Identification of transient receptor potential channel genes from the swimming crab, Portunus Trituberculatus, and their expression profiles under acute temperature stress

Background Temperature is an important environment factor that is critical to the survival and growth of crustaceans. However, the mechanisms by which crustaceans detect changes in temperature are still unclear. The transient receptor potential (TRP) channels are non-selective cation channels well known for properties in temperature sensation. However, comprehensive understandings on TRP channels as well as their temperature sensing functions are still lacking in crustaceans. Results In this study, a total of 26 TRP genes were identified in the swimming crab, Portunus trituberculatus, which can be classified into TRPA, TRPC, TRPP, TRPM, TRPML, TRPN and TRPV. Tissue expression analysis revealed a wide distribution of these TRP genes in P. trituberculatus, and antennules, neural tissues, and ovaries were the most commonly expressed tissues. To investigate the responsiveness of TRP genes to the temperature change, 18 TRPs were selected to detect their expression after high and low temperature stress. The results showed that 12 TRPs showed induced gene expression in both high and low temperature groups, while 3 were down-regulated in the low temperature group, and 3 showed no change in expression in either group. Conclusions This study characterized the TRP family genes in P. trituberculatus, and explored their involvement in response to temperature stress. Our results will enhance overall understanding of crustacean TRP channels and their possible functions. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-09973-x.


Background
Temperature is one of the main environmental variables that significantly affect the physiological characteristics of aquatic and terrestrial animals [1].Crustaceans are poikilothermic animals, so water temperature is critical to their survival and growth [2].Crabs reared at higher water temperatures often have a shorter intermolt period and a faster growth rate, and vice versa [3,4].Due to global warming and the frequent occurrence of climate extremes, shrimps and crabs are constantly exposed to temperature changes during propagating and cultivating processes [5].Investigating the mechanisms underlying the signals of a temperature change as perceived by crustaceans could provide a theoretical guidance for their scientific farming.However, such mechanisms remain largely unknown.
Transient receptor potential (TRP) channels are ideal candidates when it comes to research in this area, as they are widely known for sensing changes in temperature, osmolarity, and mechanical stimuli in mammals and insects [6].TRP channels share a common structure with six transmembrane helical segments, two variable and intracellular amino (-NH2) and a carboxy (-COOH) terminal cytosolic domain, and form a pore region between TM5 and TM6, which gives most TRP channels the nonselective cation property that have high Ca 2+ permeability [7].Based on the homology of amino acid sequences, TRP channels in animals can be classified into nine subfamilies that belong to two groups [8].Group 1 includes subfamilies TRPA (ankyrin), TRPC (canonical), TRPN (nompC, or no mechanoreceptor potential C), TRPM (melastatin), TRPS (soromelastatin), TRPV (vanilloid), and TRPVL (vanilloid-like), while Group 2 involves subfamilies TRPP (polycystin or polycystic kidney disease) and TRPML (mucolipin) [9].The TRPA, TRPV, TRPVL, TRPC, and TRPN subfamilies are characterized by variable numbers of ankyrin repeats (ARs) at their N-terminus, which are essential for their interactions with ligands and protein partners [10].Besides, TRPC, TRPN, and TRPM proteins are hallmarked with a WKxxR motif called TRP domain (or TRP box) in their C-terminals of the TM domain, which is vital for channel activation [11].There are other structural domains that also affect the function of TRP channels, such as the coiled-coil domain, nudix hydrolase domain, and EF hand, but the types of structural domains vary between different TRP channels [6].
TRP channels can be gated by temperature and have extensive thermosensory functions in the animalia [12,13].In mammals, detection of temperature variations is proposed to be attributed by some channels in TRPV, TRPM, TRPC, and TRPA subfamily [13].While in arthropods, members of TRPP and TRPN were also included [14].The directionality of these TRP channels for sensing temperature has been particular well studied in the Drosophila melanogaster.Briefly, TRPC, TRPM, TRPP, and TRPN are involved in cold-sensing [14,15], and TRPA is essential for heat avoidance [16,17].However, whether a TRP channel detects heat or cold is not conserved among species, even within the same class.For instance, the Drosophila TRPA1 was demonstrated as a heat sensor, but in rice planthoppers (Sogatella furcifera), this channel is involved in cold avoidance [18,19].It has been proposed that the inconsistency in function of TRP proteins in different species may be related to sequence discrepancy in their specific transmembrane domains [13,20].
In recent years, the presence of TRP channels have been reported in several crustaceans.Daphnia pulex is a microcrustacean that has 14 TRP channels, with all the subfamilies represented [21,22].The barnacle, Balanus improvisus, has 13 TRP channels in the transcriptomes of its antennules [23].Among the decapod, Homarus americanus has 7 TRP channels in the nervous system, while Cancer borealis has 6 TRP channels [22].TRP channels have also been identified in the transcriptomes of antennular lateral flagella and dactyl of Panulirus argus, Callinectes sapidus, and Procambarus clarkii [24].However, some homologs representing subfamilies of TRP channels are still missing currently in many crustaceans.More importantly, the temperature-sensing function of the TRP family genes in crustaceans have been barely reported, except for TRPA1, which exhibits possible temperature sensor properties in P. clarkii and Eriocheir sinensis [25,26].
The swimming crab, Portunus trituberculatus is a commercially important crustacean species that have been artificially propagated and cultivated [27].In this study, the TRP family genes of P. trituberculatus were identified, and their tissue distributions were analyzed by RT-PCR.To investigate the responsiveness of TRP genes to the temperature change, the high and low temperature stress experiments were performed.The findings of this study provide valuable information for future studies on the role of TRP genes in temperature sensing.

Identification of TRP families
A total of 26 TRP sequences were identified from the P. trituberculatus using keywords screening of our transcriptomic databases, and were named according to the annotations of highest homology sequences.They can be divided into seven TRP subfamilies, which are TRPA, TRPC, TRPN, TRPM, TRPV, TRPML, and TRPP (Table 1).The TRP genes were predicted to locate nonuniformly on sixteen chromosomes (Fig. 1).Furthermore, to assign the candidates to TRP subfamilies, we constructed maximum-likelihood phylogeny which consisted of TRPs from insect and crustacean species (Fig. 2).
The TRPA subfamily of P. trituberculatus includes 13 members consist of two TRPA1, two TRPA1-like, five Painless, three TRPA5, and one Pyrexia.Of the two TRPA1 sequences, the PtriTRPA1-1 homologues seems to be only present in crustaceans, whereas the PtriTRPA1-2 homologues were found in both insects  and TRPM subfamily, designated as PtriNompC, Ptri-Inactive, and PtriTRPM respectively.Among them, the PtriNompC has 28 ankyrin repeats (ARs) in the N-terminal region and a TRP domain in the C-terminal region (Fig. 3A).

Tissue distribution of TRP genes
The mRNA expression levels of predicted TRP genes varied in crab tissues.Considering that sequence differences between the splice variants cannot be accurately distinguished by PCR, the result of TRPML in Fig. 4 represents the common expression pattern of PtriTRPML-1 and PtriTRPML-2, and PtriPKD2-like represents the common expression pattern of PtriPKD2-like1 and PtriPKD2-like2.

Discussion
A total of 26 TRP genes were identified from the transcriptome of P. trituberculatus.These genes belonged to seven subfamilies, thirteen of which were in the TRPA subfamily, four in TRPC, four in TRPP, two in TRPML, and one each in TRPN, TRPM and TRPV.Some of the TRP members identified in insects were not found in this study, such as waterwitch, HsTRPA and TRPApw in the TRPA subfamily and Nanchung in the TRPV subfamily [28].Since homologs of TRPApw and Nanchung have been reported in P. argus and C. sapidus [24], their absence in the present study may be related to the depth of transcriptome sequencing.To date, no waterwitch and HsTRPA homologue were reported in crustaceans and whether they are insect-specific requires more investigations.
TRPA subfamily is the most abundant TRP channel identified in P. trituberculatus.Two PtriTRPA1 sequences were identified, which are also present in C. sapidus and P. clarkia [24], compared to only one TRPA1 in insects.Crustacean TRPA1-2 clustered in a branch with insect TRPA1, indicating that they might be orthologues.Five Painless genes were identified, compared to two Painless genes in C. sapidus and P. argus, and one in Drosophila [24].The novel PtriPainless-5 was clustered with crustacean Painless-2s, whereas Painless-3 and 4 form a new branch.In addition, one Pyrexia was identified, which was clustered with insects and D. pulex Pyrexia, but in a separate branch.Indeed, our phylogenetic tree showed some confusion in the nomenclature of TRPA subfamily at current time.As with many other crustacean TRP nomenclatures, the TRPs in this study were named mainly according to the sequence with the highest homology, but the accuracy needs to be further confirmed.
The TRPA subfamily has been characterized as temperature-sensitive TRP channels in many species [13].The sole member of mammalian TRPA subfamily, TRPA1, has been shown as an intrinsic bidirectional temperature sensor in human, and a cold receptor in rodents [13,29,30].Of the two TRPA1 genes in zebrafish, the TRPA1a is specialized for chemical sensing, whereas TRPA1b responds to cold and heat stimuli [31].Arthropods have more members in TRPA subfamily, among which TRPA1, Pyrexia, and Painless are the most well described thermosensors [16,19,32].Our results suggest that the P. trituberculatus TRPA subfamily genes may also be involved in temperature-sensing, as most of the identified TRPA subfamily genes had elevated expression levels in response to the acute temperature stress.However, the induction in PtriTRPA1 expression in both high and low temperature treatments differs from the observations in P. clarkii and E. sinensis: TRPA1 was only induced by high temperature in the former, while by low temperature in the latter [25,26].
It is also noteworthy that Pyrexia has been identified as a temperature-associated TRP channel in Drosophila [16], but was unresponsive to temperature stimuli in the present study.This may be due to its absence in the antennae or neural tissues of P. trituberculatus.As essential sensory organs in crustaceans, antennae are strong candidates for detecting chemical and mechanical stimuli, as well as other stimuli such as temperature and salinity [33,34].Indeed, a majority of the tested TRP genes Fig. 4 Tissue distribution of P. trituberculatus TRP transcripts.Twenty-two TRP transcripts were detected in the fifteen tissues from P. trituberculatus.M, DNA Marker; An1, Antenna 1; An2, Antenna 2; AnG, antennal gland; Br, brain; TG, thoracic ganglion; Ht, heart; Gi, gill; Hp, hepatopancreas; Ms, muscle; Es, eyestalk; Ep, epidermis; In, intestine; Ov, ovary; YO, Y-organ; Te, testis; N, negative control (representing no template in PCR).β-actin was used as the reference gene Fig. 5 Relative expression of TRP genes under low-temperature stress in P. trituberculatus.Data are presented as mean ± SD (n = 3).Significant differences among groups are indicated by different letter labels (one-way ANOVA, followed by post hoc Tukey's multiple-group comparison, P < 0.05) Fig. 6 Relative expression of TRP genes under high-temperature stress in P. trituberculatus.Data are presented as mean ± SD (n = 3).Significant differences among groups are indicated by different letter labels (one-way ANOVA, followed by post hoc Tukey's multiple-group comparison, P < 0.05).
were expressed in the antennae of P. trituberculatus, suggesting their closely involvements in sensing environmental changes.On the other hand, it was found that the two TRPA1-like genes of P. trituberculatus were present in antennae and nerve tissues, but not sensitive to acute temperature stress, thus may have the function of sensing other mechanical or chemical stimuli.
Four members were identified in the classical TRPC subfamily, designated as PtriTRPC-1, PtriTRPC-2, PtriTRPL, and PtriTRPgamma.In contrast to the broad tissue distribution of the TRPA subfamily in P. trituberculatus, the obtained TRPC genes showed rather tissuespecific expression.In particular, they are expressed in either the ovaries or the testis, which is consistent with the reproductive roles of TRPC ion channels in mammals and insects [35,36].In addition, the highest expression of PtriTRPL was observed in the eyestalk, which would be reminiscent of the characterized function of Drosophila TRPL in phototransduction [37].Although the TRPC family genes from vertebrates and invertebrates are not all orthologous, the presence of PtriTRPgammma in heart and intestine conforms with the tissue distribution of mammalian TRPC genes, which can also be detected in heart and small intestine [38,39].In both mammals and insects, the TRPC family genes have been proposed to be involved in sensing of low temperatures [15,38,40].In Drosophila larval, TRP and TRPL are required for their cool avoidance [15].Interestingly, unlike those cold-sensitive TRPs are induced by low temperature, the expression of PtriTRPL was down-regulated under low temperature stress.
The reduced expression by low temperature was also observed for PtriNompC, a TRPN member.NompC has been identified as a mechanotransduction channel in Drosophila, but it also has a thermosensory function [41][42][43].NompC is expressed in the cold-activated Class III multidendritic sensory neurons of Drosophila larval, and is required for the full-body contraction induced by near-freezing temperatures [14].Paradoxically, while the paper also revealed that TRPM and PKD-2 have the same function with NompC, orthologues of these two genes in P. trituberculatus showed an up-regulated expression pattern when treated by low temperature.As mentioned above, the directionality of a TRP channel for sensing temperature may vary among species, the involvements of PtriTRPL and PtriNompC in cold-sensing need to be explored in further depth.
Besides their sensing properties, the TRP channels have been extensively demonstrated to be implicated in various physiological processes, and are essential for the physiology of the tissues in which they are expressed [44].It is indeed that most TRP genes of P. trituberculatus identified in the present study were widely distributed among tissues, which may indicate their diverse physiological functions [45], but those TRPs that are expressed in reproductive and endocrine organs may be of interest for our future studies.In addition to the TRPC members mentioned above, many other TRP genes are also expressed in the reproductive system.We noticed that some of these TRPs showed biased expression in the gonads, for instance, PtriTRPA1-like2, PtriPainless-3, and PtriTRPL were preferentially expressed in ovaries, whereas PtriTRP-2, PtriPKD1-like1, and PtriPainless-1 were preferentially expressed in testis.Since several TRPs have been proposed as targets for sex hormones in mammals [44], the sexual regulatory role of these gonadsborne TRPs in crustaceans will be an intriguing topic.Also of interest are the TRPs expressed in the Y-organ, a pair of molting glands of crustaceans responsible for ecdysteroids synthesis [46].Ecdysteroids act as crucial coordinators of cell proliferation, differentiation, and apoptosis during the molting and reproductive processes of arthropods [47].Illustrating the relationship between TRP channels and the Y-organ ecdysteroidogenesis may give insight into the mechanisms by which temperatures affect molting process.

Conclusion
In conclusion, the present study identified and characterized 26 genes encoding for the putative TRP channels from the swimming crab, P. trituberculatus.The obtained TRPs covered all seven subfamilies known for TRP proteins, but may not represent the entire number of TRPs in this species.It was found that the number of TRPA subfamily expanded in crustaceans when compared with insects and mammals, but a conserved role in thermosensing can be suggested.Orthologues of two cold sensors in insects, TRPL and NompC, were down-regulated by low temperature stress, which may suggest a different cold-sensing mechanism.In addition to the extensive presence in antennae, the candidate sensing organs in crustaceans, many of the tested TRPs showed widespread expression in adult tissues, particularly represented by neural, reproductive, and endocrine tissues, indicating diverse physiological functions for P. trituberculatus TRPs.As shown by the acute temperature stress experiments, many of the obtained TRPs were transcriptional sensitive to temperature changes.However, it should be emphasized that many TRP proteins often form heteromultimeric channels that consist of two or more TRP subunits [48,49], and some channels may be activated by associating with other TRP channels or specific proteins.Therefore, using qPCR to detect temperature sensitivity does not, by itself, infers that these ion channels are functionally relevant temperature sensors [50].Considering the absence of data from other periods or temperature stress, further validations are needed to clarify the role of TRPs in temperature sensing.
For temperature challenge experiments, two temperature groups (34℃ and 18℃) were set up.The high and low temperatures were maintained by a thermostatic heater and chiller, respectively.During the experiment, the samples for the two temperature groups were continuously aerated and the salinity was maintained at 27 ± 1‰.Crabs at the C3 (crablet 3) stage were purchased from a crab farm that is located in Ningbo, Zhejiang Province, China.All the C3 crabs were acclimated at the temperature of 26 ± 1 ℃ for three days before the experiment.They were then transferred to chambers for the 34℃ and 18℃ groups, respectively.Samples were collected at two time points (24 and 48 h) during experiments.Three crabs were randomly selected at each time point and the surface water was gently wiped off with gauze, before they were flash-frozen in liquid nitrogen.They were ultimately stored at -80 ℃ until RNA extraction was done.

RNA extraction and cDNA synthesis
Total RNA was extracted from different samples using the RNA-Solv® Reagent (Omega Biotek, USA), according to the manufacturer's protocol.RNA was quantified using a NanoDrop 2000 UV Spectrophotometer (Thermo Fisher Scientific, USA).Total RNA (~ 1 μg RNA) was used for synthesizing cDNA using the HiScript® II Q RT SuperMix (+ gDNA wiper) (Vazyme, China).This was followed by storage at − 80 °C until use in further experiments.

Bioinformatics analysis
The sequences for P. trituberculatus were collected from transcriptomes as previously reported (SRR13870345, SRR13870346, SRR13870347).The genome annotation file for P. trituberculatus (GCA_017591435.1)was downloaded from the NCBI databases.
To search for TRP genes in P. trituberculatus, the list of annotated sequences and ORF file were scanned for keywords of previously known TRP channels such as "TRPA" and conserved amino acid sequences such as "WKFAR", respectively [51].Subsequently, the obtained TRP sequences were re-validated using BLASTp.The conserved domains were analyzed by CD search.
(http://www.cbs.dtu.dk/services/TMHMM-2.0/).The distribution of the P. trituberculatus TRP genes on the chromosomes was analyzed using the TBtools software with default parameters, according to the genome annotation file [52].Multiple sequence alignments were done using ClustalX.Multiple alignment files were imported to the Jalview 2.11.2 software to identify conserved sequence motifs.The phylogenetic tree was constructed based on transmembrane protein sequences and using the MEGA 7.0 software with the maximum likelihood method based on the LG + G amino acid model, and bootstrapped with 1000 replications.The species sequence accession numbers and the length of sequences used are listed in Additional File 1.The tree was visualized using iTOL (https://itol.embl.de/).

Reverse transcription-PCR
To detect the expression levels of TRP genes in different tissues, gene-specific primers were designed using the primer 5.0 software and synthesized by YKang Biotech (Hangzhou, China).The gene-specific primers that were designed by the primer 5.0 software are presented in Additional Information 2. RT-PCR was performed with 2 × Es Taq MasterMix (Dye) (CWBIO, China) according to the manufacturer's instructions.β-actin was used as the internal reference.Amplifications were performed as follows: 94℃ for 3 min, followed by 35 cycles of 94℃ for 30 s, 57℃ for 30 s, and 72℃ for 30 s, with a final elongation at 72℃ for 10 min.PCR products were separated on 1.5% agarose gel electrophoresis and visualized using GelRed (Biotium).

Quantitative real-time PCR
Quantitative Real-time PCR (qPCR) was performed to analyze the expression profiles of TRP genes under acute temperature stress.The specific primers that were used in this study were designed by primer 5.0 software and are presented in Additional Information 2. PCR was carried out using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China), according to the manufacturer's instructions.The cycling parameters were as follows: 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 s, 60℃ for 30 s, 72℃ for 15 s.To confirm product specificity, a melting curve analysis of the amplified DNA was performed following amplification, at temperatures between 60 and 95 °C, with the temperature increasing at a rate of 0.15℃/s.The relative mRNA expression levels were normalized to β-actin mRNA expression.Calculations for the relative mRNA expression levels were done using the comparative Ct (2 −ΔΔCt ) method.

Statistical analysis
All data were expressed as mean ± SD (standard deviation).The statistical differences were analyzed using oneway analysis of variance (ANOVA), followed by Tukey's multiple-group comparison test (SPSS 22.0 software).Significant differences were accepted at p < 0.05.

Fig. 1
Fig. 1 Distribution of TRP genes in chromosome of P. trituberculatus.Twenty-six TRP genes were mapped on the sixteen P. trituberculatus chromosomes.The scale on the left is in million bases (Mb).Chromosome numbers are indicated at the left of each vertical bar

Table 1
Putative Transient receptor potential (TRP) channels in the transcriptomes of P.