Effect of Polycyclic Aromatic Hydrocarbons on Development of the Ascidian Ciona intestinalis Type A

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that exert harmful effects on marine invertebrates; however, the molecular mechanism underlying PAH action remains unclear. We investigated the effect of PAHs on the ascidian Ciona intestinalis type A (Ciona robusta). First, the influence of PAHs on early Ciona development was evaluated. PAHs such as dibenzothiophene, fluorene, and phenanthrene resulted in formation of abnormal larvae. PAH treatment of swimming larva induced malformation in the form of tail regression. Additionally, we observed the Ciona aryl hydrocarbon receptor (Ci-AhR) mRNA expression in swimming larva, mid body axis rotation, and early juvenile stages. The time correlation between PAH action and AhR mRNA expression suggested that Ci-AhR could be associated with PAH metabolism. Lastly, we analyzed Ci-AhR mRNA localization in Ciona juveniles. Ci-AhR mRNA was localized in the digestive tract, dorsal tubercle, ganglion, and papillae of the branchial sac, suggesting that Ci-AhR is a candidate for an environmental pollutant sensor and performs a neural function. Our results provide basic knowledge on the biological function of Ci-AhR and PAH activity in marine invertebrates.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are chemicals that contain multiple aromatic rings and primarily are environmental contaminants [1,2]. PAHs are released into the air via incomplete combustion of wood, coal, and petroleum products [1,2]. Airborne PAHs enter the ocean via both dry and wet precipitation [3]. In addition, since PAHs are present in crude petroleum, accidental oil spillage from oil tankers and offshore oilfield results in severe PAH contamination in the ocean [4,5]. Polluted seawater containing PAHs affects aquatic animals. PAHs were reported to induce oncogenesis, immune system disorders, and developmental defects in aquatic animals [6,7]. Therefore, it is essential reagent (Thermo Fisher Scientific, Waltham, MA, USA) at 20, 42, and 66 h after fertilization, respectively. All specimens were stored at −80 • C until use. To obtain the whole-mount in situ hybridization (WISH) sample, juvenile specimens 1-2 mm in length were collected and fixed in buffer containing 4% paraformaldehyde at 4 • C, as described by Ogasawara et al. [31]. After dehydration using ethanol, all specimens were maintained at −20 • C in 80% ethanol until the experiment.

Chemicals
Dibenzothiophene, fluorene, and phenanthrene were purchased from Sigma-Aldrich (St. Louis, MI, USA). Each reagent was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mM to obtain stock solutions.

Exposure Experiments
Stock solutions were serially diluted with DMSO to create a dilution series. A total of 0.2% of each solution was added to artificial seawater warmed to 40 • C to produce exposure solutions that were subsequently used at 18 • C. Nominal test concentrations were 100, 50, 25, 12.5, and 6.25 µM. Artificial seawater containing 0.2% DMSO was used as a solvent reference. Exposures were carried out in 6-well plastic tissue culture plates. Each well contained 10 ml exposure solution. Twenty microliters of artificial seawater containing approximately fifty fertilized eggs or swimming larvae were added to the exposure solutions. Fertilized eggs were collected before the first cleavage and used for conducting exposure experiments from fertilized eggs to swimming larvae. Embryos were fixed 22 h after fertilization by adding paraformaldehyde solution to artificial seawater and evaluated for rates of malformation. For exposure experiments on swimming larvae to early juvenile stages, swimming larvae were collected 20 h after fertilization and used in the exposure experiments. During the exposure experiments, larvae metamorphosed into early juveniles. Early juveniles were fixed 90 h after fertilization and examined for the ratio of tail residue length to body length. The exposure experiment was repeated five times, and the average and standard deviation of the measurement results were calculated. The significance test was performed between multiple groups using one-way analysis of variance appropriately. Moreover, the Dunnett's test was used to determine post-hoc differences. The significance level was accepted at p-value < 0.05.

Reverse-Transcription (RT)-PCR of Ci-AhR mRNA
Total RNA was extracted at the swimming larvae, mid body axis rotation, and early juvenile stages using TRIZOL reagent (Thermo Fisher Scientific). cDNA was synthesized from 500 ng total RNA using Superscript III (Thermo Fisher Scientific) and oligo dT (20) primer. Ci-AhR cDNA was obtained by RT-PCR using EX-taq polymerase (Takara, Shiga, Japan). Since Ci-AhR was previously identified and annotated by Satou and Satoh [32], the primers were designed from the Ci-AhR cDNA sequence deposited in GenBank™/EBI Data Bank (Accession number, AB210300.1). Ci-EF1α (Accession number, AK113163.1) was used as an internal control for RT-PCR to confirm the quality and quantity of cDNA. Ci-AhR primers (forward primer: 5'-CCAGCAAGTCAAACCCAAGC-3'; reverse primer: 5'-AAGTCCCGGTCTTCAGCATG-3') and Ci-EF1α primers (forward primer: 5'-TTTCGCTGTCCGTGACATGA-3'; reverse primer: 5'-CCGCAACCAACCACGTTAAG-3') were selected using the primer3 Plus web site [33]. The PCR conditions were as follows: 94 • C for 1 min, 35 cycles of 94 • C for 30 s, 60 • C for 30 s, 72 • C for 1 min, and final extension at 72 • C for 7 min. The PCR products were detected using electrophoresis on 1.5% agarose gel. Sanger sequence analysis was performed to confirm that the PCR fragments isolated were Ci-AhR and Ci-EF1α.

Exposure Experiments from Fertilized Eggs to Swimming Larvae
In ascidian development, the embryo first develops into a swimming larva, which then undergoes metamorphosis to form an adult-like juvenile. Swimming larvae consist of a trunk and a tail. The trunk contains the brain vesicle and adult organ primordia, and the tail contains the notochord and muscles. During metamorphosis, the ascidian larvae attach to the substratum via the adhesive papillae on the trunk, absorb the tail, and develop adult organs in the trunk. Here, we examined the effects of PAHs (dibenzothiophene, fluorene, and phenanthrene) on early ascidian development from fertilized eggs to swimming larvae and during metamorphosis from swimming larvae to juveniles.
First, the larvae were treated with PAH, starting at the 1-cell stage to the swimming larva stage (20 h after fertilization). All three examined PAH types exerted a concentration-dependent adverse effect on larval development. While normal larvae have a straight tail, PAHs induce bending in the tail of larvae. Compared to the control, dibenzothiophene and fluorine administered at 100 µM and 50 µM significantly increased the percentage of abnormal larvae (Figure 1a,b). Phenanthrene significantly increased the percentage of abnormal larvae at concentrations of 100, 50, and 25 µM (Figure 1c).

Exposure Experiments from Swimming Larvae to Early Juvenile Stages
PAH treatment was conducted on larvae, starting with swimming larvae 20 h after fertilization to early juveniles 90 h after fertilization, and the effect of PAH on metamorphosis was examined. The effect of PAHs on the rate of normal metamorphosis could not be determined because the metamorphosis rate varied randomly. Next, we focused on the ratio of tail residue length to body length in early juveniles. The tail residue is a mass of tail cells absorbed by the trunk at the beginning of metamorphosis, and it normally shrinks as metamorphosis progresses. The ratio of the tail residue diameter to the juvenile body length was measured, and the measurement results were compared between the early juveniles treated with 100 µM of each PAH and the control early juveniles. The ratio of the diameter of the tail residues to the body length was significantly higher in the PAH-treated early juveniles than in the control early juveniles ( Figure 2). In particular, dibenzothiophene-treated early juveniles exhibited the greatest difference compared to control early juveniles. The results described above suggest that the three types of PAHs adversely affect tail absorption during metamorphosis.

Gene Expression of Ci-AhR during Early Metamorphosis
In vertebrates, AhR is a sensor protein of toxic agents such as PAHs and 2,3,7,8tetrachlorodibenzodioxin [21,22]. After AhR binds PAHs, AhR induces CYP1A1 gene transcription, which is a monooxygenase involved in PAH detoxication [21,22]. Since Ci-AhR is likely to be a putative PAH sensor as well, we evaluated Ci-AhR gene expression during metamorphosis. RT-PCR analysis revealed that Ci-AhR mRNA expression was detected in the swimming larval, mid body axis rotation, and early juvenile stages ( Figure 3).

Exposure Experiments from Swimming Larvae to Early Juvenile Stages
PAH treatment was conducted on larvae, starting with swimming larvae 20 hours after fertilization to early juveniles 90 hours after fertilization, and the effect of PAH on metamorphosis was examined. The effect of PAHs on the rate of normal metamorphosis could not be determined because the metamorphosis rate varied randomly. Next, we focused on the ratio of tail residue length dibenzothiophene-treated early juveniles exhibited the greatest difference compared to control early juveniles. The results described above suggest that the three types of PAHs adversely affect tail absorption during metamorphosis.

Gene Expression of Ci-AhR during Early Metamorphosis
In vertebrates, AhR is a sensor protein of toxic agents such as PAHs and 2,3,7,8tetrachlorodibenzodioxin [21,22]. After AhR binds PAHs, AhR induces CYP1A1 gene transcription, which is a monooxygenase involved in PAH detoxication [21,22]. Since Ci-AhR is likely to be a putative PAH sensor as well, we evaluated Ci-AhR gene expression during metamorphosis. RT-PCR analysis revealed that Ci-AhR mRNA expression was detected in the swimming larval, mid body axis rotation, and early juvenile stages ( Figure 3).

Gene Expression of Ci-AhR during Early Metamorphosis
In vertebrates, AhR is a sensor protein of toxic agents such as PAHs and 2,3,7,8tetrachlorodibenzodioxin [21,22]. After AhR binds PAHs, AhR induces CYP1A1 gene transcription, which is a monooxygenase involved in PAH detoxication [21,22]. Since Ci-AhR is likely to be a putative PAH sensor as well, we evaluated Ci-AhR gene expression during metamorphosis. RT-PCR analysis revealed that Ci-AhR mRNA expression was detected in the swimming larval, mid body axis rotation, and early juvenile stages (Figure 3).

Localization Analysis of Ci-AhR mRNA Using WISH
To further evaluate the expression pattern of Ci-AhR, we performed WISH analysis in Ciona juveniles. A strong Ci-AhR mRNA signal was observed in the digestive tract (Figure 4a). Ci-AhR mRNA was expressed in the anterior part of the esophagus (Figure 4c). The Ci-AhR transcript was detected in the entire stomach (Figure 4d). Ci-AhR mRNA was localized in the middle and posterior part of the intestine (Figure 4e,f). Moreover, the Ci-AhR gene was transcribed in the ganglion and dorsal tubercle (Figure 4g). Ci-AhR mRNA expression was detected in the gill papillae (Figure 4h). Lack of signal detection in the experiment using sense probe indicates that signals developed using antisense probe are specific (Figure 4b).
mRNA was expressed in the anterior part of the esophagus (Figure 4c). The Ci-AhR transcript was detected in the entire stomach (Figure 4d). Ci-AhR mRNA was localized in the middle and posterior part of the intestine (Figure 4e,f). Moreover, the Ci-AhR gene was transcribed in the ganglion and dorsal tubercle (Figure 4g). Ci-AhR mRNA expression was detected in the gill papillae (Figure 4h). Lack of signal detection in the experiment using sense probe indicates that signals developed using antisense probe are specific (Figure 4b).

Discussion
Marine invertebrates inhabit various marine environments, such as the intertidal zone, shallow sea, and deep-sea regions, and they are consumed as aquatic resources. The influence of marine pollutants on marine invertebrates is a vital subject in research on environmental preservation and marine resource conservation. PAH is a pollutant that is used in the investigation of biological effects owing to its carcinogenic, mutagenic, endocrine disruptive, and immunotoxic properties. However, the biological action and metabolic pathway of PAHs in marine invertebrates is yet to be characterized completely. Therefore, we have focused on these issues using Ciona intestinalis type A as a model animal for marine invertebrates.
We first evaluated the effect of PAHs on early development. Treatment of the fertilized egg with dibenzothiophene, fluorene, and phenanthrene resulted in the formation of abnormal larvae in a dose-dependent manner (Figure 1). Bellas et al. [13] examined the effects of PAHs on invertebrates, including Ciona intestinalis. They treated Ciona embryos with phenanthrene at stages ranging from fertilized eggs to swimming larvae and did not observe any toxic effects. Since the concentrations of phenanthrene they used were lower than the concentrations we used, their results are consistent with our results. Considered together, these results revealed that PAHs affect early C. intestinalis type A development. Furthermore, this study is the first to denote the toxic effect of dibenzothiophene on early development. Next, the effect of PAHs on metamorphosis was elucidated. Consistent with the results for early development, an abnormality was observed in early juveniles treated with dibenzothiophene, fluorene, and phenanthrene. Even though the metamorphosis defect was weaker than that in early development, there was an increase in the ratio of cell debris formed by tail regression to body length ( Figure 2). Ciona larvae swim for several hours to several days before they attach to a substrate, following which metamorphosis begins. Larval tail regression and body axis

Discussion
Marine invertebrates inhabit various marine environments, such as the intertidal zone, shallow sea, and deep-sea regions, and they are consumed as aquatic resources. The influence of marine pollutants on marine invertebrates is a vital subject in research on environmental preservation and marine resource conservation. PAH is a pollutant that is used in the investigation of biological effects owing to its carcinogenic, mutagenic, endocrine disruptive, and immunotoxic properties. However, the biological action and metabolic pathway of PAHs in marine invertebrates is yet to be characterized completely. Therefore, we have focused on these issues using Ciona intestinalis type A as a model animal for marine invertebrates.
We first evaluated the effect of PAHs on early development. Treatment of the fertilized egg with dibenzothiophene, fluorene, and phenanthrene resulted in the formation of abnormal larvae in a dose-dependent manner (Figure 1). Bellas et al. [13] examined the effects of PAHs on invertebrates, including Ciona intestinalis. They treated Ciona embryos with phenanthrene at stages ranging from fertilized eggs to swimming larvae and did not observe any toxic effects. Since the concentrations of phenanthrene they used were lower than the concentrations we used, their results are consistent with our results. Considered together, these results revealed that PAHs affect early C. intestinalis type A development. Furthermore, this study is the first to denote the toxic effect of dibenzothiophene on early development. Next, the effect of PAHs on metamorphosis was elucidated. Consistent with the results for early development, an abnormality was observed in early juveniles treated with dibenzothiophene, fluorene, and phenanthrene. Even though the metamorphosis defect was weaker than that in early development, there was an increase in the ratio of cell debris formed by tail regression to body length ( Figure 2). Ciona larvae swim for several hours to several days before they attach to a substrate, following which metamorphosis begins. Larval tail regression and body axis rotation occur successively [37]. The larval body plan converts to a juvenile body plan [37]. Debris derived from the larval tail is absorbed during the early juvenile stage [37]. The PAH-induced phenotype is expected to be induced by the suppression of larval tail absorption. Overall, we unveiled that PAHs disturb Ciona metamorphosis. Collectively, dibenzothiophene, fluorene, and phenanthrene exerted a toxic effect on Ciona development.
The metabolic pathway of PAHs has been characterized in vertebrates. Vertebrate AhRs bind to PAHs in the cytoplasm, following which AhR activates the transcription of the CYP1 family gene. PAHs are metabolized by the CYP1 family protein and eventually discharged from cells. Conversely, the PAHs metabolic pathway via AhR in marine invertebrates remains elusive and hence is a subject of significant interest. Recent genomic research has revealed that the AhR gene broadly exists in eumetazoa, including bilaterians, cnidarians, and placozoans [38]. Further, the expression of both AhR and CYP genes is induced by PAH treatment of marine invertebrates [39,40]. Since there are no reports of PAH binding to AhR [38], it is unclear how PAHs activate AhR in marine vertebrates. Induction of AhR and CYP genes by PAHs suggests that PAHs activate AhR by mechanisms yet to be determined, and AhR controls expression level of CYP genes as a transcription factor.
We selected Ciona as a model animal to investigate the biological and molecular functions of AhR in marine invertebrates as there are experiment systems to investigate the molecular function of transcriptional factors like Ci-AhR [41]. Gain-of-function and loss-of-function experiments using overexpression and genome-editing systems are available, respectively [27][28][29]. One AhR gene and five CYP1 family genes were identified in Ciona intestinalis type A [42]. The upstream nucleotide sequence of these CYP1 homolog genes contains the xenobiotic responsive element that acts as the putative binding site of Ci-AhR, suggesting that Ci-AhR might transactivate CYP1 homolog genes [42]. However, whether Ci-AhR activates transcription of Ci-CYP1 genes is yet to be evaluated. Moreover, the detailed expression pattern of Ci-AhR and CYP1 family genes remains unclear thus far.
We performed the expression analysis of AhR in Ciona intestinalis type A to reveal basic information on Ci-AhR function and the relationship between PAH action and AhR expression. Maternal AhR mRNA expression has been observed in the fertilized egg to the late tailbud stages in early development [41]. In this stage, PAHs disturbed normal development ( Figure 1). These results suggest that the defects induced by these chemicals in early development might be mediated by AhR. During the metamorphosis stage, Ci-AhR was expressed in the larval, mid body axis rotation, and early juvenile stages, implying that Ci-AhR might be associated with the tail regression defect (Figure 3). Lastly, we evaluated Ci-AhR transcript localization in the juvenile stage. Ci-AhR mRNA was expressed in the digestive organ, branchial sac papillae, and dorsal tubercle, which open to the exterior of the body (Figure 4). A similar expression pattern was observed in other marine invertebrates as well. For instance, in pearl oyster Pinctada martensii, the AhR gene transcript ortholog was detected in the mantle, gill, hepatopancreas, and adductor muscle [43]. It is hypothesized that Ci-AhR is involved in metabolism of PAHs entering from seawater because these organs directly connect to the body exterior. Ci-AhR mRNA localization in the ganglion, also known as the ascidian central nerve, is expected to correspond to neural development and neural function observed in Caenorhabditis elegans [44][45][46]. Qin et al. reported that the C. elegans AhR homolog is involved in the regulation of aggregation behavior [46]. Collectively, the results revealed the expression pattern of Ci-AhR mRNA in the developmental and juvenile stages. These expression analyses were performed under normal conditions. We intend to further analyze PAH-induced changes in the level and localization of Ci-AhR mRNA by using quantification PCR and WISH in the next study, respectively. Such information will help to further understand the role of Ci-AhR in PAH metabolism.

Conclusions
Our study revealed that fluorene and phenanthrene affect early development and metamorphosis in Ciona intestinalis type A. We identified dibenzothiophene as a toxic agent in Ciona development as well. To the best of our knowledge, this is first report of its kind on Ciona intestinalis type A. Moreover, we denoted the expression of Ci-AhR during metamorphosis; in particular, tissue distribution of Ci-AhR was denoted in juvenile Ciona. These findings provide basic information on the molecular mechanism underlying PAH action and the biological function of AhR in marine invertebrates.