moment of weakness: Male spiders escape sexual cannibalism by copulating with moulting females. Sci Rep. 5(July):1–7. doi:10.1038/srep16928.</span></span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">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.</span></span></p>
<p><span style="font-size:14px">Wang Y, Fan HW, Huang HJ, Xue J, Wu WJ, Bao YY, Xu HJ, Zhu ZR, Cheng JA, Zhang CX. 2012. Chitin synthase 1 gene and its two alternative splicing variants from two sap-sucking insects, <em>Nilaparvata lugens</em> and <em>Laodelphax striatellus</em> (Hemiptera: Delphacidae). Insect Biochem Mol Biol. 42(9):637–646. doi:10.1016/j.ibmb.2012.04.009. http://dx.doi.org/10.1016/j.ibmb.2012.04.009.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Wang Y, Fan HW, Huang HJ, Xue J, Wu WJ, Bao YY, Xu HJ, Zhu ZR, Cheng JA, Zhang CX. 2012. Chitin synthase 1 gene and its two alternative splicing variants from two sap-sucking insects, <em>Nilaparvata lugens</em> and <em>Laodelphax striatellus</em> (Hemiptera: Delphacidae). Insect Biochem Mol Biol. 42(9):637–646. doi:10.1016/j.ibmb.2012.04.009. http://dx.doi.org/10.1016/j.ibmb.2012.04.009.</span></span></p>
<p><span style="font-size:14px">Wang Z, Yang H, Zhou C, Yang WJ, Jin DC, Long GY. 2019. Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, <em>Sogatella furcifera</em> (Hemiptera: Delphacidae). Sci Rep. 9(1):1–14. doi:10.1038/s41598-018-37488-5. http://dx.doi.org/10.1038/s41598-018-37488-5.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Wang Z, Yang H, Zhou C, Yang WJ, Jin DC, Long GY. 2019. Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, <em>Sogatella furcifera</em> (Hemiptera: Delphacidae). Sci Rep. 9(1):1–14. doi:10.1038/s41598-018-37488-5. http://dx.doi.org/10.1038/s41598-018-37488-5.</span></span></p>
<p><span style="font-size:14px">Yang WJ, Xu KK, Cong L, Wang JJ. 2013. Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, <em>Bactrocera dorsalis</em>. Int J Biol Sci. 9(4):331–342. doi:10.7150/ijbs.6022.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Yang WJ, Xu KK, Cong L, Wang JJ. 2013. Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, <em>Bactrocera dorsalis</em>. Int J Biol Sci. 9(4):331–342. doi:10.7150/ijbs.6022.</span></span></p>
<p><span style="font-size:14px">Ye C, Jiang Y Di, An X, Yang L, Shang F, Niu J, Wang JJ. 2019. Effects of RNAi-based silencing of chitin synthase gene on moulting and fecundity in pea aphids (<em>Acyrthosiphon pisum</em>). Sci Rep. 9(1):1–10. doi:10.1038/s41598-019-39837-4. http://dx.doi.org/10.1038/s41598-019-39837-4.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Ye C, Jiang Y Di, An X, Yang L, Shang F, Niu J, Wang JJ. 2019. Effects of RNAi-based silencing of chitin synthase gene on moulting and fecundity in pea aphids (<em>Acyrthosiphon pisum</em>). Sci Rep. 9(1):1–10. doi:10.1038/s41598-019-39837-4. http://dx.doi.org/10.1038/s41598-019-39837-4.</span></span></p>
<p><span style="font-size:14px">Zhai Y, Fan X, Yin Z, Yue X, Men X, Zheng L, Zhang W. 2017. Identification and Functional Analysis of Chitin Synthase A in Oriental Armyworm, <em>Mythimna separata</em>. Proteomics. 17(21):1–11. doi:10.1002/pmic.201700165.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Zhai Y, Fan X, Yin Z, Yue X, Men X, Zheng L, Zhang W. 2017. Identification and Functional Analysis of Chitin Synthase A in Oriental Armyworm, <em>Mythimna separata</em>. Proteomics. 17(21):1–11. doi:10.1002/pmic.201700165.</span></span></p>
<p><span style="font-size:14px">Zhang J, Liu X, Zhang Jianqin, Li D, Sun Y, Guo Y, Ma E, Zhu KY. 2010. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen). Insect Biochem Mol Biol. 40(11):824–833. doi:10.1016/j.ibmb.2010.08.001. http://dx.doi.org/10.1016/j.ibmb.2010.08.001.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Zhang J, Liu X, Zhang Jianqin, Li D, Sun Y, Guo Y, Ma E, Zhu KY. 2010. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen). Insect Biochem Mol Biol. 40(11):824–833. doi:10.1016/j.ibmb.2010.08.001. http://dx.doi.org/10.1016/j.ibmb.2010.08.001.</span></span></p>
<p><span style="font-size:14px">Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, <em>Anopheles gambiae</em>. Insect Sci. 20(2):158–166. doi:10.1111/j.1744-7917.2012.01568.x.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, <em>Anopheles gambiae</em>. Insect Sci. 20(2):158–166. doi:10.1111/j.1744-7917.2012.01568.x.</span></span></p>
<p><span style="font-size:14px">Zhang X, Zhang J, Zhu KY. 2010. Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (<em>Anopheles gambiae</em>). Insect Mol Biol. 19(5):683–693. doi:10.1111/j.1365-2583.2010.01029.x.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Zhang X, Zhang J, Zhu KY. 2010. Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (<em>Anopheles gambiae</em>). Insect Mol Biol. 19(5):683–693. doi:10.1111/j.1365-2583.2010.01029.x.</span></span></p>
<p><span style="font-size:14px">Zhu KY, Heise S, Zhang J, Anderson TD, Starkey SR. 2007. Comparative Studies on Effects of Three Chitin Synthesis Inhibitors on Common Malaria Mosquito (Diptera: Culicidae). J Med Entomol. 44(6):1047–1053. doi:10.1093/jmedent/44.6.1047.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Zhu KY, Heise S, Zhang J, Anderson TD, Starkey SR. 2007. Comparative Studies on Effects of Three Chitin Synthesis Inhibitors on Common Malaria Mosquito (Diptera: Culicidae). J Med Entomol. 44(6):1047–1053. doi:10.1093/jmedent/44.6.1047.</span></span></p>
<p><span style="font-size:14px">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 <em>Bombyx mori</em>. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">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 <em>Bombyx mori</em>. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.</span></span></p>
<!-- Evidence for Perturbation of This Event by Stressors -->
<h3>Evidence for Perturbation by Stressor</h3>
<hr>
<h4>Overview for Molecular Initiating Event</h4>
<p><span style="font-size:14px">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).</span></p>
<br>
<br>
<!-- end Evidence for Perturbation of This Event by Stressors -->
<h4>Domain of Applicability</h4>
<br>
<!-- loop to find taxonomic applicability under event -->
<p><span style="font-size:14px">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).</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>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, <a href="https://seqapass.epa.gov/seqapass/info.xhtml">https://seqapass.epa.gov/seqapass</a>) 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.</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>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, <a href="https://seqapass.epa.gov/seqapass/info.xhtml">https://seqapass.epa.gov/seqapass</a>) 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 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 this MIE is applicable to the whole phylum of arthropods.</span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>This MIE is applicable for organisms undergoing continuous molt cycles. Namely larval stages of insects and all life stages of crustaceans and arachnids.</span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>The MIE is applicable to all sexes.</span></p>
<p><span style="font-size:14px"><strong>Chemical:</strong> 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 <em>in vitro</em> (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.</span></p>
<br>
</div>
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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).</span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">Since the purification or even recombinant production of CHS-1 has not been achieved yet, the most common way is to use crude enzyme preparations for CHS-1 activity assays. It is noteworthy that in crude enzyme preparations of whole organisms both CHS isoforms, CHS-1 and CHS-2, are present. However, the expression of CHS-1 was shown to be much higher than CHS-2 in <em>Anopheles gambiae </em>(Zhang et al. 2012), therefore the effect of CHS-2 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). Chitin synthase activity can also be measured in a non-radioactive way after the addition of precursors to a crude enzyme extract. There, the detection of CHS-1 activity involves the binding of 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.</span></p>
<!-- event text -->
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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).</span></p>
<br>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">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 <em>Anopheles gambiae </em>(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.</span></p>
<br>
<h4>References</h4>
<p><span style="font-size:14px">Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost M, Beeman RW. 2005. The <em>Tribolium </em> 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.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost M, Beeman RW. 2005. The <em>Tribolium </em> 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.</span></p>
<p><span style="font-size:14px">Cohen E. 1982. In vitro chitin synthesis in an insect: formation and structure of microfibrils. Eur J Cell Biol. 26(2):289–294.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Kuwano E, Cohen E. 1984. The use of a <em>Tribolium</em> 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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Merzendorfer H. 2006. Insect chitin synthases: A review. J Comp Physiol B Biochem Syst Environ Physiol. doi:10.1007/s00360-005-0005-3.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, <em>Anopheles gambiae</em>. Insect Sci. 20(2):158–166. doi:10.1111/j.1744-7917.2012.01568.x.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Effect data for the occurrence of this KE exist from <em>Pieris brassicae</em>, <em>Lucilia cuprina</em>, <em>Bombyx mori</em>, <em>Artemia salina</em> and <em>Ostrinia nubilalis</em>, 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.</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Effect data for the occurrence of this KE exist from <em>Pieris brassicae</em>, <em>Lucilia cuprina</em>, <em>Bombyx mori</em>, <em>Artemia salina</em> and <em>Ostrinia nubilalis</em>, 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.</span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>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.</span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>This KE is applicable to all sexes.</span></p>
<p><span style="font-size:14px"><strong>Chemical:</strong> 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 <em>in vitro</em> (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.</span></p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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 <!--[if supportFields]><span
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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 <!--[if supportFields]><span
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. 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 <!--[if supportFields]><span
Structure and Development","id":"ITEM-1","issue":"3","issued":{"date-parts":[["2004"]]},"page":"187-199","title":"Design
style='mso-element:field-end'></span></span><![endif]-->. 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.<br />
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 <!--[if supportFields]><span
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. 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) <!--[if supportFields]><span lang=EN-US style='font-size:11.0pt;
of Insect Physiology","id":"ITEM-4","issue":"1","issued":{"date-parts":[["1961"]]},"page":"36-51","title":"The
variation of blood volume with age in the desert locust (<i>Schistocerca
style='mso-element:field-end'></span></span><![endif]-->. 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.</span><br />
</p>
<br>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">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 <!--[if supportFields]><span lang=EN-US style='font-size:11.0pt;
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">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 <em>N</em>-Acetylclucosamine (GlcNAc) amount can be determined colorimetrically by a modified Morgan-Elson assay <!--[if supportFields]><span lang=EN-US style='font-size:11.0pt;
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. Alternatively, one can also quantify glucosamine colorimetrically after deacetylation and hydrolysis of chitin <!--[if supportFields]><span lang=EN-US
Biochemistry and Molecular Biology","id":"ITEM-2","issue":"9","issued":{"date-parts":[["2006"]]},"page":"712-725","title":"Characterization
of a chitin synthase cDNA and its increased mRNA level associated with
decreased chitin synthesis in <i>Anopheles quadrimaculatus</i>
style='mso-element:field-separator'></span></span><![endif]-->(Lehmann and White 1975; Zhang and Zhu 2006)<!--[if supportFields]><span lang=EN-US style='font-size:
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 <!--[if supportFields]><span
Chitin can also be quantified using radioactively labelled precursors (e.g. 14C-UDP-GlcNAc) which are incorporated into <em>in vitro</em> cultured integument pieces or into the cuticle of whole organisms <!--[if supportFields]><span
Journal of Biological Sciences","id":"ITEM-2","issue":"5","issued":{"date-parts":[["1982"]]},"page":"491-504","title":"Effects
of several larvicidal compounds on chitin biosynthesis by isolated larval
style='mso-element:field-separator'></span></span><![endif]-->(Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986)<!--[if supportFields]><span
Another possibility is to use the non-radioactive assay developed to measure chitin synthase activity (Lucero et al. 2002; Zhang and Yan Zhu 2013). Instead of adding an enzyme extract and chitin precursors to the reaction, one could simply add homogenized chitin containing material to the reaction to quantify its chitin content.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost M, Beeman RW. 2005. The <em>Tribolium </em> 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.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost M, Beeman RW. 2005. The <em>Tribolium </em> 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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Clarke KU. 1957. On the Increase in Linear Size During Growth in <em>Locusta Migratoria</em> L. Proc R Entomol Soc London Ser A, Gen Entomol. 32(1–3):35–39. doi:10.1111/j.1365-3032.1957.tb00361.x.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">deFur PL, Mangum CP, McMahon BR. 1985. Cardiovascular and Ventilatory Changes During Ecdysis in the Blue Crab <em>Callinectes Sapidus</em> Rathbun. J Crustac Biol. 5(2):207–215. doi:10.2307/1547867.</span></p>
<p><span style="font-size:14px">Ewer J. 2005. How the ecdysozoan changed its coat. PLoS Biol. 3(10):1696–1699. doi:10.1371/journal.pbio.0030349.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Lee RM. 1961. The variation of blood volume with age in the desert locust (<em>Schistocerca gregaria</em> Forsk.). J Insect Physiol. 6(1):36–51. doi:10.1016/0022-1910(61)90090-7.</span></p>
<p><span style="font-size:14px">Lehmann PF, White LO. 1975. Chitin Assay Used to Demonstrate Renal Localization and Cortisone-Enhanced Growth of <em>Aspergillus fumigatus</em> Mycelium in Mice. Infect Immun. 12(5):987–992.</span></p>
<p><span style="font-size:14px">Lucero HA, Kuranda MJ, Bulik DA. 2002. A nonradioactive, high throughput assay for chitin synthase activity. Anal Biochem. 305(1):97–105.<br />
doi:10.1006/abio.2002.5594.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Reissig JL, Strominger JL, Leloir LF. 1955. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem.:959–966.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly <em>Lucilia cuprina</em>. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Zhang J, Zhu KY. 2006. Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in <em>Anopheles quadrimaculatus</em> exposed to diflubenzuron. Insect Biochem Mol Biol. 36(9):712–725. doi:10.1016/j.ibmb.2006.06.002.</span></p>
<p><span style="font-size:14px">Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles<br />
<p><span style="font-size:14px">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 <em>Bombyx mori</em>. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Effect data for the occurrence of this KE exist from <em>Pieris brassicae</em> and <em>Lucilia cuprina</em>, 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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Taxonomic: </strong>Effect data for the occurrence of this KE exist from <em>Pieris brassicae</em> and <em>Lucilia cuprina</em>. However, all arthropods undergo molting, so it is highly likely that this KE is applicable to the whole phylum of arthropods.</span></span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>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.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Life stage: </strong>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.</span></span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>This KE is applicable to all sexes.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Sex: </strong>This KE is applicable to all sexes.</span></span></p>
<p><span style="font-size:14px"><strong>Chemical:</strong> 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).</span></p>
<br>
</div>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px"><strong>Chemical:</strong> 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).</span></span></p>
<!-- event text -->
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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.</span></p>
<h4>Key Event Description</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">This key event is measured on the level of the individual. In order to grow and develop, arthropods need to shed their exoskeleton periodically (molting) (Heming 2018). 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). If arthropods are not able to molt properly, the organism will eventually die. Premature molting 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. Specific effects observed are animals stuck in their exuviae (Wang et al., 2019), and if molting can be completed despite an immature cuticle, animals might be smaller and die at subsequent molts (Arakawa et al., 2008; Chen et al., 2008; Mohammed et al., 2017).</span></span></p>
<br>
<h4>How it is Measured or Detected</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Premature molting can be determined by observation. No standardized tests for the endpoint of molting exist to date. However, during an OECD 202 <em>Daphnia</em> sp. Acute immobilization test (OECD 2004), the cumulative number of molts can be assessed as an additional endpoint. Molting can also be assessed during a OECD 211 <em>Daphnia</em> sp. Reproduction test (OECD 2012), as proposed previously (OECD 2003). 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).</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">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).</span></p>
<br>
<h4>References</h4>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<h4>References</h4>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></span></p>
<p><span style="font-size:14px">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Chen, X.; Tian, H.; Zou, L.; Tang, B.; Hu, J.; Zhang, W. Disruption of Spodoptera Exigua Larval Development by Silencing Chitin Synthase Gene A with RNA Interference. Bull. Entomol. Res. 2008, 98 (6), 613–619. https://doi.org/10.1017/S0007485308005932.</span></span></p>
<p><span style="font-size:14px">Heming BS. 2018. Insect development and evolution. Ithaca: Cornell University Press.</span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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–<br />
<p><span style="font-size:14px">OECD. 2004. Test No. 202: <em>Daphnia sp.</em> 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.</span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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-<br />
0981(78)90074-6.</span></span></p>
<p><span style="font-size:14px">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.</span></p>
<br>
<!-- end event text -->
</div>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">deFur PL, Mangum CP, McMahon BR. 1985. Cardiovascular and Ventilatory Changes During Ecdysis in the Blue Crab Callinectes Sapidus<br />
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Ewer J. 2005. How the ecdysozoan changed its coat. PLoS Biol. 3(10):1696–1699. doi:10.1371/journal.pbio.0030349.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Heming BS. 2018. Insect development and evolution. Ithaca: Cornell University Press.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Mohammed, A. M. A.; DIab, M. R.; Abdelsattar, M.; Khalil, S. M. S. Characterization and RNAi-Mediated Knockdown of Chitin Synthase A in the Potato Tuber Moth, Phthorimaea Operculella. Sci. Rep. 2017, 7 (1), 1–12. https://doi.org/10.1038/s41598-017-09858-y.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Lee RM. 1961. The variation of blood volume with age in the desert locust (Schistocerca gregaria Forsk.). J Insect Physiol. 6(1):36–51.<br />
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">OECD (2003), Proposal for an Enhanced Test Guideline. Daphnia magna Reproduction Test. Draft OECD Guidel. Test. Chem. Enhanc. Tech. Guid. Doc. 211 21.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">OECD (2004), <em>Test No. 202: Daphnia sp. Acute Immobilisation Test</em>, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264069947-en" title="">https://doi.org/10.1787/9789264069947-en</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">OECD (2012), <em>Test No. 211: Daphnia magna Reproduction Test</em>, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264185203-en" title="">https://doi.org/10.1787/9789264185203-en</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Wang, Z.; Yang, H.; Zhou, C.; Yang, W. J.; Jin, D. C.; Long, G. Y. Molecular Cloning, Expression, and Functional Analysis of the Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in the White-Backed Planthopper, Sogatella Furcifera (Hemiptera: Delphacidae). Sci. Rep. 2019, 9 (1), 1–14. https://doi.org/10.1038/s41598-018-37488-5.</span></span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>This AO is applicable to all living organisms.</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>This AO is applicable to all living organisms.</span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>This AO is applicable to all life stages.</span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>This AO is applicable to all sexes.</span></p>
<p><span style="font-size:14px"><strong>Chemical:</strong> 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).</span></p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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.</span></p>
<br>
<h4>Key Event Description</h4>
<p><span style="font-size:14px">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.</span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">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 </span><!--[if supportFields]><span lang=EN-US
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">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 </span><!--[if supportFields]><span lang=EN-US
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]--><span style="font-size:14px"> which can also be modified depending on the effect one expects.</span></p>
<br>
<h4>Regulatory Significance of the AO</h4>
<p><span style="font-size:14px">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.</span></p>
<h4>Regulatory Significance of the AO</h4>
<p><span style="font-size:14px">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.</span></p>
<br>
<h4>References</h4>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<p><span style="font-size:14px">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></p>
<p><span style="font-size:14px">OECD. 2004. Test No. 202: <em>Daphnia sp.</em> 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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<br>
<!-- end event text -->
</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<!-- Evidence for relationship links section, this lists the relationships and then supports them -->
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Likely, this KER is applicable to the whole phylum of arthropods as they all depend on the synthesis of chitin.</span></p>
</div>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Likely, this KER is likely applicable to the whole phylum of arthropods as they all depend on the synthesis of chitin.</span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>This KER 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.</span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>This KER is applicable to all sexes.</span></p>
<p><span style="font-size:14px"><strong>Chemical: </strong>Substances inducing both, the inhibition of CHS-1 and the decrease in cuticular chitin content are of the family of pyrimidine nucleosides (e.g. polyoxin D, polyoxin B and nikkomycin Z) (Gijswijt et al. 1979; Cohen and Casida 1982; Turnbull and Howells 1982; Calcott and Fatig 1984; Kuwano and Cohen 1984; Cohen and Casida 1990; Zhang and Yan Zhu 2013; Zhuo et al. 2014; Osada 2019). The phthalimide captan was also shown to induce CHS-1 inhibition and a decrease in cuticular chitin content (Cohen and Casida 1982; Gelman and Borkovec 1986). However, studies assessing both endpoints in sequence are lacking.</span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:14px">Chitin in the arthropod cuticle is synthesized by the chitin synthase isoform 1 (CHS-1) which spans the plasma membrane on the apical plasma membrane of epithelial cells (Locke and Huie 1979; Binnington 1985; Merzendorfer and Zimoch 2003; Merzendorfer 2006). Since CHS-1 is the enzyme to polymerize chitin from UDP-<em>N</em>-Acetylglucosamine (UDP-GlcNAc) (Merzendorfer 2006), it is solely responsible for the content of chitin in the exoskeleton. Consequently, the inhibition of CHS-1 leads to a decrease in chitin content in the arthropod cuticle.</span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">The process of chitin synthesis in arthropods is well characterized. Although the exact mechanism of the polymerization reaction remains elusive, CHS-1 is known to be the key enzyme in the biosynthesis of chitin and therefore, responsible for the cuticular chitin content (Merzendorfer and Zimoch 2003; Merzendorfer 2006). Therefore, the biological plausibility of this KER can be regarded as high.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:14px">Empirical evidence for the occurrence of both KEs, the inhibition of CHS-1 and the decrease in cuticular chitin content exist. For example, the occurrence of chitin synthase inhibition was characterized using cell free crude enzyme preparations <em>in vitro </em>from coleopteran, lepidopteran and dipteran insect species upon treatment with polyoxin B, polyoxin D and nikkomycin Z (Cohen and Casida 1982; Kuwano and Cohen 1984; Cohen and Casida 1990; Zhang and Yan Zhu 2013). The cuticular chitin content was characterized <em>in vivo</em> in <em>Artemia salina </em>or using cultured integumental tissue from lepidopteran and dipteran species after exposure to polyoxin D and nikkomycin Z as well as the phthalimides captan, captafol, and folpet (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986; Zhuo et al. 2014). Data from studies with specific stressors assessing both endpoints and therefore supporting dose concordance of the KER are lacking. However, results from studies where CHS-1 was knocked down by RNA interference support temporal concordance of the KER (Arakane et al. 2005, Li et al. 2017, Zhang X. et al. 2010). Given the support for temporal concordance and the lack of studies showing dose concordance, the empirical evidence for this KER was judged as moderate.</span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:14px">The major uncertainty in this KER is the absence of studies which assess both endpoints, the inhibition of the chitin synthase and the decrease in cuticular chitin content after exposure to specific stressors.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:14px">Due to the lack of studies linking the inhibition of CHS-1 to the decrease in cuticular chitin content, it is not possible to describe the nature of the response-response relationship.</span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:14px">Chitin in the arthropod cuticle is synthesized by the chitin synthase isoform 1 (CHS-1) which spans the plasma membrane on the apical plasma membrane of epithelial cells (Locke and Huie 1979; Binnington 1985; Merzendorfer and Zimoch 2003; Merzendorfer 2006). Since CHS-1 is the enzyme to polymerize chitin from UDP-<em>N</em>-Acetylglucosamine (UDP-GlcNAc) (Merzendorfer 2006), it is solely responsible for the content of chitin in the exoskeleton. Consequently, the inhibition of CHS-1 leads to a decrease in chitin content in the arthropod cuticle.</span></p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">The process of chitin synthesis in arthropods is well characterized. Although the exact mechanism of the polymerization reaction remains elusive, CHS-1 is known to be the key enzyme in the biosynthesis of chitin and therefore, responsible for the cuticular chitin content (Merzendorfer and Zimoch 2003; Merzendorfer 2006). Therefore, the biological plausibility of this KER can be regarded as high.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:14px">Empirical evidence for the occurrence of both KEs, the inhibition of CHS-1 and the decrease in cuticular chitin content exist. For example, the occurrence of chitin synthase inhibition was characterized using cell free crude enzyme preparations <em>in vitro </em>from coleopteran, lepidopteran and dipteran insect species upon treatment with polyoxin B, polyoxin D and nikkomycin Z (Cohen and Casida 1982; Kuwano and Cohen 1984; Cohen and Casida 1990; Zhang and Yan Zhu 2013). The cuticular chitin content was characterized <em>in vivo</em> in <em>Artemia salina </em>or using cultured integumental tissue from lepidopteran and dipteran species after exposure to polyoxin D and nikkomycin Z as well as the phthalimides captan, captafol, and folpet (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986; Zhuo et al. 2014). However, studies assessing both endpoints and therefore linking both KEs are lacking.</span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:14px">The major uncertainty in this KER is the absence of studies which assess both, the inhibition of the chitin synthase and the decrease in cuticular chitin content.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:14px">Due to the lack of studies linking the inhibition of CHS-1 to the decrease in cuticular chitin content, it is not possible to describe the nature of the response-response relationship.</span></p>
<strong>Time-scale</strong>
<p><span style="font-size:14px">Due to the lack of studies assessing the inhibition of CHS-1 and the decrease in cuticular chitin content, it is not possible to make a statement on the timescale of the relationship. However, the expression of CHS-1 peaks at the time of ecdysis <!--[if supportFields]><span lang=EN-US style='font-size:11.0pt;
<strong>Time-scale</strong>
<p><span style="font-size:14px">Due to the lack of studies assessing the inhibition of CHS-1 and the decrease in cuticular chitin content, it is not possible to make a statement on the timescale of the relationship. However, the expression of CHS-1 peaks at the time of ecdysis <!--[if supportFields]><span lang=EN-US style='font-size:11.0pt;
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->, indicating the highest rate of chitin synthesis at this timepoint. Hence it can be assumed that a decrease in chitin content in the newly synthesized cuticle should become apparent shortly after.</span></p>
<strong>Known modulating factors</strong>
<p><span style="font-size:14px">CHS is dependent on bivalent ions as cofactor such as Mg<sup>2+</sup> or Mn<sup>2+ </sup>(Merzendorfer 2006). Both low and high levels of Mg<sup>2+ </sup>inhibited CHS activity <em>in vitro</em> (Zhang and Yan Zhu 2013).</span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:14px">Upon knockdown of CHS-1 in the salmon louse <em>Lepeophtheirus salmonis</em>, upregulation of the UDP-GlcNAc pyrophosphorylase (UAP), which catalyzes the conversion of GlcNAc to UDP-GlcNAc, was observed (Braden et al. 2020). The knockdown of UAP also led to upregulation of CHS-1 demonstrating a clear dependence of the two enzymes. Most likely, the upregulation of UAP is a compensatory mechanism with the goal to restore homeostasis in absence of CHS-1. The exact regulation of the feedback, however, remains to be investigated.</span></p>
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->, indicating the highest rate of chitin synthesis at this timepoint. Hence it can be assumed that a decrease in chitin content in the newly synthesized cuticle should become apparent shortly after. In studies where CHS-1 was knocked down, chitin contents were assessed after 3 and 7 days and found to be decreased (Arakane et al. 2005, Li et al. 2017, Zhang X. et al. 2010).</span></p>
<strong>Known modulating factors</strong>
<p><span style="font-size:14px">CHS is dependent on bivalent ions as cofactor such as Mg<sup>2+</sup> or Mn<sup>2+ </sup>(Merzendorfer 2006). Both low and high levels of Mg<sup>2+ </sup>inhibited CHS activity <em>in vitro</em> (Zhang and Yan Zhu 2013).</span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:14px">Upon knockdown of CHS-1 in the salmon louse <em>Lepeophtheirus salmonis</em>, upregulation of the UDP-GlcNAc pyrophosphorylase (UAP), which catalyzes the conversion of GlcNAc to UDP-GlcNAc, was observed (Braden et al. 2020). The knockdown of UAP also led to upregulation of CHS-1 demonstrating a clear dependence of the two enzymes. Most likely, the upregulation of UAP is a compensatory mechanism with the goal to restore homeostasis in absence of CHS-1. The exact regulation of the feedback, however, remains to be investigated.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Ampasala DR, Zheng S, Zhang D, Ladd T, Doucet D, Krell PJ, Retnakaran A, Feng Q. 2011. An epidermis-specific chitin synthase cDNA in Choristoneura fumiferana: Cloning, characterization, developmental and hormonal-regulated expression. Arch Insect Biochem Physiol. 76(2):83–96. doi:10.1002/arch.20404.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Ampasala DR, Zheng S, Zhang D, Ladd T, Doucet D, Krell PJ, Retnakaran A, Feng Q. 2011. An epidermis-specific chitin synthase cDNA in Choristoneura fumiferana: Cloning, characterization, developmental and hormonal-regulated expression. Arch Insect Biochem Physiol. 76(2):83–96. doi:10.1002/arch.20404.</span></p>
<p><span style="font-size:14px">Arakane, Y.; Muthukrishnan, S.; Kramer, K. J.; Specht, C. A.; Tomoyasu, Y.; Lorenzen, M. D.; Kanost, M.; Beeman, R. W. The Tribolium Chitin Synthase Genes TcCHS1 and TcCHS2 Are Specialized for Synthesis of Epidermal Cuticle and Midgut Peritrophic Matrix. Insect Mol. Biol. 2005, 14 (5), 453–463. https://doi.org/10.1111/j.1365-2583.2005.00576.x.</span></p>
<p><span style="font-size:14px">Binnington KC. 1985. Ultrastructural changes in the cuticle of the sheep blowfly, <em>Lucilia</em>, induced by certain insecticides and biological inhibitors. Tissue Cell. 17(1):131–140. doi:10.1016/0040-8166(85)90021-7.</span></p>
<p><span style="font-size:14px">Braden L, Michaud D, Igboeli OO, Dondrup M, Hamre L, Dalvin S, Purcell SL, Kongshaug H, Eichner C, Nilsen F, et al. 2020. Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity. Int J Parasitol. 50(10–11):873–889. doi:10.1016/j.ijpara.2020.06.007. https://doi.org/10.1016/j.ijpara.2020.06.007.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></p>
<p><span style="font-size:14px">Kuwano E, Cohen E. 1984. The use of a <em>Tribolium</em> 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.</span></p>
<p><span style="font-size:14px">Li, T.; Chen, J.; Fan, X.; Chen, W.; Zhang, W. MicroRNA and DsRNA Targeting Chitin Synthase A Reveal a Great Potential for Pest Management of the Hemipteran Insect Nilaparvata Lugens. Pest Manag. Sci. 2017, 73 (7), 1529–1537. https://doi.org/10.1002/ps.4492.</span></p>
<p><span style="font-size:14px">Locke M, Huie P. 1979. Apolysis and the Turnover of Plasmamembrane Plaques during Cuticle formation in an Insect. Tissue Cell. 11(2):277–291. doi:10.1016/0040-8166(79)90042-9.</span></p>
<p><span style="font-size:14px">Merzendorfer H. 2006. Insect chitin synthases: A review. J Comp Physiol B Biochem Syst Environ Physiol. doi:10.1007/s00360-005-0005-3.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly <em>Lucilia cuprina</em>. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.</span></p>
<p><span style="font-size:14px">Wang Y, Fan HW, Huang HJ, Xue J, Wu WJ, Bao YY, Xu HJ, Zhu ZR, Cheng JA, Zhang CX. 2012. Chitin synthase 1 gene and its two alternative splicing variants from two sap-sucking insects, <em>Nilaparvata lugens</em> and <em>Laodelphax striatellus</em> (Hemiptera: Delphacidae). Insect Biochem Mol Biol. 42(9):637–646. doi:10.1016/j.ibmb.2012.04.009. http://dx.doi.org/10.1016/j.ibmb.2012.04.009.</span></p>
<p><span style="font-size:14px">Zhang, X.; Zhang, J.; Zhu, K. Y. Chitosan/Double-Stranded RNA Nanoparticle-Mediated RNA Interference to Silence Chitin Synthase Genes through Larval Feeding in the African Malaria Mosquito (Anopheles Gambiae). Insect Mol. Biol. 2010, 19 (5), 683–693. https://doi.org/10.1111/j.1365-2583.2010.01029.x.</span></p>
<p><span style="font-size:14px">Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, <em>Anopheles gambiae</em>. Insect Sci. 20(2):158–166. doi:10.1111/j.1744-7917.2012.01568.x.</span></p>
<p><span style="font-size:14px">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 <em>Bombyx mori</em>. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.</span></p>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>In all likelihood, this KER is applicable to the whole phylum of arthropods as they all depend on the synthesis of chitin and molting in order to develop.</span></p>
</div>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>In all likelihood, this KER is applicable to the whole phylum of arthropods as they all depend on the synthesis of chitin and molting in order to develop.</span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>This KER is applicable for organisms synthesizing chitin and molting in order to grow and develop, namely larval stages of insects and all life stages of crustaceans and arachnids.</span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>This KER is applicable to all sexes.</span></p>
<p><span style="font-size:14px"><strong>Chemical: </strong>Occurrence of a decrease in cticular chitin content as well as premature molting was observed after treatment with the pyrimidine nucleosides polyoxin D, polyoxin B and nikkomycin Z (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986; Tellam et al. 2000; Arakawa et al. 2008; Zhuo et al. 2014). However, studies causally linking both endpoints are lacking.</span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:14px">As the arthropod cuticle is a central part in the molting process, its proper composition is indispensable for a proper molt. The ecdysis motor program, the behavioral part of ecdysis, constitutes a distinct motor pattern to split and shed the old cuticle <!--[if supportFields]><span lang=EN-US
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:14px">As the arthropod cuticle is a central part in the molting process, its proper composition is indispensable for a proper molt. The ecdysis motor program, the behavioral part of ecdysis, constitutes a distinct motor pattern to split and shed the old cuticle <!--[if supportFields]><span lang=EN-US
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. As the cuticle supports muscular function <!--[if supportFields]><span
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->, it needs to possess a certain integrity in order to successfully molt. The integrity of the cuticle is also important after ecdysis as arthropods, such as insects and crustaceans, expand the new cuticle by swallowing air or water in order to build up pressure to split the old and expand the new exoskeleton and provide stability to the soft new cuticle <!--[if supportFields]><span
uptake at ecdysis in the western rock lobster","type":"article-journal","volume":"35"},"uris":["http://www.mendeley.com/documents/?uuid=7de2ab94-c05e-4646-957b-a900dc162056"]},{"id":"ITEM-3","itemData":{"DOI":"10.1016/0022-1910(61)90090-7","ISSN":"00221910","abstract":"Two
methods of estimating insect blood volume are discussed. A method based on
haemocyte counts before and after injection of a measured volume of saline is
shown to be invalid, whereas a method based on the dilution of amaranth dye by
the haemolymph gave repeatable and consistent results. The blood volume of
Schistocerca gregaria Forsk. rises during the latter half of an instar, and
attains its highest level just prior to ecdysis. This high blood volume is
maintained for about 24 hr after ecdysis, then falls sharply to a mid-instar or
adult value, which is constant under the conditions described herein. The
increase in blood volume is shown to be due partly to changes in the
distribution of water within the body, and not merely to an intake of water
from the exterior. During periods of dietary water deficiency, the blood of the
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. The arthropod cuticle mostly consists of chitin embedded in and crosslinked with a matrix of proteins <!--[if supportFields]><span
Molecular Biology and Biochemistry","id":"ITEM-1","issued":{"date-parts":[["2012"]]},"number-of-pages":"193-235","publisher":"Elsevier
B.V.","title":"Chitin Metabolism in
EN-US;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. If the chitin content is too low, the cuticle may not possess enough integrity to support muscular function or withstand the beforementioned stresses of ecdysis, which leads to the organism being stuck in the old cuticle or the rupture of the new cuticle.</span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">The ecdysis motor program, the behavioral part of ecdysis, constitutes a distinct motor pattern to split and shed the old cuticle (Ayali 2009). As the cuticle supports muscular function (Vincent and Wegst 2004), it needs to possess a certain integrity in order to successfully molt. The integrity of the cuticle is also important after ecdysis as arthropods, such as insects and crustaceans, expand the new cuticle by swallowing air or water in order to build up pressure to expand the new exoskeleton and provide stability to the soft new cuticle (Clarke 1957; Lee 1961; Dall et al. 1978; deFur et al. 1985). The arthropod cuticle mostly consists of chitin embedded in and crosslinked with a matrix of proteins (Muthukrishnan et al. 2012). Given the well biological understanding of the processes, the biological plausibility can be regarded as high.</span></p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Empirical Evidence</strong>
<p><span style="font-size:14px">The cuticular chitin content was characterized <em>in vivo</em> in <em>Artemia salina </em>or using cultured integumental tissue from lepidopteran and dipteran insect species after exposure to polyoxin D and nikkomycin Z as well as the phthalimides captan, captafol, and folpet (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986; Zhuo et al. 2014). The event of premature molting was not assessed as endpoint in studies involving specific stressors rather than mentioned after exposure to polyoxin D, polyoxin B and nikkomycin Z (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008). However, results from studies where CHS-1 was knocked down by RNA interference support temporal concordance of the KER (Arakane et al. 2005, Li et al. 2017, Zhang X. et al. 2010). Given the support for temporal concordance and the lack of studies showing dose concordance, the empirical evidence for this KER was judged as moderate.</span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">The ecdysis motor program, the behavioral part of ecdysis, constitutes a distinct motor pattern to split and shed the old cuticle (Ayali 2009). As the cuticle supports muscular function (Vincent and Wegst 2004), it needs to possess a certain integrity in order to successfully molt. The integrity of the cuticle is also important after ecdysis as arthropods, such as insects and crustaceans, expand the new cuticle by swallowing air or water in order to build up pressure to expand the new exoskeleton and provide stability to the soft new cuticle (Clarke 1957; Lee 1961; Dall et al. 1978; deFur et al. 1985). The arthropod cuticle mostly consists of chitin embedded in and crosslinked with a matrix of proteins (Muthukrishnan et al. 2012). Given the well biological understanding of the processes, the biological plausibility can be regarded as high.</span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:14px">The absence of studies (quantitatively) assessing premature molting constitutes a major data gap. A further data gap is the absence of studies which assess both, the decrease in cuticular chitin content and the increase in premature molting.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:14px">Due to the lack of studies linking the decrease in cuticular chitin content with the increase in premature molting, it is not possible to describe the nature of the response-response relationship.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:14px">The cuticular chitin content was characterized <em>in vivo</em> in <em>Artemia salina </em>or using cultured integumental tissue from lepidopteran and dipteran insect species after exposure to polyoxin D and nikkomycin Z as well as the phthalimides captan, captafol, and folpet (Gijswijt et al. 1979; Turnbull and Howells 1982; Calcott and Fatig 1984; Gelman and Borkovec 1986; Zhuo et al. 2014). The event of premature molting was not assessed as endpoint in studies involving specific stressors rather than mentioned after exposure to polyoxin D, polyoxin B and nikkomycin Z (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008). Evidence from studies which assess and link both endpoints is lacking.</span></p>
<strong>Time-scale</strong>
<p><span style="font-size:14px">Due to the nature of the process, premature molting onsets at the time of ecdysis after the decrease in cuticular chitin content.</span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:14px">The absence of studies (quantitatively) assessing premature molting constitutes a major data gap. A further data gap is the absence of studies which assess both, the decrease in cuticular chitin content and the increase in premature molting.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:14px">Due to the lack of studies linking the decrease in cuticular chitin content with the increase in premature molting, it is not possible to describe the nature of the response-response relationship.</span></p>
<strong>Time-scale</strong>
<p><span style="font-size:14px">Due to the nature of the process, premature molting onsets at the time of ecdysis after the decrease in cuticular chitin content.</span></p>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Arakane, Y.; Muthukrishnan, S.; Kramer, K. J.; Specht, C. A.; Tomoyasu, Y.; Lorenzen, M. D.; Kanost, M.; Beeman, R. W. The Tribolium Chitin Synthase Genes TcCHS1 and TcCHS2 Are Specialized for Synthesis of Epidermal Cuticle and Midgut Peritrophic Matrix. Insect Mol. Biol. 2005, 14 (5), 453–463. https://doi.org/10.1111/j.1365-2583.2005.00576.x.</span></p>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<p><span style="font-size:14px">Ayali A. 2009. The role of the arthropod stomatogastric nervous system in moulting behaviour and ecdysis. J Exp Biol. 212(4):453–459. doi:10.1242/jeb.023879.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Clarke KU. 1957. On the Increase in Linear Size During Growth in <em>Locusta Migratoria</em> L. Proc R Entomol Soc London Ser A, Gen Entomol. 32(1–3):35–39. doi:10.1111/j.1365-3032.1957.tb00361.x.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">deFur PL, Mangum CP, McMahon BR. 1985. Cardiovascular and Ventilatory Changes During Ecdysis in the Blue Crab <em>Callinectes Sapidus</em> Rathbun. J Crustac Biol. 5(2):207–215. doi:10.2307/1547867.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></p>
<p><span style="font-size:14px">Lee RM. 1961. The variation of blood volume with age in the desert locust (<em>Schistocerca gregaria</em> Forsk.). J Insect Physiol. 6(1):36–51. doi:10.1016/0022-1910(61)90090-7.</span></p>
<p><span style="font-size:14px">Li, T.; Chen, J.; Fan, X.; Chen, W.; Zhang, W. MicroRNA and DsRNA Targeting Chitin Synthase A Reveal a Great Potential for Pest Management of the Hemipteran Insect Nilaparvata Lugens. Pest Manag. Sci. 2017, 73 (7), 1529–1537. https://doi.org/10.1002/ps.4492.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly <em>Lucilia cuprina</em>. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Zhang, X.; Zhang, J.; Zhu, K. Y. Chitosan/Double-Stranded RNA Nanoparticle-Mediated RNA Interference to Silence Chitin Synthase Genes through Larval Feeding in the African Malaria Mosquito (Anopheles Gambiae). Insect Mol. Biol. 2010, 19 (5), 683–693. https://doi.org/10.1111/j.1365-2583.2010.01029.x.</span></p>
<p><span style="font-size:14px">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 <em>Bombyx mori</em>. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.</span></p>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/1744">Relationship: 1744: Increase, Premature molting leads to Increase, Mortality</a></h4>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Likely, this KER is applicable to the whole phylum of arthropods as they all depend on molting in order to develop.</span></p>
</div>
<p><span style="font-size:14px"><strong>Taxonomic: </strong>Likely, this KER is applicable to the whole phylum of arthropods as they all depend on molting in order to develop.</span></p>
<p><span style="font-size:14px"><strong>Life stage: </strong>This KER is applicable for organisms molting in order to grow and develop, namely larval stages of insects and all life stages of crustaceans and arachnids.</span></p>
<p><span style="font-size:14px"><strong>Sex: </strong>This KER is applicable to all sexes.</span></p>
<p><span style="font-size:14px"><strong>Chemical: </strong>Occurrence of premature molting and an increase in mortality observed after treatment with the pyrimidine nucleosides ( e.g. polyoxin D, polyoxin B and nikkomycin Z) (Gijswijt et al. 1979; Tellam et al. 2000; Tellam and Eisemann 2000; Arakawa et al. 2008; New Zealand Environmental Protection Authority 2015). However, studies causally linking both endpoints are lacking.</span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:14px">During molting, arthropods pause food uptake and in certain cases also respiration (Camp et al. 2014; Song et al. 2017a). If molting is disrupted and the organism is not able to shed the old exoskeleton, the organism may eventually die of starvation, suffocation or the rupture of the exoskeleton.</span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">In order to grow and develop, arthropods need to molt periodically (Heming 2018). Since molting is a determining point in arthropod development, the disruption of molting leads to increased mortality (Arakawa et al. 2008; Merzendorfer et al. 2012; Song et al. 2017a; Song et al. 2017b). During ecdysis, arthropods pause food intake and respiration (Camp et al. 2014; Song et al. 2017a). Therefore, if the molt cannot be completed, the organism may die of starvation or suffocation. Additionally, if the cuticle is immature, it may not withstand the stresses associated with ecdysis (Clarke 1957; Lee 1961; Dall et al. 1978; deFur et al. 1985), and the organism may die of desiccation or increased susceptibility to pathogens. Given the well understood biological processes, the biological plausibility of this KER was rated as high.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:14px">The event of premature molting is not well characterized. It gets mentioned as cause of death in studies with <em>Pieris brassicae, Spodoptera litura</em>, <em>Bombyx mori </em>and <em>Lucilia cuprina </em>after treatment with polyoxin D, polyoxin B, polyoxin AL (a mixture of polyoxins) and nikkomycin Z (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008). The increase in mortality was reported in studies with <em>Lucilia cuprina</em>,<em> Spodoptera litura</em> and <em>Bombyx mori </em>(Tellam et al. 2000; Tellam and Eisemann 2000; Arakawa et al. 2008). Evidence from studies which assess and link both endpoints, and therefore would support dose concordance, is lacking. However, results from studies where CHS-1 was knocked down by RNA interference support temporal concordance of the KER (Arakane et al. 2005, Li et al. 2017, <span style="font-family:Calibri,sans-serif">Chen et al., 2008; Mohammed et al., 2017; Shang et al., 2016; Wang et al., 2012, 2019; Yang et al., 2013; Ye et al., 2019; Zhai et al., 2017; Zhang et al., 2010</span>). Given the support for temporal concordance and the lack of studies showing dose concordance, the empirical evidence for this KER was judged as moderate.</span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:14px">The absence of studies (quantitatively) assessing premature molting constitutes a major data gap. A further data gap is the absence of studies which assess both, increase in premature molting and the increase in mortality are lacking.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:14px">Due to the lack of studies linking the increase in premature molting with the increase in mortality, it is not possible to describe the nature of the response-response relationship.</span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:14px">During molting, arthropods pause food uptake and in certain cases also respiration (Camp et al. 2014; Song et al. 2017a). If molting is disrupted and the organism is not able to shed the old exoskeleton, the organism may eventually die of starvation, suffocation or the rupture of the exoskeleton.</span></p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">In order to grow and develop, arthropods need to molt periodically (Heming 2018). Since molting is a determining point in arthropod development, the disruption of molting leads to increased mortality (Arakawa et al. 2008; Merzendorfer et al. 2012; Song et al. 2017a; Song et al. 2017b). During ecdysis, arthropods pause food intake and respiration (Camp et al. 2014; Song et al. 2017a). Therefore, if the molt cannot be completed, the organism may die of starvation or suffocation. Additionally, if the cuticle is immature, it may not withstand the stresses associated with ecdysis (Clarke 1957; Lee 1961; Dall et al. 1978; deFur et al. 1985), and the organism may die of desiccation or increased susceptibility to pathogens. Given the well understood biological processes, the biological plausibility of this KER was rated as high.</span></p>
<strong>Time-scale</strong>
<p><span style="font-size:14px">Death occurs after premature molting. However, an exact time frame in which death occurs cannot be defined yet.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:14px">The event of premature molting is not well characterized. It gets mentioned as cause of death in studies with <em>Pieris brassicae, Spodoptera litura</em>, <em>Bombyx mori </em>and <em>Lucilia cuprina </em>after treatment with polyoxin D, polyoxin B, polyoxin AL (a mixture of polyoxins) and nikkomycin Z (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008). The increase in mortality was reported in studies with <em>Lucilia cuprina</em>,<em> Spodoptera litura</em> and <em>Bombyx mori </em>(Tellam et al. 2000; Tellam and Eisemann 2000; Arakawa et al. 2008). However, evidence of studies which assess and link both endpoints is lacking.</span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:14px">The absence of studies (quantitatively) assessing premature molting constitutes a major data gap. A further data gap is the absence of studies which assess both, increase in premature molting and the increase in mortality are lacking.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:14px">Due to the lack of studies linking the increase in premature molting with the increase in mortality, it is not possible to describe the nature of the response-response relationship.</span></p>
<strong>Time-scale</strong>
<p><span style="font-size:14px">Death occurs after premature molting. However, an exact time frame in which death occurs cannot be defined yet.</span></p>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<h4>References</h4>
<p><span style="font-size:14px">Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of <em>Bombyx mori</em> (Lepidoptera: Bombycidae), <em>Mamestra brassicae</em>, <em>Mythimna separata</em>, and <em>Spodoptera litura</em> (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173.</span></p>
<p><span style="font-size:14px">Camp AA, Funk DH, Buchwalter DB. 2014. A stressful shortness of breath: Molting disrupts breathing in the mayfly <em>Cloeon dipterum</em>. Freshw Sci. 33(3):695–699. doi:10.1086/677899.</span></p>
<p><span style="font-size:14px">Chen, X.; Tian, H.; Zou, L.; Tang, B.; Hu, J.; Zhang, W. Disruption of Spodoptera Exigua Larval Development by Silencing Chitin Synthase Gene A with RNA Interference. Bull. Entomol. Res. 2008, 98 (6), 613–619. https://doi.org/10.1017/S0007485308005932.</span></p>
<p><span style="font-size:14px">Mohammed, A. M. A.; DIab, M. R.; Abdelsattar, M.; Khalil, S. M. S. Characterization and RNAi-Mediated Knockdown of Chitin Synthase A in the Potato Tuber Moth, Phthorimaea Operculella. Sci. Rep. 2017, 7 (1), 1–12. https://doi.org/10.1038/s41598-017-09858-y.</span></p>
<p><span style="font-size:14px">Clarke KU. 1957. On the Increase in Linear Size During Growth in <em>Locusta Migratoria</em> L. Proc R Entomol Soc London Ser A, Gen Entomol. 32(1–3):35–39. doi:10.1111/j.1365-3032.1957.tb00361.x.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">deFur PL, Mangum CP, McMahon BR. 1985. Cardiovascular and Ventilatory Changes During Ecdysis in the Blue Crab <em>Callinectes Sapidus</em> Rathbun. J Crustac Biol. 5(2):207–215. doi:10.2307/1547867.</span></p>
<p><span style="font-size:14px">Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in <em>Pieris brassicae</em> (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.</span></p>
<p><span style="font-size:14px">Heming BS. 2018. Insect development and evolution. Ithaca: Cornell University Press.</span></p>
<p><span style="font-size:14px">Lee RM. 1961. The variation of blood volume with age in the desert locust (<em>Schistocerca gregaria</em> Forsk.). J Insect Physiol. 6(1):36–51. doi:10.1016/0022-1910(61)90090-7.</span></p>
<p><span style="font-size:14px">Merzendorfer H, Kim HS, Chaudhari SS, Kumari M, Specht CA, Butcher S, Brown SJ, Robert Manak J, Beeman RW, Kramer KJ, et al. 2012. Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species <em>Tribolium castaneum</em>. Insect Biochem Mol Biol. 42(4):264–276. doi:10.1016/j.ibmb.2011.12.008. http://dx.doi.org/10.1016/j.ibmb.2011.12.008.</span></p>
<p><span style="font-size:14px">New Zealand Environmental Protection Authority. 2015. Application for approval to import ESTEEM for release. https://www.epa.govt.nz/assets/FileAPI/hsno-ar/APP202334/fbce9a39e6/APP202334-APP202334-Staff-Report-Final-updated.pdf.</span></p>
<p><span style="font-size:14px">Shang, F.; Xiong, Y.; Xia, W. K.; Wei, D. D.; Wei, D.; Wang, J. J. Identification, Characterization and Functional Analysis of a Chitin Synthase Gene in the Brown Citrus Aphid, Toxoptera Citricida (Hemiptera, Aphididae). Insect Mol. Biol. 2016, 25 (4), 422–430. https://doi.org/10.1111/imb.12228.</span></p>
<p><span style="font-size:14px">Song Y, Evenseth LM, Iguchi T, Tollefsen KE. 2017b. Release of chitobiase as an indicator of potential molting disruption in juvenile <em>Daphnia magna</em> exposed to the ecdysone receptor agonist 20-hydroxyecdysone. J Toxicol Environ Heal - Part A Curr Issues. 80(16–18):954–962. doi:10.1080/15287394.2017.1352215. https://doi.org/10.1080/15287394.2017.1352215.</span></p>
<p><span style="font-size:14px">Song Y, Villeneuve DL, Toyota K, Iguchi T, Tollefsen KE. 2017a. Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development. Environ Sci Technol. 51(8):4142–4157. doi:10.1021/acs.est.7b00480.</span></p>
<p><span style="font-size:14px">Tellam RL, Eisemann C. 2000. Chitin is only a minor component of the peritrophic matrix from larvae of <em>Lucilia cuprina</em>. Insect Biochem Mol Biol. 30(12):1189–1201. doi:10.1016/S0965-1748(00)00097-7.</span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:14px">Wang, Z.; Yang, H.; Zhou, C.; Yang, W. J.; Jin, D. C.; Long, G. Y. Molecular Cloning, Expression, and Functional Analysis of the Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in the White-Backed Planthopper, Sogatella Furcifera (Hemiptera: Delphacidae). Sci. Rep. 2019, 9 (1), 1–14. https://doi.org/10.1038/s41598-018-37488-5.</span></p>
<p><span style="font-size:14px">Wang, Y.; Fan, H. W.; Huang, H. J.; Xue, J.; Wu, W. J.; Bao, Y. Y.; Xu, H. J.; Zhu, Z. R.; Cheng, J. A.; Zhang, C. X. Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants from Two Sap-Sucking Insects, Nilaparvata Lugens and Laodelphax Striatellus (Hemiptera: Delphacidae). Insect Biochem. Mol. Biol. 2012, 42 (9), 637–646. https://doi.org/10.1016/j.ibmb.2012.04.009.</span></p>
<p><span style="font-size:14px">Yang, W. J.; Xu, K. K.; Cong, L.; Wang, J. J. Identification, mRNA Expression, and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly, Bactrocera Dorsalis. Int. J. Biol. Sci. 2013, 9 (4), 331–342. https://doi.org/10.7150/ijbs.6022.</span></p>
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