The BLI-3/TSP-15/DOXA-1 Dual Oxidase Complex Is Required for Iodide Toxicity in Caenorhabditis elegans

Iodine is an essential trace element for life. Iodide deficiency can lead to defective biosynthesis of thyroid hormones and is a major cause of hypothyroidism and mental retardation. Excess iodide intake, however, has been linked to different thyroidal diseases. How excess iodide causes harmful effects is not well understood. Here, we found that the nematode Caenorhabditis elegans exhibits developmental arrest and other pleiotropic defects when exposed to excess iodide. To identify the responsible genes, we performed a forward genetic screen and isolated 12 mutants that can survive in excess iodide. These mutants define at least four genes, two of which we identified as bli-3 and tsp-15. bli-3 encodes the C. elegans ortholog of the mammalian dual oxidase DUOX1 and tsp-15 encodes the tetraspanin protein TSP-15, which was previously shown to interact with BLI-3. The C. elegans dual oxidase maturation factor DOXA-1 is also required for the arresting effect of excess iodide. Finally, we detected a dramatically increased biogenesis of reactive oxygen species in animals treated with excess iodide, and this effect can be partially suppressed by bli-3 and tsp-15 mutations. We propose that the BLI-3/TSP-15/DOXA-1 dual oxidase complex is required for the toxic pleiotropic effects of excess iodide.

Iodine is an essential trace element for life and a key component for the biogenesis of thyroid hormones. Iodine is unevenly distributed in nature. Insufficient intake of iodide is a major cause of thyroid hormone deficiency and can lead to severe developmental defects, including hypothyroidism and mental retardation (Nussey and Whitehead 2001). Mutations in enzymes involved in the generation of H 2 O 2 , the iodination of the thyroglobulin protein, and the recycling of iodide from mono-iodotyrosine and di-iodotyrosine are commonly found in congenital hypothyroidisms (de Vijlder 2003;Moreno et al. 2008;Park and Chatterjee 2005;Weber et al. 2013).
Although iodide deficiency is a major cause of human diseases, excess iodide intake has severe health consequences as well. Decades ago, Wolff and Chaikoff (1948) found that rats injected with an excess amount of iodide salt could develop an acute symptom that resembles hypothyroidism (the Wolff-Chaikoff effect). Excess iodide intake has been identified as a major risk factor for autoimmune thyroiditis (Bagchi et al. 1985;Rose et al. 1999;Rose et al. 1997;Teng et al. 2006), hyperthyroidism (Nussey and Whitehead 2001), hypothyroidism (Rose et al. 1999;Teng et al. 2006), and thyroid cancers (Blomberg et al. 2012;Dong et al. 2013;Guan et al. 2009;Lind et al. 1998). To date, the molecular mechanism that mediates the deleterious effects of excess iodide in humans is largely unknown.
The nematode Caenorhabditis elegans has provided a powerful system for dissecting the genetic mechanisms controlling animals' intrinsic biology and interaction with environmental factors. By studying the C. elegans response to excess iodide, we might gain new insights into the biological effects of excess iodide on other animals. In this study, we examined how excess iodide affects C. elegans development and identified three genes required for the effects. CB767 bli-3(e767) I (Brenner 1974) CB769 bli-1(e769) I (Brenner 1974) CB937 bli-4(e937) I (Brenner 1974) SP2275 tsp-15(sv15) I (Moribe et al. 2004) CSM421 duox-2(ok1775) I (this study, backcrossed five times) CSM296 bli-3(mac37) I (this study) CSM297 bli-3(mac38) I (this study) CSM299 bli-3(mac40) I (this study) CSM300 bli-3(mac41) I (this study) CSM292 tsp-15(mac33) I (this study) CSM291 mac32 I (this study) CSM294 mac35 I (this study) CSM295 mac36 I (this study) CSM301 mac42 I (this study) CSM302 mac43 I (this study) CSM303 mac44 I (this study) CB768 bli-2(e768) II (Brenner 1974) CB518 bli-5(e518) III (Brenner 1974) MT1655 bli-6(n776) IV (Park and Horvitz 1986) BE16 bli-6(sc16) IV (Park and Horvitz 1986) CSM418 ZK822.5(ok2281) IV (this study, backcrossed six times) CSM298 mac39 IV (this study) CB4856 (Hawaiian) (Wicks et al. 2001) C. elegans survival assay Five young adults were grown in an OP50-seeded NGM plate supplemented with chemicals (NaI, KI, or NaCl) at different concentrations and their F 1 progeny were observed. When the concentration of I 2 is high (e.g., at 5 mM), the F 1 progeny of wild-type P 0 animals would arrest at the larval stages and the bacterial food would be barely consumed on day 8. Animals that could survive and propagate in 5 mM I 2 would generate numerous progeny of mixed developmental stages and consume most or all of the bacterial food by day 8. We found that adding NaI stock solution to a premade NGM agar plate to a final concentration of 5 mM has the same effect on animal development as does a plate to which an equal amount of NaI was added during the preparation of the NGM media.
Genetic screen and mapping of mutations Synchronized L4 wild-type animals (P 0 ) were mutagenized with EMS (ethyl methanesulfonate) as described (Brenner 1974). F 1 progeny were allowed to grow to young adults in NGM plates seeded with OP50 bacteria and were transferred to plates containing 5 mM NaI. After 8 d, F 2 progeny were observed under a dissecting microscope to identify surviving adult animals. From $5000 F 1 ($10,000 haploid genomes) animals screened, we obtained 12 independent F 2 isolates.
We mapped the mutations to chromosomes using single nucleotide polymorphisms (SNPs) (Wicks et al. 2001) and previously described SNPs (Davis et al. 2005). Mutations mapped near each other were tested by genetic complementation for the survival of trans heterozygous animals in 5 mM NaI.
Observation of gonad development, cuticle shedding defect, and intestinal autofluorescence Bright field pictures of C. elegans gonads and cuticle blisters were taken using a digital camera attached to a Leica microscope. Pictures of unshedded cuticles were taken using a digital camera attached to an Olympus SZX16 dissecting microscope. Intestinal autofluorescence of excess iodide-treated animals were observed under an Olympus SZX16 dissecting microscope using either a GFP filter (excitation wavelength 460-495 nm) or an RFP filter (excitation wavelength 530-550 nm).
Hoechst 33258 staining Hoechst 33258 staining was performed as described (Moribe et al. 2004) with minor modifications. Synchronized young adult animals were washed off plates and incubated at room temperature with gentle shaking for 15 min with 1 mg/ml Hoechst 33258 (Sigma) diluted in M9. After staining, animals were washed three times with M9 and observed under a Leica DM5000B fluorescence microscope.

Measuring general reactive oxygen species
Whole-animal reactive oxygen species (ROS) production was measured using 29, 79-dichlorodihydrofluorescein diacetate (DCFDA) (D6883, Sigma), a fluorescence-base probe for general ROS (Halliwell and Gutteridge 2007) based on a method described previously (Gruber et al. 2011) with modifications. Synchronized animals at the L1 larval stage were allowed to recover on NGM agar plates for 8 hr with food and washed three times with H 2 O afterward. Approximately 150 to 300 animals were transferred to each well of a dark-walled 96-well plate containing ample bacterial food, 200 ml H 2 O, and 50 mM DCFDA with or without 5 mM NaI. DCFDA fluorescence intensity was measured using a fluoremeter (Synergy 2; BioTek) at the excitation wavelength of 485 nm and the emission wavelength of 528 nm every 10 min for 12 hr at room temperature. After fluorescence measurement, the exact number of animals in each well was counted. Fluorescence readings were normalized to bacterial controls on the same plate.
We normalized all raw control and experimental values against the mean of the control values (wild-type animals without iodide treatment) of the biological replicates. Statistics were performed using raw values.

Excess iodide causes pleiotropic defects in C. elegans
The effect of iodide on the model organism Caenorhabditis elegans is largely unknown, except that it can act as an attractant (Ward 1973). To investigate whether iodide affects C. elegans development, we examined F 1 progeny of wild-type young adults grown on OP50-seeded NGM plates containing different concentrations of NaI. We found that at concentrations below 1 mM, NaI had no apparent effect on animal development (Table 1). A stepwise increase of NaI concentrations from 1 mM to 10 mM incrementally suppressed the development of animals. When exposed to high concentrations of NaI, animals never reached adulthood and instead were arrested at or before the L3 (5 mM NaI) or L2 (10 mM NaI) larval stages based on body sizes. This result suggests that NaI has a dose-dependent development-arresting effect on C. elegans.
We treated synchronized animals of the L1 larval stage with 5 mM NaI (excess iodide hereafter) and identified the gonads of arrested animals ( Figure 1A). We found that the arms of most gonads extended less than halfway along the ventral sides, similar to the morphology of wild-type gonads between the L2 and L3 larval stages (Hirsh et al. 1976) and consistent the notion that these animals arrested at or before the L3 larval stage based on body size analysis.
To determine whether Na + plays a role in causing the developmental arrest, we replaced NaI with NaCl in the culture media. At comparable concentrations as that of NaI, NaCl had no apparent effect on animal development, suggesting that Na + does not cause the developmental arrest (Table 1). To understand whether this effect is limited to NaI, we tested KI. At comparable concentrations, KI is equally efficient as NaI in causing the developmental arrest (Table 1).
To examine whether biological activities of the OP50 bacteria might be involved in the development-arresting effect of NaI on C. elegans, we fed animals OP50 bacteria killed by heating (75°for 1 hr) and found that excess iodide caused a developmental arrest indistinguishable from that of animals grown on live OP50 (Z. Xu and L. Ma, unpublished observations), suggesting that iodide acts directly on animals. The effect of excess iodide on animal development is reversible, as some arrested larva could resume development and grow to healthy adults when placed in a regular culture plate without NaI (Z. Xu and L. Ma, unpublished observations). Taken together these results suggest that excess iodide is solely responsible for the development-arresting effect on C. elegans.
In animals treated with excess iodide, we identified two other apparent defects: a cuticle shedding defect and a premature accumulation of intestinal autofluorescence ( Figure 1). The cuticle shedding defect left some animals trapped in old cuticles ( Figure 1B, arrows), and approximately 8% of animals had this defect after 48-hr exposure n  to excess iodide ( Figure 1C). Animals exposed to excess iodide also developed strong intestinal autofluorescence ( Figure 1D), which reached $80% penetrance after 72-hr exposure ( Figure 1E). Animals not exposed to NaI rarely exhibited unshedded old cuticles (Z. Xu and L. Ma, unpublished observations) or intestinal autofluorescence (Figure 1D) at comparable time intervals. Therefore, excess iodide caused pleiotropic defects in C. elegans that include developmental arrest, cuticle shedding defect, and premature accumulation of intestinal autofluorescence.
A forward genetic screen identifies mutants that survive in excess iodide To investigate whether any genes are involved in the developmentarresting effect of excess iodide, we mutagenized P 0 animals with ethyl methanesulfonate and screened the progeny of $5000 F 1 animals for mutants that could survive in 5 mM NaI. We obtained 12 independent isolates from the screen. We mapped all 12 mutations using SNPs (see Materials and Methods). Genetic complementation tests suggest that these mutations might affect at least four genes ( Table 2).

Mutants that survive in excess iodide have defective cuticles
The mac33 mutation representing complementation group 1 (Table 2) caused an obviously blistered (Bli) phenotype ( Figure 2A). The mac40 mutants of complementation group 2 occasionally exhibited the Bli phenotype as well ( Figure 2A). The majority of the mutants, however, did not exhibit an obviously Bli phenotype. To determine whether these mutants had microscopic cuticle lesions that were not obvious under dissecting microscope, we stained animals with the nuclearbinding fluorescence dye Hoechst 33258, which was previously used as a sensitive detection for cuticle integrity (Moribe et al. 2004;Thein et al. 2009). As shown in Figure 2B, five isolates from complementation groups 1, 2, and 3, including mac33, mac38, mac40, mac42, and mac43, exhibited defective cuticle integrity (Figure 2A and B), suggesting that these mutants carry genetic lesions affecting C. elegans cuticle formation. This result raises the possibility that genes known to affect cuticle biogenesis might be involved in the development-arresting effect of excess iodide. Mutations affecting C. elegans cuticles generally cause several phenotypes that include blisters on cuticle (Bli), dumpy (Dpy), long (Lon), roller (Rol), and squat (Sqt) (Kramer 1997). Because we only detected an obviously Bli phenotype in the mac isolates, we postulate that some previously characterized Bli mutants might behave similarly as our isolates for survival in excess iodide. We tested a series of Bli mutants (Table 3), including bli-1(e769), bli-2(e768), bli-3(e767), bli-4 (e937), bli-5(e518) (Brenner 1974), bli-6(n776, sc16), (Park and Horvitz 1986) and tsp-15(sv15) (Moribe et al. 2004), and found that only bli-3 (e767) and tsp-15(sv15) mutants could survive in excess iodide (Table 3). Hence, a subset of genes involved in cuticle formation is also required for the development-arresting effect of excess iodide.
Three of our complementation groups were mapped to chromosome I (Table 2), on which both bli-3 and tsp-15 locate (www. wormbase.org). Genetic complementation tests between bli-3(e767) or tsp-15(sv15) and a mutation representing each of the three groups (Table 2, alleles in bold) indicate that mac40 is an allele of bli-3 and mac33 is an allele of tsp-15, respectively, whereas mac32 represents an unknown gene. Consistent with this finding, the blisters of mac33 and mac40 mutants are filled with cellular components ( Figure 2A) and resemble those of tsp-15(sv15) and bli-3(e767) mutants but are different from the clear blisters of bli-1(e769) mutants ( Figure 2A).
We determined the coding sequences of bli-3 in the isolates of the mac40 complementation group and identified a missense mutation in each of the four mutants ( Figure 3A). These mutations caused a G44S (mac41) change in the peroxidase domain, an S694F (mac38) change in the region between the first transmembrane domain and the EF hand domain, and an A1263T (mac40) change and an A1330V (mac37) change in the NADPH oxidase domain ( Figure 3A).
Similarly in mac33 mutants, we identified a missense mutation in tsp-15 that changes a nonconserved Gly249 to Arg in the fourth transmembrane domain of TSP-15 ( Figure 3B).
To verify the functions of bli-3 and tsp-15 in mediating the arresting effect of excess iodide, we reduced the expression of bli-3 or tsp-15 in animals using feeding RNAi. We found that these RNAi-treated animals exhibited the Bli phenotype (Z. Xu and L. Ma, unpublished observations) and acquired the ability for surviving in 5 mM NaI (Table 3). Together with genetic and molecular analyses above, this result suggests that functional losses in bli-3 or tsp-15 can suppress the development-arresting effect of excess iodide and the mac mutations in bli-3 or tsp-15 are loss-of-function mutations.
In addition to developmental arrest, excess iodide also caused cuticle shedding defect and premature accumulation of intestinal autofluorescence in animals (Figure 1). We found that bli-3(e767) and tsp-15(sv15) mutants treated with excess iodide did not exhibit these phenotypes (Z. Xu and L. Ma, unpublished observations), suggesting that these defects also require the activities of BLI-3 and TSP-15.
The C. elegans dual oxidase maturation factor DOXA-1 is required for the development-arresting effect of excess iodide DOXA-1 is the C. elegans ortholog of the mammalian dual oxidase maturation factor (Grasberger and Refetoff 2006;Morand et al. 2009) and forms a complex with BLI-3 and TSP-15 to regulate the biogenesis of H 2 O 2 (Moribe et al. 2012). We found that animals fed dsRNAs targeting doxa-1 were Bli (Z. Xu and L. Ma, unpublished observations) and could survive in 5 mM NaI (Table 3). Hence, each component of the BLI-3/TSP-15/DOXA-1 dual oxidase complex is required for the development-arresting effect of excess iodide in C. elegans.
Besides MLT-7, the C. elegans genome also encodes three other ShkT-domain-containing peroxidases named SKPO-1, SKPO-2, and SKPO-3 (Tiller and Garsin 2014), among which SKPO-3 is the most identical to MLT-7 (BLAST, www.wormbase.org). SKPO-1 is expressed in the hypodermis and protects C. elegans from the infection by bacterial pathogen Enterococcus faecalis, whereas SKPO-2 and SKPO-3 do not appear to have such a function (Tiller and Garsin 2014). We examined whether MLT-7 might function redundantly with the SKPO proteins in affecting the survival of animals in excess iodide (Supporting Information, Table S1). We used RNAi to reduce the expression of mlt-7 and each skpo gene individually or in combination. We did not observe survival of these RNAi-treated animals in excess iodide (Table S1). Therefore, MLT-7 and each of the SKPO proteins might not be individually or redundantly required for the development-arresting effect of excess iodide.
In C. elegans, duox-2 encodes a barely expressed paralog of BLI-3 that has a truncated C-terminal NADPH oxidase domain (Edens et al. 2001). A duox-2(ok1775) deletion mutant failed to survive in excess iodide ( Table 3), suggesting that duox-2 is not required for the development-arresting effect of excess iodide.
Finally, we tested two C. elegans genes (F52H2.4 and ZK822.5) that encode homologs (BLAST search, www.wormbase.org) of the mammalian Na/I symporter, a protein that actively transports iodide from the blood into the thyroid gland (Dai et al. 1996). We found that a ZK822.5(ok2281) deletion mutant (Table 3) and animals treated with F52H2.4(RNAi), ZK822.5(RNAi), or both RNAis (Table 3) were n Table 3 Differential effects of excess iodide on the survival of mutants and animals treated with RNAis

Excess iodide promotes ROS biogenesis
Because a conserved function of the BLI-3/TSP-15/DOXA-1 complex is to generate the reactive oxygen species H 2 O 2 (Moribe et al. 2012), we tested whether excess iodide would cause an altered ROS biogenesis, which might contribute to the aforementioned pleiotropic defects. We treated synchronized animals at the L1 larval stage with excess iodide and measured relative ROS biogenesis using 29, 79dichlorodihydrofluorescein diacetate (DCFDA), a fluorescence-base probe for general ROS (Gruber et al. 2011;Halliwell and Gutteridge 2007) (see Materials and Methods). As shown in Figure 4A, with a stepwise increase of the NaI concentration we detected a dose-dependent increase of the rate of ROS production in wild-type animals. It is interesting to note that 2.0 mM NaI caused a more than four-fold increase of the rate of ROS production ( Figure 4A). Animals were scrawny but can still survive in 2.0 mM NaI (Table 1), suggesting that C. elegans might have a high tolerance for ROS levels.
We next compared the rate of ROS production in the wild-type with that in bli-3 or tsp-15 mutant strains. Without iodide treatment, the rate of ROS production in the wild-type was indistinguishable from that in each mutant strain ( Figure 4B). This result is consistent with a previous finding that the loss of bli-3 function did not obviously affect ROS production in C. elegans (Chavez et al. 2009). With excess iodide, we detected a multiple-fold increase of the rate of ROS pro-duction in all strains ( Figure 4B). However, the increase in each bli-3 or tsp-15 mutant strain (six-fold to seven-fold) was significantly less than that in the wild-type (approximately nine-fold) ( Figure 4B). The total production of ROS (normalized DCFDA fluorescence intensity of the last measurement point) ( Figure 4C) also showed a significant reduction in each bli-3 or tsp-15 mutant strain compared with that in the wild-type. Therefore, bli-3 and tsp-15 mutations might partially suppress the increased ROS production caused by excess iodide.

DISCUSSION
In this study, we suggest that C. elegans might be an efficient genetic system for studying the biological effects of excess iodide. We found that excess iodide caused pleiotropic defects in C. elegans that include reversible developmental arrest, defective cuticle shedding, premature accumulation of intestinal autofluorescence, and dramatically increased production of ROS. We identified the conserved BLI-3/TSP-15/DOXA-1 dual oxidase complex to be required for the toxic effects of excess iodide.
BLI-3-dependent ROS production might contribute to iodide-induced ROS biogenesis in C. elegans The increase of iodide-induced ROS production was significantly less in bli-3 or tsp-15 mutants than that in wild-type animals (Figure 4, B and C). Nevertheless, excess iodide still caused several-fold increase of ROS production in these mutants, suggesting that BLI-3-independent mechanism(s) might also be involved in iodide-induced ROS biogenesis. We propose a model to explain the biological effects of excess iodide ( Figure 4D). In this model, iodide triggers an increased ROS biogenesis that is partially dependent on activity of the BLI-3/TSP-15/DOXA-1 complex and partially on unknown molecules. Together with other possible iodide forms (e.g., HOI, see below), the increased ROS biogenesis leads to the pleiotropic defects in C. elegans. Question marks in the model indicate that we have yet to provide experimental evidence supporting the involvement of HOI in the toxic effects of excess iodide.

Excess iodide might cause increased ROS biogenesis across species
That excess iodide causes increased ROS biogenesis is not a C. elegansspecific phenomenon. For example, several studies showed that treating thyroid tissues or thyrocytes with excess iodide resulted in increased ROS biogenesis (Corvilain et al. 2000;Golstein and Dumont 1996;Many et al. 1992;Serrano-Nascimento et al. 2014;Sharma et al. 2008;Vitale et al. 2000;Yao et al. 2012). Therefore, a coupled increase of ROS biogenesis in response to excess iodide might be a conserved biological phenomenon across species.
The chemical property of iodide (as an electron donor) suggests that it can function as a mild reducing agent. For example, iodide is a scavenger of a variety of ROS in Brown algae of the Laminariales (kelps) and appears to have similar functions in human blood cells (Kupper et al. 2008). It is intriguing that ROS biogenesis is dramatically increased in C. elegans treated with excess iodide. It is possible that the extra reducing activity of excess iodide might perturb C. elegans endogenous redox state, the homeostasis of which is likely essential for animal survival. In response, C. elegans increases ROS biogenesis accordingly to rebalance the endogenous redox state. Apparently this hypothesis raises a series of questions, e.g., how a cell detects iodide, how the signals are transmitted, what cells and genes are involved, etc. Future studies are warranted to address these questions.
Increased ROS biogenesis and other factors might function together to cause iodide-induced pleiotropic defects in C. elegans Our findings suggest that the dramatically increased ROS biogenesis might be a major cause of the pleiotropic defects in animals treated with excess iodide, as functional losses in the BLI-3/TSP-15/DOXA-1 complex could significantly reduce iodide-induced ROS biogenesis and suppress these defects. However, we could not exclude the possibility that other iodide forms, molecules, or pathways are also involved in mediating the toxic effects of excess iodide. Figure 4 General ROS biogenesis in animals treated with or without 5 mM NaI. (A) Hourly rate of ROS production (relative DCFDA fluorescence intensity unit, RFU) in wild-type animals treated with different concentrations of NaI. Statistics: different from animals without iodide treatment (control). Bars: SEs of nine biological replicates. Ã P , 0.05, ÃÃÃ P , 0.001 (Bonferroni test with one-way ANOVA). (B) Hourly rate of ROS production in different strains. Without iodide treatment, no significant difference was observed between the wild-type and each bli-3 or tsp-15 mutant. With 5 mM NaI treatment, the increase of the rate of ROS production in each mutant strain was significantly less than that in wild-type animals. Statistics: different from wild-type with the same treatment or difference of the same strain with no NaI (control) or 5 mM NaI treatment. Bars: SEs of five biological replicates. Ã P , 0.05, ÃÃ P , 0.01, ÃÃÃ P , 0.001 (two-tailed unpaired Student's t test). (C) Total ROS production per animal 12 hr after iodide treatment. Statistics: different from wild-type with the same treatment or difference of the same strain with no NaI (control) or 5 mM NaI treatment. Bars: SEs of five biological replicates. Ã P , 0.05, ÃÃ P , 0.01, ÃÃÃ P , 0.001 (two-tailed unpaired Student's t test). (D) A graphic model describing the toxic effect of excess iodide in C. elegans (see Discussion).
It was shown that iodide could be catalyzed by peroxidases into the toxic hypoiodous acid (HOI) in vitro (Magnusson et al. 1984a, b). HOI has bactericidal and antiviral activity (Fischer et al. 2011;Klebanoff 1967) and could inactivate proteins by iodination (Philpot and Small 1939). The mammalian airway mucosa uses dual oxidase-generated H 2 O 2 to convert iodide to HOI by lactoperoxidase-catalyzed oxidation (Fischer et al. 2011). The C. elegans genome encodes two close homologs of lactoperoxidase (Gotenstein et al. 2010) and several other similar proteins (Tiller and Garsin 2014), raising the possibility that in C. elegans iodide might be converted to HOI as well. Therefore, the toxicity of excess iodide could be a combined consequence of excess HOI and ROS in vivo (see Figure 4D).
C. elegans cuticle formation requires activities of the H 2 O 2 -generating BLI-3/TSP-15/DOXA-1 dual oxidase complex, the MLT-7 peroxidase that reduces H 2 O 2 to crosslink cuticle collagens, and the participation of numerous extracellular matrix components (Edens et al. 2001;Moribe et al. 2012;Page and Johnstone 2007;Thein et al. 2009). Functional losses in the BLI-3 dual oxidase complex or the MLT-7 peroxidase could cause cuticle crosslinking defects (Edens et al. 2001;Moribe et al. 2012;Thein et al. 2009). However, little is known about the effects of functional gains in these proteins. Excess iodide can cause a dramatically increased ROS biogenesis in C. elegans, which might mimic a functional gain in BLI-3 activity and shift the collagen-crosslinking reaction catalyzed by the MLT-7 peroxidase toward over-crosslinking. Over-crosslinked cuticles might not be degraded during molting, resulting in unshedded cuticles in animals treated with excess iodide.
Is it possible that bli-3, tsp-15, or doxa-1 mutants survive in excess iodide because the mutants have defective cuticles and therefore can shed them more easily? We found that bli-1, bli-2, bli-4, bli-5, bli-6, and mlt-7 mutants and/or RNAi-treated animals all had defective cuticles and were still arrested by excess iodide (Table 3). Therefore, the cuticle defect is not sufficient for these animals to survive in excess iodide.
The in vivo function of iodine in C. elegans remains to be understood Wild C. elegans animals are found in gardens, compost piles, and rotting fruits (Felix and Braendle 2010;Kiontke and Sudhaus 2006), and might have access to iodine by feeding on iodine-containing bacteria or environmental iodide compounds. The C. elegans genome contains genes encoding sodium/iodide symporter-like proteins (see above) and an ortholog of the iodotyrosine deiodinase (IYD) (de la Cruz et al. 2014;Friedman et al. 2006;Gnidehou et al. 2004;Moreno 2003), suggesting that iodide might function biologically in the animal. Iodide is not an essential ingredient in the standard C. elegans culture medium (Brenner 1974). However, the agar, a phycocolloid extracted from marine algae (Hitchens and Leikind 1939) used for growing the bacterial food (Brenner 1974) and other medium ingredients might contain residual iodide that is sufficient for animals to survive. Future studies might answer the question whether iodine is essential for C. elegans development.
In short, we provide evidence that C. elegans could serve as an efficient genetic system for studying the biological effect of excess iodide. We found that excess iodide has toxic pleiotropic effects on C. elegans and could cause a dramatic increase of in vivo ROS biogenesis. The BLI-3/TSP-15/DOXA-1 dual oxidase complex is required for the toxicity and is partially responsible for the increased ROS biogenesis. We suggest that genes interacting with the BLI-3/TSP-15/DOXA-1 dual oxidase complex might be identified, e.g., by screening for mutants that can survive in or are hypersensitive to excess iodide.