Introduction
Marine invertebrates are exposed to both acute and long-term thermal stress in intertidal and shallow waters during the juvenile and adult periods in their life histories (Pinsky et al., Reference Pinsky, Eikeset, McCauley, Payne and Sunday2019). Migration and acclimation are the main strategies for these thermally stressed animals to avoid decrease in function or mortality (Murren et al., Reference Murren, Auld, Callahan, Ghalambor, Handelsman, Heskel, Kingsolver, Maclean, Masel, Maughan, Pfennig, Relyea, Seiter, Snell-Rood, Steiner and Schlichting2015; Stillman & Armstrong, Reference Stillman and Armstrong2015; Veilleux et al., Reference Veilleux, Ryu, Donelson, Herwerden, Seridi, Ghosheh, Berumen, Leggat, Ravasi and Munday2015). Notably, both strategies are based on thermal perception. Thus, it is essential to explore the relationship between temperature stimuli and thermosensory systems under short- and long-term temperature stress in ecologically important marine invertebrates in intertidal and shallow waters.
Transient receptor potential (TRP) ion channels are key molecules in thermosensory systems and consequently important for the fitness of various organisms (Patapoutian et al., Reference Patapoutian, Peier, Story and Viswanath2003; Tominaga & Caterina, Reference Tominaga and Caterina2004). The temperature-responsive TRPs are designated thermo-TRPs. They consist of TRPV, TRPM and TRPA subfamilies (Patapoutian et al., Reference Patapoutian, Peier, Story and Viswanath2003; Jordt et al., Reference Jordt, Bautista, Chuang, McKemy, Zygmunt, Högestätt, Meng and Julius2004). TRPA1 is a non-selective cation channel that belongs to a TRPA subfamily with pivotal roles in initiating signal transduction of thermal perception (Roessingh & Stanewsky, Reference Roessingh and Stanewsky2017). Activated TRPA1 probably leads to Ca2+ signalling and neuronal excitability after cold detection (Jordt et al., Reference Jordt, Bautista, Chuang, McKemy, Zygmunt, Högestätt, Meng and Julius2004; Chen, Reference Chen2015). TRPA1 highly expresses in cold-sensitive neurons, and was proposed to function as a sensor of noxious cold (painful sensation caused by cold) in mice and humans (Story et al., Reference Story, Peier, Reeve, Eid, Mosbacher, Hricik, Earley, Hergarden, Andersson and Hwang2003; Bandell et al., Reference Bandell, Story, Hwang, Viswanath, Eid, Petrus, Earley and Patapoutian2004). In addition, TRPA1 is highly expressed in thermal sensory neurons of other vertebrates (Yun et al., Reference Yun, Son, Kim, Sang, Yi, Noguchi, Dong and Yong2010; Yonemitsu et al., Reference Yonemitsu, Kuroki, Takahashi, Mori, Kanmura, Kashiwadani, Ootsuka and Kuwaki2013) and invertebrates (Lee, Reference Lee2013; Luo et al., Reference Luo, Shen and Montell2016).
Despite the importance of TRPA1 in the thermal detection of terrestrial animals, it has been rarely proposed as a thermal detector in marine invertebrates. Saito et al. (Reference Saito, Hamanaka, Kawai, Furukawa, Gojobori, Tominaga, Kaneko and Satta2017) found that TRPA1 highly expressed in the neural ciliary band, as a thermal sensor, of the planktonic larvae of the starfish Patiria pectinifera. Acute and long-term thermal stimuli, however, were not involved in this study, leaving the role of thermal detection by TRPA1 uninvestigated. Furthermore, to our knowledge, TRPA1 has not been investigated in any intertidal or shallow water marine invertebrate.
Sea urchins are ecologically important in structuring marine benthic communities, both as grazers and prey (Pearse, Reference Pearse2006). Studies have reported that temperature changes significantly impact the fitness related traits of sea urchins (Wolfe et al., Reference Wolfe, Dworjanyn and Byrne2013; Uthicke et al., Reference Uthicke, Liddy, Nguyen and Byrne2014; Bodmer et al., Reference Bodmer, Wheeler, Hendrix, Cesarano, East and Exton2017). Further, these impacts on sea urchin fitness can extend to the whole marine benthic communities, highlighting their research value for marine ecology. The sea urchin Strongylocentrotus intermedius, as a typical cold-water invertebrate, is distributed in intertidal and shallow waters of Hokkaido of Japan, Korea, China and Far East Russia (Agatsuma, Reference Agatsuma and Lawrence2013). Considering its ecological importance and exposure to thermal stress, S. intermedius is an ideal research model to investigate the TRPA1 expression in response to temperature changes. Tube feet have strong sensory functions and have a complicated nerve plexus, responding to a variety of environmental stimuli (Sharp & Gray, Reference Sharp and Gray1962; Florey & Cahill, Reference Florey and Cahill1977; Ullrich-Lüter et al., Reference Ullrich-Lüter, Dupont, Arboleda, Hausen and Arnone2011).
The main aim of the present study is to investigate the distribution and temporal expression of TRPA1 of S. intermedius after acute and long-term temperature stimuli in order to provide new insights into thermal detection of marine invertebrates. We asked (1) whether SiTRPA1 expressed significantly more in tube feet than in other analysed tissues; (2) whether SiTRPA1 expression significantly responds to high and/or low temperature; and (3) whether SiTRPA1 expression significantly responds to acute temperature changes and/or long-term temperature treatments.
Materials and methods
Sea urchins
Strongylocentrotus intermedius were obtained from a local aquaculture farm and taken to the Key Laboratory of Mariculture & Stock Enhancement in the North China's Sea, Ministry of Agriculture and Rural Affairs at Dalian Ocean University. Nine sea urchins were used for analysis of SiTRPA1 in different tissues. Analysis of acute and long-term temperature stimuli were performed with 108 individuals in total (body weight = 9.99 ± 0.18 g). In order to ensure that the sea urchins were randomly selected for each experiment, individual sea urchins were placed into a labelled plastic container. A random number generator was used to obtain the sea urchins corresponding to the random number. The method was repeated until 108 individuals were randomly selected. The sea urchins were maintained at 16–17°C in the laboratory in tanks with inflowing ambient sand filtered sea water. The salinity was 28–30‰, the pH was 8.03–8.26 and dissolved oxygen was 6.2–7.0 mg l−1. The photoperiod was 12 h light: 12 h dark. Aeration was provided continuously. Sea urchins were not fed during the experiments
Sample collection for transcriptional analysis of SiTRPA1
In order to investigate the expression pattern of SiTRPA1 transcripts in different tissues of S. intermedius, tube feet from the aboral side (Lesser et al., Reference Lesser, Carleton, Böttger, Barry and Walker2011), coelomocytes, gonads and gut were carefully collected from nine S. intermedius (body weight = 23.41 ± 0.63 g). Tissues of 3 individuals were mixed as one sample (N = 3) (Li et al., Reference Li, Liu, Shang, Wang, Zhan, Song, Zhang and Chang2017). Coelomic fluid samples were centrifuged immediately at 3000 × rpm for 2 min (4°C). The tissue samples and the coelomocytes were immediately snap-frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction.
Acute temperature rise and decrease treatments
Generally, for S. intermedius the upper lethal temperature and optimal temperatures are ~23 and ~15°C, respectively (Chang et al., Reference Chang, Wang and Wang1999). Thus, two target temperatures were set as 12 and 22°C. Water temperatures in acute temperature increase and decrease treatments were increased from 17 to 22°C and decreased from 17 to 12°C within 2 h at a rate of 2.5°C h−1. Four independent samples were taken at 1, 3, 6 and 12 h after achieving the target temperatures (12 and 22°C). According to the experimental design, 72 sea urchins (body weight = 10.09 ± 0.22 g) were randomly selected and equally distributed into eight temperature-controlled tanks (750 × 430 × 430 mm; Huixin Co., Dalian, China) before the temperature change. Each temperature-controlled tank contained nine containers made of a plastic screen (200 × 200 × 400 mm, mesh size: 8 mm), each with an individual sea urchin. In addition, nine sea urchins (body weight = 9.45 ± 0.51 g) were individually placed into nine containers made of a plastic screen (200 × 200 × 400 mm, mesh size: 8 mm) in a temperature-controlled tank (17°C) as the control group. Tube feet were dissected from the aboral side (Lesser et al., Reference Lesser, Carleton, Böttger, Barry and Walker2011) at 1, 3, 6 and 12 h after the target had been reached (three individuals mixed as one sample, N = 3) (Li et al., Reference Li, Liu, Shang, Wang, Zhan, Song, Zhang and Chang2017). All samples were immediately frozen by liquid nitrogen and stored at −80°C for subsequent RNA extraction.
Long-term high and low temperature treatments
Three treatments were set in the long-term temperature stimuli experiment as: high temperature (20°C), control temperature (15°C) and low temperature (10°C). Twenty-seven sea urchins (body weight = 9.92 ± 0.34 g) were randomly selected and equally distributed into three treatments. Nine individuals of each treatment were placed into individual containers made of a plastic screen (200 × 200 × 400 mm, mesh size: 8 mm) inside three temperature-controlled tanks (750 × 430 × 430 mm; Huixin Co., Dalian, China). Prior to the experiments, the sea urchins were maintained under 17°C for 24 h. After this period, seawater temperature was increased or decreased at a rate of 0.5°C day−1 to the target temperatures (20, 15 and 10°C). After the target temperatures were reached, the sea urchins were cultured for 6 weeks. At the end of the experiment, tube feet of each individual were collected from the aboral side (Lesser et al., Reference Lesser, Carleton, Böttger, Barry and Walker2011) (three individuals mixed as one sample, N = 3) (Li et al., Reference Li, Liu, Shang, Wang, Zhan, Song, Zhang and Chang2017). All samples were immediately frozen in liquid nitrogen and stored at −80°C before RNA extraction.
Total RNA extraction and cDNA synthesis
Total RNA was extracted using the RNAprep Pure Tissue Kit (Tiangen, China) according to the manufacturer's instructions. The integrity of RNA was assessed by agarose gel electrophoresis. The concentration and quality of RNA were determined using a NanoPhotometer (Implen GmbH, Germany). The cDNA was synthesized using PrimeScript™ RT reagent Kit (TaKaRa, Japan). The PCR reaction system was carried out in a total volume of 20 µl composed of 1000 ng total RNA, 4 µl 5 × PrimeScript™ buffer, 50 pmol Oligo dT Primer, 100 pmol Random 6 mers and 1 µl PrimeScript RT Enzyme Mix I. The thermal condition of reaction was incubated at 37°C for 15 min, and then at 85°C for 5 s to deactivate the enzyme. All cDNA products were stored at −20°C for subsequent SYBR Green fluorescent quantitative real-time PCR.
Transcriptional analysis of SiTRPA1 by quantitative real-time PCR
The SiTRPA1 expression levels were analysed with quantitative real-time PCR (qRT-PCR), which was conducted with the Applied Biosystem 7500 Real-time System (Applied Biosystems, Foster City, CA, USA). Gene-specific primers were designed for the SiTRPA1 fragments using Primer Premier 5.0 software. The 18 S gene was used as the reference gene in this study (Table 1) (Zhou et al., Reference Zhou, Bao, Dong, Wang, He and Liu2008; Han et al., Reference Han, Ding, Wang, Zuo, Quan, Fan, Liu and Chang2019). According to the manufacturer's instructions, real-time PCR was carried out in a total volume of 20 µl containing 1 µl of 1:5 diluted original cDNA, 7 µl ddH2O, 10 µl of 2 × SYBR Green Master mix (SYBR PrimeScript × RT-PCR kit II, TaKaRa, Japan), 0.4 µl of ROX Reference Dye II, 0.4 µM of each primer (Table 1). The qRT-PCR cycling protocol was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 32 s. At the end of the PCR reactions, melting curve analysis of the amplification products confirmed that a single PCR product was present in these reactions (Han et al., Reference Han, Ding, Wang, Zuo, Quan, Fan, Liu and Chang2019). The comparative Ct method (2−△△CT method) was used to calculate the relative expression levels of the target genes (Livak & Schmittgen, Reference Livak and Schmittgen2001).
PCR primers used in this study
Statistical analysis
Data were tested for normal distribution and homogeneity of variance before statistical analysis. Gene expression was analysed using one-way ANOVA. When a significant effect was found, Duncan multiple comparisons were conducted to compare significant differences among treatments. All data are expressed as mean values ± standard error (mean ± SE). All statistical analysis was performed using SPSS 16.0 statistical software. The level of significance was considered as P < 0.05.
Results
Tissues distribution of SiTRPA1 mRNA
The expression of SiTRPA1 in tube feet was 235.7-fold, 450.0-fold and 3299.7-fold significantly higher than those in the coelomocytes, gonads and gut (df = 3, F = 47.382, P < 0.001, Figure 1). But the SiTRPA1 expression were not significantly different among coelomocytes, gonads and gut (df = 3, F = 47.382, P = 0.972, Figure 1).
Gene expression of SiTRPA1 in different tissues (N = 3, mean ± SE).
Expression pattern of SiTRPA1 after acute temperature increase stimuli
No significant difference of SiTRPA1 expression was found among all groups in the acute temperature increase experiment (df = 4, F = 0.25, P = 0.902, Figure 2).
Gene expression of SiTRPA1 of tube feet (from the aboral side) in different time points after acute temperature rise treatment (N = 3, mean ± SE).
Expression pattern of SiTRPA1 after acute temperature decrease stimuli
The expression of SiTRPA1 showed no significant difference among all groups in the acute temperature decrease experiment (df = 4, F = 1.802, P = 0.205) (Figure 3).
Gene expression of SiTRPA1 of tube feet (from the aboral side) in different time point of acute temperature decrease treatment (N = 3, mean ± SE).
Long-term effects of high and low temperatures on SiTRPA1 expression pattern
In the long-term temperature experiment, SiTRPA1 expression in the low temperature group (10°C) was significantly higher than those in the high temperature (20°C) and control (15°C) groups (df = 2, F = 9.57, P = 0.014, Figure 4). However, there was no significant difference in the expression of SiTRPA1 between the high temperature (20°C) and control group (15°C) (df = 2, F = 9.57, P = 0.808, Figure 4).
Gene expression of SiTRPA1 of tube feet (from the aboral side) in different long-term temperature treatments (N = 3, mean ± SE).
Discussion
During the process of thermal perception, expression of TRPA1 is significantly highest in the organs that contain the most abundant sensory neurons (Lee, Reference Lee2013; Luo et al., Reference Luo, Shen and Montell2016) in a number of animals, including the antennae of the insect Apolygus lucorum (Fu et al., Reference Fu, Yang, Liu and Wang2015) and the ciliary band of the starfish P. pectinifera larvae (Saito et al., Reference Saito, Hamanaka, Kawai, Furukawa, Gojobori, Tominaga, Kaneko and Satta2017). In the present study, the highest expression of SiTRPA1 was detected in the tube feet among the experimented tissues of S. intermedius. Similar to antennae of A. lucorum and ciliary band of starfish P. pectinifera, tube feet as a functional tissue are essential for the sea urchin's locomotion and feeding behaviour (Ullrich-Lüter et al., Reference Ullrich-Lüter, Dupont, Arboleda, Hausen and Arnone2011). This indicates that tube feet play an essential role in thermal perception of S. intermedius, although radial nerves and nerve ring tissues were not involved in the present study. However, those tissues lack well-developed musculature and sensory neurons would be insensitive to external temperature changes (Sharp & Gray, Reference Sharp and Gray1962; Florey & Cahill, Reference Florey and Cahill1977). This would account for the very low level of SiTRPA1 expression in coelomocytes, gonads and gut compared with tube feet.
Generally, TRPA1 is well documented as a cold receptor rather than a heat receptor (Bandell et al., Reference Bandell, Story, Hwang, Viswanath, Eid, Petrus, Earley and Patapoutian2004; Jordt et al., Reference Jordt, Bautista, Chuang, McKemy, Zygmunt, Högestätt, Meng and Julius2004; Caspani & Heppenstall, Reference Caspani and Heppenstall2009). Consistently, SiTRPA1 expression was insensitive to high temperature stimuli (acute temperature and long-term increase temperature) in S. intermedius. Chen et al. (Reference Chen, Kang, Xu, Lake, Hogan, Sun, Walter, Yao and Kim2013) reported that the Glycine (G) residue is critical for cold-activation of TRPA1 (e.g. rat), whereas an equivalent Valine (V) residue is preserved in heat-insensitive (e.g. human), as well as in heat-activated TRPA1 channel (e.g. rattlesnake). This suggests that the structure of the TRPA1 channel itself exhibits divergence between different species. SiTRPA1 expression was insensitive to high temperature stimuli in S. intermedius. It is essential to sequence transcripts of SiTRPA1 to further test this phenomenon in the future. Another possibility is that other members of the thermo-TRPs rather than SiTRPA1 may be involved in the heat perception. Caterina et al. (Reference Caterina, Schumacher, Tominaga, Rosen, Levine and Julius1997) reported that heat-activated nociceptors are characterized by the expression of the heat- and capsaicin-activated channel TRPV1. In addition, TRPV1−/− mice significantly reduced or even lost their perception of heat (Caterina, Reference Caterina2007). TRPV1 blockers could increase the body temperature in animals and humans, suggesting that TRPV1 is a necessary thermosensor involved in body temperature regulation (Caterina, Reference Caterina2007; Cheng et al., Reference Cheng, Yang, Takanishi and Zheng2007; Gavva et al., Reference Gavva, Treanor, Garami, Fang, Surapaneni, Akrami, Alvarez, Bak, Darling, Gore, Jang, Kesslak, Ni, Norman, Palluconi, Rose, Salfi, Tan, Romanovsky and Davar2008). Further, Chen (Reference Chen2015) inferred that the gain or loss of heat sensitivity of TRPA1 may coincide with the emergence of other thermal sensitive TRP ion channels such as TRPV1. Consistent with Chen's (Reference Chen2015) finding, Vandewauw et al. (Reference Vandewauw, De Clercq, Mulier, Held, Pinto, Van Ranst, Segal, Voet, Vennekens, Zimmermann, Vriens and Voets2018) proved that TRPA1, TRPV1 and TRPM3 of mice had critical but redundant roles in heat perception, in which the functionality of at least one of the three channels was necessary and sufficient to sustain robust heat responses in sensory neurons. The presence of three redundant molecules in the nociceptors with overlapping expressions of heat-sensing mechanism neurons represents a powerful fail-safe mechanism (Vandewauw et al., Reference Vandewauw, De Clercq, Mulier, Held, Pinto, Van Ranst, Segal, Voet, Vennekens, Zimmermann, Vriens and Voets2018). Based on these findings, we infer that the similar powerful fail-safe mechanism may exist in S. intermedius. This information provides new insights into our understanding of why SiTRPA1 expression was not affected by high temperature stimuli (acute temperature increase and long-term elevated temperature). Further investigations are essential to test it.
Although SiTRPA1 expression was influenced by long-term cold stimulation, it did not respond to acute cold stimulation. Story et al. (Reference Story, Peier, Reeve, Eid, Mosbacher, Hricik, Earley, Hergarden, Andersson and Hwang2003) found that TRPA1 and TRPM8 act as primary cold sensors in two distinct subsets of cold-sensitive trigeminal ganglion neurons of mice. Consistently, Chen (Reference Chen2015) inferred that the establishment of TRPM8 may allow TRPA1 to diverge from acute cold sensing and concentrate on another core function of sensing of noxious, reactive electrophiles. Further, Vandewauw et al. (Reference Vandewauw, De Clercq, Mulier, Held, Pinto, Van Ranst, Segal, Voet, Vennekens, Zimmermann, Vriens and Voets2018) reported that the TRPM8 was preserved in the trigeminal ganglia of TRPV1−/−TRPM3−/−TRPA1−/− triple knockout mice, thus accepting acute cold stimuli. All the findings above support our finding that SiTRPA1 expression is insensitive to acute cold stimulation and may be related to the presence of SiTRPM8, although the mechanism remains unknown.
In conclusion, the present study is the first investigation on the distribution and expression of SiTRPA1 after acute and long-term temperature stimuli in adult marine invertebrates. Unsurprisingly, the highest expression of SiTRPA1 was detected in tube feet among the tissues analysed. SiTRPA1 expression was insensitive to high temperature stimuli (acute temperature increase and long-term high temperature exposure). Further, SiTRPA1 expression significantly responded to long-term low temperature treatment, although it did not significantly respond to acute temperature decrease stimulation. This present study provides new insights into the thermal detection of sea urchins.
Acknowledgements
We thank Prof. John Lawrence and Prof. Hong Liang for academic and editorial suggestions. All authors have no conflict of interest.
Financial support
This work was supported by the National Natural Science Foundation of China (41506177), Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, China P.R. (2017-1B05), Key Research and Development Program of Liaoning Province (2017203006), Ocean and Fisheries research grants of Liaoning Province (201826, 201818) and a grant from Dalian Key Laboratory of Genetic Resources for Marine Shellfish.
