Expression,of,mapk1,and,egr1,genes,in,Onchidium,reevesii,under,tidal,stimulation

Rongyu Wu, Rongcheng Rao, Xiaoming Zhang, Heding Shen,＊

aKey Laboratory of Aquatic Germplasm Resources Exploration and Utilization of Ministry of Education, Shanghai Ocean University, Shanghai, 201306, China

bNational Experimental Teaching Demonstration Center of Aquaculture Science, Shanghai Ocean University, Shanghai, 201306, China

cClassification and Evolution of Marine Animal System, Shanghai Ocean University Key Laboratory of Shanghai Universities, Shanghai, 201306, China

Keywords:

Onchidium reevesii

Tide rhythm

mapk1

egr1

A B S T R A C T

Previous studies have speculated that tidal rhythm of Onchidium reevesii was linked to its memory ability, which has not been well explained.Mapk1 and egr1 genes are closely related to memory formation in the MAPK signaling pathway that have been extensively studied in vertebrates.These two genes are involved in Long-term potentiation (LTP), which is generally regarded as one of the main molecular mechanisms underlying learning and memory.To investigate the relationship between tidal rhythm of O.reevesii and its memory ability, we studied the molecular mechanism of mapk1 and egr1 genes cooperating with tidal rhythm.The full-length cDNA sequence of mapk1 gene was cloned by RACE method and analyzed by bioinformatics, then qRT-PCR was used to analyze its expression levels in each tissue.A homology analysis and phylogenetic tree reconstruction revealed that O.reevesii is most closely related to the Biomphalaria glabrata.The qRT-PCR showed that mapk1 was expressed in all the tissues, but the highest expression was in the ganglion.We speculated that mapk1 is related to memory formation of O.reevesii.Then we used qRT-PCR to detect the expression of mapk1 and egr1 in ganglion of O.reevesi under tidal stimulation.The expression of mapk1 and egr1 at the rising tide points were significantly higher (P ＜ 0.05) than that at the previous one lowest tide points on May 15–21, except the rising tide point on May 20 and May 21 of mapk1.The expression of mapk1 and egr1 genes in the ganglion sampled for 7-day were basically the same as that of the tide change of the same day.The expression of the two genes were upregulated at raising tide and downregulated at the lowest tide, it is presumed that the O.reevesii was affected by the tide for a long time and formed the tide memory.

Onchidium reevesiiis widely distributed in the coastal areas of southeast China, and its feeding rhythm and reproductive cycle are influenced by local tides in the intertidal environments such as estuaries and tidal flats (Wang et al., 2005; Wu et al., 2010; Liu and Huang, 2018).Back in 1918, Arey and Crozier of the Bermuda Institute of Marine Biology reported on the tidal rhythm ofOnchidium floridanum, which lives in nests, caves and crevices, emerging at low tide to ingest food exposed to the surface, and returning home at the same time from different nests in an hour before the tide rises again, they speculated that was linked to its memory ability (Arey & Crozier, 1918).Some studies have speculated that there is also a relationship between the tidal rhythm ofO.reevesiiand its memory ability (Liang et al., 2019; Xu,2019).The distribution ofO.reevesiiis extensive, and the tidal variations of the sea area is complex and changeable (Yang et al., 2018; Wen et al.,2019; Tessmar-Raible., 2015).The problem of howO.reevesiifeels the tidal change and forms the tidal rhythm has not been well explained for a long time.

Previous studies have speculated that the tidal rhythms ofO.reevesiimight be related to its ability memory.Mapk1andegr1in MAPK signal pathway are closely related to learning and memory.The Mitogenactivated Protein Kinase 1 (mapk1), also known as erk2, is a kind of serine-threonine protein kinase that transmits extracellular and intracellular signals to cell responses (Jia, 2015).It exists widely in eukaryotes and is an important component of MAPK signal pathway,participate in the growth, differentiation, apoptosis and other cell physiological processes (Roisin et al., 2000; Robert-Gangneux et al.,2000; Gong et al., 2015).Long-term potentiation (LTP) is thought to be one of the important molecular mechanisms of learning and memory.LTP can inducemapk1(erk2) rapid phosphorylation, and then regulate the phosphorylation of two downstream transcription factors,ElkandCREB, the transcription ofegr1is regulated by the combination of Serum Response Elements SRE and cAMP response elements CRE inegr1promoter region, respectively, which affects the consolidation of long-term memory(Davis et al., 2000; Fang & Wu., 2006).Mapk1(erk2) regulates the expression of downstreamegr1gene through the MAPK signaling pathway.Inhibition and blocking of this process with MEK kinase inhibitors will inhibit the expression of downstreamegr1gene and ultimately hinder the formation of long-term memory (Adams & Sweatt,2002).Theegr1, also known asZenk,Zif268andkrox-24, is a transcription factor with three zinc finger structures.It not only participates in the growth, development, and differentiation of nerve cells, but also plays an important role in learning and memory of animals (Davis et al.,2003; Knapska & Kaczmarek, 2004).The role ofegr1in memory formation is to promote the change from short-term memory to long-term memory.Mice that lacked outegr1gene showed normal short-term memory and impaired long-term memory such as spatial recognition(Bozon et al., 2002; Jones et al., 2001).

Fig.1.The full-length cDNA and predicted amino acid sequence of the mapk1 gene of O.reevesii.Start codon and stop codon are indicated by thin line boxes.Light gray indicates the conserved region of the catalytic domain of S_TKc domain.Double-underscore indicates the TEY double phosphorylation site.Light blue indicates the HRD catalytic site.The wave line indicates the glycine enrichment area.Tailed signal sequences AATAAA and Poly(A) are indicated in red font.(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

In the present study, we cloned the full-length cDNA ofmapk1by the RACE method and analyzed the expression patterns ofmapk1and its downstreamegr1genes at the lowest tide point, rising tide point and ebb tide point under 7-day tidal cycle.The research results will provide a valuable reference for the investigation on the mechanism of tidal rhythm inO.reevesii.

2.1.Experimental sample collection

O.reevesiiindividuals used in this study were collected from Yancheng in Jiangsu province and were cultured in a plastic incubator at the Lab, where they were fed regularly with corn flour and the dead individuals and feces were cleared.The indoor temperature was maintained at (26 ± 0.5) ℃ andO.reevesiiwere temporarily cultured for 2 weeks (Shen et al., 2004), and then we selected 250 healthy and adultO.reevesiiwith a body weight of 15.12 ±0.13 g and a body length of 5.80 ±0.11 cm for following experiment.

2.2.Cloning the full-length mapk1 cDNA

In the experiment, 6 healthy adult individuals with the similar body(Shanghai, China) and the sequences obtained were assembled into complete cDNA.All the primers used are listed in Table 1.

Table 1Primers used in the present study.

2.3.Bioinformatic analysis of the mapk1

Based on the full-lengthmapk1cDNA ofO.reevesii, ORF Finder(https://www.ncbi.nlm.nih.gov/orffinder/) was used to identify the open reading frame (ORF) encoded by gene.The physicochemical properties of the encoded protein were predicted by Expasy (http://web.expasy.org/protparam/).Signal 4.1 (https://www.cbs.dtu.dk/services/SignalP-4.1/) was used to predict potential signal peptide cleavage sites.Protein secondary and tertiary structures were predicted by Phyre2(http://www.sbg.bio.ic.ac.uk/phyre2/).The amino acid sequences of themapk1protein from other species were obtained from the NCBI database (https://blast.ncbi.nlm.nih.gov/) and DNAMAN software was used to constrict a multiple alignment of the amino acid sequences.Similarly, Phylogenetic tree was constructed by Mrbayes software with 1,000,000 generations, the other parameters used were the default values.

2.4.Analysis of mapk1 mRNA expression in different tissues using qRTPCR

Nine healthy adultO.reevesiiwere divided into three groups as repeat group and Trizol was used to extract mRNA from different tissues,including the dorsal skin, mouthparts, ganglion, hermaphroditic organ and pleopod.The extracted mRNA was reversed into a cDNA and store in-20 ℃ until used.weight and length were selected, and the complete ganglion were collected used for total RNA extraction.Complete ganglion tissue should include cerebral ganglion, visceral ganglion, pleural ganglion and pedal ganglion.The extracted ganglion should to be stored in liquid nitrogen immediately to ensure the quality of RNA.Total RNA was extracted using Trizol reagent (Takara, China), and the quality of RNA was detected by 1% Agarose gel electrophoresis.The RNA concentration was determined using a NanoDrop 2000instrument (Thermo Scientific,USA).The RNA was then reverse transcribed into cDNA by HiScript QRT SuperMix for qPCR (Vazyme, USA) and stored at -20 ℃.

An incomplete cDNA sequence of themapk1gene obtained from the transcriptome of O.reevesii.Based on this partial fragment, we designed multiple primers to amplify the incomplete sequence ofmapk1.The 5′and 3′ends of the chains by rapid-amplification of cDNA ends method using SMARTER® RACE5′/3′Kit User Manual (Takara, Japan).The amplified product was ligated with pGEM-T vector (Promega, USA) and transformed intoE.Coli(DH5α, Tiangen).The positive clones were picked and cultured, and then sent to Sangon Biotech for sequencing

2.5.Tide stimulation experiment

In advance, 200 adultO.reevesiiwere randomly selected to be released to the net enclosure of east Sea beach of Luchao Port, Nanhui New Town, Pudong New Area, Shanghai for culturing, so as to expose them to the environment of tidal cycle for 30 days.Based on the tide table (http://ocean.cnss.com.cn/）, on May 15–20,2019, select the ebb tide point (when the tide has just ceased to submerge the net enclosure),the lowest tide point and the rising tide point (when the tide is about to submerge the net enclosure).At each time point, the ganglion tissue of threeO.reevesiiwere collected for the analysis of mRNA expression.All tissues were immediately frozen in liquid nitrogen and stored -80 ℃ until used.

2.6.Analysis of the mRNA expression of mapk1 and egr1 by qRT-PCR

Theegr1gene’s primers for qRT-PCR were obtained from the published (Wu et al., 2020).Themapk1gene’s primers for qRT-PCR were designed by Genscript (https://www.genscript.com/tools/real-time-pcr-tagman-primer-tool), with 18s rRNA selected as the internal reference.ChamQ™ Universal SYBR qPCR Master Mix reagent (Vazyme, USA) was used to carry out qRT-PCR on Abi Q6 instrument.The reaction System was 20 μL: 0.4 μL of each primer, 10 μL of ChamQ Universal SYBR® Mix Master PCR, 2 μL of cDNA, 7.2 μL of ddH2O.The two-step qRT-PCR reaction was used: 95 ℃ for 30 s, 40 cycles of 95 ℃ for 10 s, and 60 ℃ for 30 s.

The 2-△△CTmethod was used to calculate the relative expression of genes (Schmittgen & Livak, 2002).Statistical analyses were performed using One-Way ANOVA in SPSS 22.0 software.Differences were considered significant at P＜0.05.GraphPad Prism 8 was used to drawn figures.

3.1.Analyses of the sequences of mapk1

The complete cDNA sequence ofmapk1(GenBank accession no.

3.2.Multiple sequence alignment and phylogenetic analysis of mapk1 gene

The amino acid sequence ofmapk1fromO.reevesiishowed highly homology with other invertebrates (Fig.3).What’s more, the homologs ofmapk1protein betweenO.reevesiiandB.glabrataare 94%.In the phylogenetic tree show that themapk1fromO.reevesiiwas first gathered with amapk1protein fromB.glabrata, and then gathered with other invertebrates (Fig.4).

Fig.3.Multiple sequence alignment of O.reevesii mapk1 compared with other species.The high conserved cysteine residues were indicated in black and similar residues were shown in pink.(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig.4.Phylogenetic tree indicating homology between amino acid sequences of mapk1 and other species.The numbers after each branch represent a posteriori probability.

3.3.Expression of mapk1 mRNA in different tissues

The qRT-PCR result showed thatmapk1was expressed in all tissues.The expression ofmapk1was highest in ganglion, followed by hepatopancreas, pleopod and hermaphroditic organ.The expression of themapk1in the ganglion was significantly higher than other issue (P ＜0.05).The least expression of themapk1in the dorsal skin and mouthparts, and the difference was not significantly (P ＞0.05) (Fig.5).MW296142) consisted of 1918 bp and included an ORF of 1095 bp, a 5′UTR of 68 bp and a 3′UTR of 755 bp, and encoding a protein for 364 amino acids residues in length.The 3′UTR contained a typical polyadenylation signal (AATAAA) and Poly(A) tail (Fig.1).The predicted molecular weight (MW) was 41.82 kDa, the theoretical isoelectric point(pI) was 6.28, and the molecular formula was C1876H2949N503O545S17.The predicted protein domain had the highly conserved S_TKc domain at the C-terminus (amino acids 29-317) and no signal peptide and transmembrane region.The amino acid sequence contains 43 positively charged residues (Arg +Lys) and 47 negatively charged residues (Asp +Glu).Phyre2 analysis showed that the secondary structure of mapk1 was mainly Included α helix (40%) and β folding (13%).The tertiary structure was predicted as shown in Fig.2.

Fig.5.The distribution of mapk1 mRNA expression in different tissues.Different letters indicate significant difference (P ＜0.05) and the same letters indicate no significant difference (P ＞0.05).

3.4.The effects of tide on the expression of mapk1 and egr1 mRNA

O.reevesiiwas exposed to the tide cycle and sampled in the order of ABCDEF.The expression ofmapk1andegr1genes were measured after exposure to tide cycle on May 15–21, 2019.The results showed compared with the previous one lowest tide point, the expression ofmapk1andegr1in ganglion ofO.reevesiiwas significantly upregulated(P ＜0.05)at the rising tide point on May 15–21, 2019, except the sampled C on May 20–21 ofmapk1.In most cases, the expression ofmapk1andegr1in ganglion ofO.reevesiiwas significantly upregulated(P ＜0.05)at the ebb tide point compared with the latter one lowest tide point (P ＜0.05) such as sampled C on May 16, sampled A and D on May 17, sampled A on May 18 and so on(Fig.6).

Fig.6.The a, c, e, g, i, k and n are the tide table of Shanghai Luchao Harbor on May 15–21,2019 (https://www.cnss.com.cn/).These time points in, c, e, g, j and k are the highest and lowest tide time in a natural day; The A, B, C, D, E and F are sampling points in a natural day; b, d, h, f, j, m and o are the expression level of mapk1 and egr1 in the ganglion of O.reevesii at each sampled point.Different letters indicate significant difference (P ＜0.05) and the same letters indicate no significant difference (P ＞0.05).

Fig.6.(continued).

The present study successfully clonedmapk1genes inO.reevesii.Themapk1, also known aserk2, belongs to the ERK subfamily of MAPK family (Jia and Xiamen, 2015).Domain prediction showed that the coding region contains S_TKc domain and TEY double phosphorylation site, which correspond to the main characteristics of MAPK family(Krens et al., 2006).The conserved glycine enrichment (GXGXXG) ATP phosphorylation binding ring plays a key role in the activation of mapk1(Roskoski, 2012).In addition, we found that there is a conserved HRD catalytic site in themapk1gene ofO.reevesii, which indicates thatmapk1gene has the conservative catalytic function of the MAPK family (Hanks et al., 1988).The phylogenetic tree show that theO.reevesiiwas closely related toB.Glabrata, which is consistent with the traditional morphological classification and showed thatmapk1gene was conservative in evolution.

In the present study,mapk1gene was highest expressed in the ganglion ofO.reevesii, which is similar to that ofEpinephelus coioidesandParalichthys olivaceous(Wei et al., 2015; Lee et al., 2009).In the study ofA.Californica, it was found that phosphorylated MAPK (pMAPK) was produced by memory training, and the pMAPK could significantly improve the memory ability (Abel et al., 1998).These indicated thatmapk1gene may play an important role in the formation of memory in both invertebrates and vertebrates.Secondly,mapk1gene was highly expressed in hepatopancreas ofO.reevesii.Hepatopancreas are important organs of innate immunity in shell fish, which indicated thatmapk1is involved in the immunoregulatory process of this species (Hou et al.,2019; Liang et al., 2020).

Ganglion is the nerve center that regulates invertebrate behavior,and it plays an important role in regulating learning and memory,reproduction, foraging and other advanced life activities (Balaban,1979; Samygin & Karpenko, 1980; Chase, 2015).The expression changes of MAPK signal pathway-related genes in the ganglion of this species under tidal stimulation may indirectly reflect the tidal rhythm of this species.The expression ofmapk1andegr1at the rising tide points were higher than the previous one lowest tide points on May 15–21.The tidal rhythm ofO.reevesiiis as follows: foraging at the lowest tide,entering caves or climbing on reeds at the rising tide.We found that the expression of the two genes were upregulated at the rising tide point and downregulated at the lowest tide point, which is consistent with the tidal rhythm ofO.reevesii(Liang et al., 2019).Mapk1relies on the MAPK signaling pathway to regulate the expression of downstreamegr1(Fang& Wu, 2006; Xu, 2019), so the expression trend of two genes appears to conform to the tidal variations.Intertidal organisms affected by tidal rhythm, such asCellana RotaandEmerita talpoida, also show increase activity during rising tide (Forward et al., 2005; Schnytzer et al., 2018),which is consistent with relatively high expression ofmapk1andegr1genes at the rising tide points.In the study of mice, it was found that when the mice that formed the long-term memory of the water maze explore the water maze again, the long-term memory retrieval could increase the expression ofegr1in the mouse hippocampus (Zhou, 2013).We speculated that expression of the two genes were upregulated during rising tide may be the result of the tidal memory retrieval inO.reevesii.After 30 days captivity in tidal flat,O.reevesiimay be affected by the tide to form the tidal memory, and was able to escape from the rising tide in time by tidal memory retrieval.There was no significant difference ofmapk1between the rising tide points and the previous one lowest tide points (P ＞0.05) on sampled C of May 20–21.We hypothesize that this is due to the circadian rhythm ofO.reevesii.TheO.reevesiiis a nocturnal organism, and the sampled point of the rising tide on May 20–21 is in the process of changing from night to day, during which the expression ofmapk1gene will be temporarily suppressed (Iglesia et al., 1994; Xu,2019).

The full-length cDNA ofmapk1inO.reevesiiwere obtained for the first time.Under the stimulation of tide, the memory-related genes ofmapk1andegr1in the ganglion ofO.reevesiishowed similar expression trend to the tidal rhythm, which may be verified the previous speculated that the tidal rhythm ofO.reevesiiwas linked to its memory ability.These results provide a new way for studying the molecular mechanism of tidal rhythm in other intertidal organisms.

CRediT authorship contribution statement

Rongyu Wu: Writing – original draft, Conceptualization, Investigation.Rongcheng Rao: Writing – original draft, Conceptualization,Investigation.Xiaoming Zhang: Conceptualization, Investigation.Heding Shen: Resources, Conceptualization, Supervision.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Expression ofmapk1andegr1genes inOnchidium reevesiiunder tidal stimulation”.

Acknowledgments

This work was supported by the Construction Project of the Double First-class Disciplines of Fisheries of Shanghai Ocean University.