Genome-wide investigation and expression analysis of TLR gene family reveals its immune role in Vibrio tolerance challenge of Manila clam

Abstract

Toll-like receptors (TLRs) play key roles in activating immune responses during infection. In this study, we identified TLR genes in Manila clam at the genome-wide level and characterized it into 9 types according to the Ruditapes philippinarum genome annotation, including TLR1 (1–10), TLR2 (1–10), TLR2–2 (1–5), TLR3 (1–3), TLR4 (1–9), TLR5, TLR6 (1–5), TLR7 (1–2), and TLR13 (1–4). The length of TLR proteins varied from 128 to 1257 amino acids. The molecular weights and theoretical isoelectric point (pI) values ranged from 14.63 to 143.32 kDa and 4.47 to 9.45, respectively. TLR genes showed universal expression levels in adductor muscle (AM), mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO) and labial palp (L). Transcriptome analysis revealed that the expression level of TLR4, TLR5, TLR7 and TLR13 genes are significantly highly expressed in resistant individuals of Manila clam under Vibrio anguillarum challenge, indicating these TLR genes may play significant roles in response to invading pathogens. The results obtained in this work will provide valuable insights into the immune function of TLR gene in R. philippinarum.

1. Introduction

Toll-like receptor (TLR) gene family mediates innate immunity to a wide variety of pathogenic threats through recognition of conserved pathogen-associated molecular motifs [1]. TLRs belong to pattern-recognition receptors PRRs and ancient membrane-bound sensors in animals that detect and defend against the invasion of pathogenic microorganisms, which plays a crucial role in innate immunity in vertebrates [2,3]. In invertebrates, the innate immune system relies on PRRs that recognize microbial components known as pathogen associated molecular patterns (PAMPs), which are special structures expressed on the cell surfaces of pathogens [3,4]. Like other invertebrates, shellfish depend on cellular and humoral defense for protection against infection [4,5]. The shellfish innate immune system recognizes PAMPs that are not present in higher animals and are essential and unique to almost all microorganisms [4,5]. These are recognized by a series of well-conserved PAMPs, and activated PRRs directly or indirectly trigger cellular or humoral responses such as phagocytosis, nodulation and encapsulation, and synthesis of antimicrobial peptide [5], [6], [7]. As an important PRRs, TLRs were first discovered in the Drosophila melanogaster [8]. TLRs are similar to the intracellular domain of the mammalian interleukin 1 receptor (IL-1R), and has a certain homology [9]. The structures of TLRs are evolutionary conserved and possess the typical characteristics comprised of a series of extracellular leucine-rich repeats (LRRs) and endocellular Toll/ interleukin-1 receptor (TIR) domains [10,11,12]. However, not all identified TLR proteins are prototypical TLRs, and some basal metazoans also express separate LRRs and TIR domain-only proteins defined as TLR-like proteins [1,13,14,15]. Therefore, it is hypothesized that prototypical TLRs emerged from the fusion of LRR and TIR domain-only genes [16]. It has been reported that LRR and TIR regions contribute to pathogens recognition and stimulate the downstream signal transduction, respectively [17,18]. The immune activation of TLR signaling pathway originates from the interaction between the cytoplasmic TIR domain of TLR and one of the five TIR containing adaptors, such as MyD88, MAL, TRIF, TRAM and SARM [19,20,21].

In vertebrates, LRR plays an immune role by directly binding to PAMPs, while in invertebrates LRR interacts with cytokine-like molecules, which play an important role in pathogen recognition [5]. A recent phylogenetic and evolutionary analysis in Sebastes schlegelii showed that these TLR genes were divided into five subfamilies and exhibited different selection pressures [17]. It was reported that TLR genes could be induced by lipopolysaccharide or bacterial infection, indicating that TLRs play an important role in innate immunity in teleost fish [17]. In addition, there are also some reports suggesting that TLRs play important roles in crustaceans, such as shrimps [22,23]. Moreover, recent genome-wide analyses have suggested that TLRs or TLR-like/related genes and other genes involved in TLR signaling pathway are conserved in the genome of non-mammalian organisms, including some marine invertebrates such as tunicate Ciona intestinalis [19,24], Pearl oyster Pinctada fucata martensii [25], Zhikong scallop Chlamys farreri [26], and Pacific oyster Crassostrea gigas [27].

Ruditapes philippinarum, a traditionally commercial clam, is widely distributed along the coasts of China, Japan, and Korea [28]. It has many advantages as an aquaculture species, including wide salinity and temperature resistance, rapid growth, and pollution tolerance [29]. In recent years, however, clam farming in China has been seriously hampered by environmental deterioration and pathogenic microorganisms [30]. Therefore, understanding the immune defense and immune responses of R. philippinarum against bacterial challenge is helpful to the sustainable development of Manila clam culture [28]. Previous study reported that eight cDNA sequences of TLRs (RpTLRs) were identified from the transcriptome libraries of V. anguillarum infection R. philippinarum and then classified into four groups, namely, P-TLR, V-TLR, Ls-TLR and sP- TLR, based on the corresponding LRR domain arrangement of their protein structures within the typical TLR motifs [31]. However, there was still limited understanding on TLR gene family at genome level in R. philippinarum.

In the present study, the genome-wide TLR family members were identified from the R. philippinarum genome and the molecular characterization were investigated through bioinformatics analysis. The TLR family gene expression analyses were conducted in different developmental stages and different tissues of Manila clam, and the dynamic expression characteristics of TLR genes in resistant individuals and susceptible individuals under V. anguillarum challenge. The results obtained here will provide valuable information for evolutionary classification and further functional investigations of TLR family members in R. philippinarum.

2. Material and methods

2.1. Gene identification and phylogenetic analysis

The whole genome data of R. philippinarum refer to the previous research results of our laboratory [32]. Screening of candidate TLR genes using the Hidden Markov Model (HMM) [30]. The HMM profile of TIR (PF01582) was downloaded from the Pfam 33.1 protein family database (http://pfam.xfam.org/), and then it was used to search TLR protein sequence in R. philippinarum genome. After removing the redundant sequences, the output putative TLR protein sequences were submitted to check the conserved TLR domain using CDD (https://www.ncbi.nlm. nih.gov/Structure/bwrpsb/bwrpsb.cgi), Pfam 33.1, and SMART version 9 (http://smart.embl-heidelberg.de/) [30]. The putative isoelectric (PI) point and molecular weight was computed using the Compute pl/Mw tool (http://web.expasy.org/compute_pi/, http://web.expasy.org/protparam/) [33]. Gene sequences in different species were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). The BioProject number and database accession of 15 genomes were shown in Table S1. The phylogenetic tree was constructed based on the neighbor-joining (NJ) and maximum likelihood (ML) method involved 572 amino acid sequences from 8 species (Table S2) by MEGA 7.0 software (http://www.megasoftware.net), with 1000 bootstrap replicates [34].

2.2. Gene structure and chromosomal location analysis

Gene Structure Display Server (GSDS) 2.0 (http://gsds.gao-lab.org/ index.php) was used to analyze the TLR gene structures basis on the coding sequences (exons), untranslated regions, and introns from the gene annotation file [30,35]. MEME (http://meme-suite.org/) scanned the hidden protein sequence to search for the motifs of the TLR protein domain with the following parameters: any number of repetitions, maximum of 10 misfits, and an optimum motif width of 6–50 amino acid residues. The MEME Suite Web server provides a unified portal for online analysis of sequence motifs representing features and analyzing their function and evolution [30,36]. The chromosomal location data of TLR were obtained from the clam genome annotation files. MG2C 1.1 (http://mg2c.iask.in/mg2c_v1.1/) was used to display the TLR genes’ chromosomal positions and relative distances [30,37].

2.3. Transcriptome-based gene expression analysis

RNA-seq data generated from the sampled different development stages (PRJNA808620), different tissues (PRJNA664867), and clams with different resistance after V. anguillarum challenge (PRJNA811359) were used to examine the expression profiles of TLR genes in R. philippinarum [28,32,38]. Eight tissues including adductor muscle, mantle, foot, gill, pipe, digestive gland, gonad, and labial palp were collected from 3 individuals [32]. RNA-seq data of the different developmental stages including fertilized egg (FE), 1st polar body (PB1), 2st polar body (PB2), 2-cell (TC), 8-cell (EC), blastula (B), gastrula (G), trochophora (T), D shape larva (D), umbo veliger (U), pediveliger (P), single pipe juvenile (S), and juvenile(J) [39]. The expression characteristics of the TLR genes were normalized and represented in the form of RPKM (Reads per Kilobase of exon model per Million mapped reads). The P-values were adjusted using the Benjamini and Hochberg method [30]. Differences were considered significant at P< 0.05. To render the data suitable for cluster displays, absolute Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) values were divided by the mean of all of the values, and the ratios were transformed by log2 (ratio) [30]. A heat map was generated using R-4.0.2 software (https://www.rproject.org/) with the fold-change values [30, 40].

2.4. V. anguillarum challenge experiment in manila clam

The wild adult Manila clam was collected from Jinshitan, Dalian, China. The clams were cleaned to remove any fouling and were acclimated in 50-L aerated plastic tanks (water temperature: 21 ± 0.3 °C, pH 8.2 ± 0.1, salinity 32 ± 0.2 ppt). The concentration of V. anguillarum used 10⁷ colony-forming units (CFU)/mL. The R. philippinarum were randomly divided into V. anguillarum challenge group and an untreated control group (each group n = 100). According to the 16-day number of deaths statistical data, the highest mortality occurred on day 7 of the experiment. Therefore, the clams were sampled and sequenced on day 7 of the experiment. Three individuals were selected for the susceptible group (VaS, i.e. adductor muscle was not closed, dying state/moribund), and the resistant group (VaR, normal survival individuals) after 7d V. anguillarum challenge, respectively. Three individuals were randomly selected in the control group (Con). The selected nine individuals were dissected and the hepatopancreatic tissues were collected. The samples were immediately frozen in liquid nitrogen and stored at −80 °C. For details of the RNA-seq analysis of the V. anguillarum challenged R. philippinarum, please refer to our previous work [38].

3. Results

3.1. Identification of the tlr genes

The detailed information of TLR genes, including gene name, gene ID, chromosomal location, genestand, protein length (aa), molecular weight (kDa), and theoretical PI were listed in Table 1. The length of TLR protein sequence varied from 128 to 1257 amino acids. The molecular weights and theoretical PI values ranged from 14.63 kDa to 143.32 kDa and 4.47 to 9.45 respectively. The genome sequences and amino acid sequence of TLR genes in other organisms, including 62 TLR from snail (Biomphalaria glabrata), 7 from Mediterranean mussel (Mytilus galloprovincialis), 133 from Pacific oyster (Crassostrea gigas), 52 from owl limpet (Lottia gigantea), 86 from Golden apple snail (Pomacea canaliculata), 119 from Yesso scallop (Mizuhopecten yessoensis), 56 from coral (Pocillopora damicornis), and 71 from Manila clam (R. philippinarum).

Table 1.

Summary of TLR genes in Manila clam.

Gene name	Gene ID	Chr	Chromosomal location	Gene stand	Number of amino acids	Molecular weight(kDa)	Theoretical PI
Tl.1	Sc0000006.14	8	CM018529.1:6,589,388–6,601,941	–	874	99.78	6.69
Tl.2	Sc0000120.5	9	CM018530.1:7,752,498–7,759,133	+	858	97.84	4.63
Tl.3	xfSc0002047.2	3	CM018524.1:33,705,412–33,721,558	+	632	71.28	6.16
Tl.4	xfSc0000165.22	8	CM018529.1:14,099,094–14,102,883	–	1096	126.69	6.58
Tl.5	xfSc0000165.21	8	CM018529.1:14,083,213–14,087,582	–	1187	137.22	5.17
Tl.6	xfSc0000325.10	13	CM018534.1:33,819,541–33,828,601	+	820	95.24	5.63
Tl.7	xfSc0000338.2	19	CM018540.1:19,561,106–19,563,181	–	692	79.63	8.38
Tl.8	xfSc0000338.3	19	CM018540.1:19,530,816–19,532,906	+	697	79.46	7.48
Tl.9	xfSc0000338.7	19	CM018540.1:19,473,257–19,475,317	–	687	79.04	7.83
Tl.10	xfSc0000338.1	19	CM018540.1:19,575,066–19,577,129	–	688	79.28	7.54
Tl.11	xfSc0000338.6	19	CM018540.1:19,501,539–19,502,783	+	415	47.03	7.09
Tl.12	xfSc0000386.10	11	CM018532.1:29,429,200–29,431,290	+	697	79.65	6.79
Tl.13	xfSc0000066.37	19	CM018540.1:30,734,040–30,740,025	+	695	80.86	5.56
Tl.14	xfSc0003053.1	0		–	644	73.24	7.35
Tl.15	xfSc0001308.3	1	CM018522.1:10,541,751–10,544,811	+	913	105.30	6.79
Tl.16	xfSc0000511.10	1	CM018522.1:42,703,214–42,707,014	+	810	92.88	5.33
Tl.17	xfSc0001334.5	0		+	854	96.99	4.95
Tl.18	xfSc0006789.1	0		–	308	36.14	7.20
Tl.19	xfSc0000511.9	1	CM018522.1:42,710,610–42,713,773	–	849	98.09	6.41
Tl.20	xpSc0185134.7	19	CM018540.1:23,289,004–23,291,439	+	615	71.53	6.81
Tl.21	xfSc0002434.4	0		–	782	89.21	6.49
Tl.22	xfSc0001864.4	1	CM018522.1:4,220,901–4,224,671	+	1257	143.32	6.77
TLR1.1	xfSc0001251.4	1	CM018522.1:2,257,355–2,257,843	–	163	18.91	4.75
TLR1.2	xfSc0004241.1	2	CM018523.1:9,692,881–9,693,495	–	166	19.35	4.91
TLR1.3	xfSc0004062.4	0		+	430	49.92	5.23
TLR1.4	xfSc0000331.15	3	CM018524.1:46,955,688–46,966,786	–	427	49.55	6.19
TLR1.5	xfSc0004503.3	0		+	205	23.62	4.76
TLR1.6	xfSc0001960.2	8	CM018529.1:19,723,664–19,731,904	+	407	46.46	5.74
TLR1.7	xfSc0000331.3	3	CM018524.1:46,580,671–46,594,565	–	1122	130.23	7.78
TLR1.8	xfSc0001896.7	0		–	205	23.07	7.71
TLR1.9	Sc0000144.3	19	CM018540.1:25,994,329–25,995,036	+	236	27.46	6.67
TLR1.10	xfSc0000337.32	15	CM018536.1:7,587,377–7,592,920	+	735	83.61	8.19
TLR2.1	xfSc0003053.2	0		+	210	24.51	5.67
TLR2.2	Sc0000062.14	16	CM018537.1:29,286,484–29,301,577	+	573	65.24	4.49
TLR2.3	xfSc0001145.13	17	CM018538.1:54,477,291–54,479,519	–	743	85.43	6.97
TLR2.4	xfSc0001251.5	1	CM018522.1:2,303,739–2,304,206	–	156	18.27	4.91
TLR2.5	xfSc0001121.18	10	CM018531.1:12,858,822–12,859,205	+	128	14.63	5.64
TLR2.6	xfSc0006508.1	0		–	148	17.17	4.47
TLR2.7	xfSc0001464.4	1	CM018522.1:1,507,423–1,509,537	–	496	58.32	8.86
TLR2.8	xpSc0185119.18	8	CM018529.1:9,057,420–9,060,242	–	941	108.58	7.90
TLR2.9	xfSc0000009.24	6	CM018527.1:23,885,473–23,888,375	–	858	98.67	8.36
TLR2.10	xfSc0001121.16	10	CM018531.1:12,865,133–12,866,383	+	417	48.52	7.21
TLR2–2.1	Sc0000128.8	10	CM018531.1:27,586,801–27,590,423	+	886	102.06	8.39
TLR2–2.2	xfSc0002019.2	3	CM018524.1:42,675,427–42,678,682	–	873	101.02	6.51
TLR2–2.3	xfSc0000243.2	5	CM018526.1:56,356,196–56,360,747	–	890	102.20	8.27
TLR2–2.4	xfSc0000325.9	13	CM018534.1:33,813,540–33,820,746	+	670	77.54	6.41
TLR2–2.5	Sc0000019.14	2	CM018523.1:7,058,903–7,075,655	+	813	94.13	8.15
TLR3.1	Sc0000100.12	14	CM018535.1:31,008,643–31,011,129	–	829	96.07	7.68
TLR3.2	xfSc0000854.5	15	CM018536.1:41,142,861–41,146,169	+	877	99.21	9.45
TLR3.3	xfSc0002001.4	9	CM018530.1:29,099,312–29,100,487	–	392	45.02	7.16
TLR4.1	Sc0000029.23	6	CM018527.1:41,995,704–41,998,694	–	867	99.66	7.14
TLR4.2	xfSc0000057.16	6	CM018527.1:4,834,299–4,836,944	–	882	101.93	7.64
TLR4.3	xfSc0000578.18	10	CM018531.1:17,718,921–17,722,226	+	797	90.80	6.7
TLR4.4	xfSc0000520.18	10	CM018531.1:26,621,966–26,624,863	–	749	85.47	8.58
TLR4.5	xfSc0001121.14	10	CM018531.1:12,877,213–12,879,999	–	929	107.10	7.19
TLR4.6	xfSc0000057.15	6	CM018527.1:4,826,749–4,829,325	–	859	99.30	7.58
TLR4.7	xfSc0000009.21	6	CM018527.1:23,916,942–23,919,731	–	874	100.77	6.89
TLR4.8	xpSc0185173.1	6	CM018527.1:14,649,257–14,652,869	+	865	100.16	9.19
TLR4.9	xfSc0001121.15	10	CM018531.1:12,871,065–12,873,866	+	925	107.66	7.14
TLR5	xfSc0000666.2	6	CM018527.1:14,437,325–14,439,280	+	527	60.21	6.05
TLR6.1	Sc0000007.18	18	CM018539.1:4,768,536–4,772,000	+	864	99.76	6.36
TLR6.2	xfSc0000009.23	6	CM018527.1:23,901,914–23,904,520	–	869	100.51	8.15
TLR6.3	xfSc0000215.21	3	CM018524.1:45,261,897–45,270,227	+	366	41.43	8.92
TLR6.4	xfSc0000331.1	3	CM018524.1:46,574,441–46,576,036	–	532	61.96	7.77
TLR6.5	xfSc0002742.4	8	CM018529.1:10,941,144–10,941,674	+	177	20.21	5.53
TLR7.1	Sc0000080.22	13	CM018534.1:20,010,528–20,017,684	–	643	74.23	6.55
TLR7.2	Sc0000080.15	13	CM018534.1:19,897,525–19,899,424	+	562	65.48	7.51
TLR13.1	xfSc0000118.4	4	CM018525.1:13,018,043–13,019,617	–	525	61.04	7.06
TLR13.2	xfSc0000972.6	1	CM018522.1:8,736,829–8,739,747	–	711	82.47	7.49
TLR13.3	xfSc0000972.7	1	CM018522.1:8,701,670–8,703,688	–	673	78.17	8.04
TLR13.4	xfSc0000520.19	10	CM018531.1:26,625,621–26,629,118	–	917	105.89	8.10

Chr 0 is the chromosome position not detected.

3.2. Genomic structure of tlr genes

In the R. philippinarum genome, we identified 71 TLR genes including T1, TLR1-TLR7, TLR2–2, and TLR13. Fig. 1 shows nine types of TLR and one T1 gene with conserved domain properties. All genes contain a highly conserved TIR domain and a helical single-pass transmembrane region. The intron and exon information of TLR genes was shown in Fig. 2. The number of exons is one in TLR4, TLR5, TLR13, and 0 to 10 in other TLRs. A search on MEME program identified 10 motifs in TLR genes (Fig. 3). The details of the conserved motifs are shown in Fig. S1.The MEME results indicated that the motifs number of T1 genes ranged from 2 to 25, TLR1 genes ranged from 2 to 8, and TLR2 ranged from 2 to 18, TLR2–2 genes ranged from 13 to 18, TLR3 genes ranged from 7 to 25, TLR4 genes ranged from 5 to 13, TLR6 genes ranged from 3 to 16, and TLR13 genes ranged from 4 to 10.The motifs number of TLR5 and TLR7 genes was 6 and 9, respectively.

Fig. 1.

Representative partial domains of 10 TLR proteins in Manila clam.

Fig. 2.

Exon-intron structure of TLR genes. Yellow boxes indicate untranslated region (UTR), green boxes indicate exons, black lines represent introns.

Fig. 3.

. The conserved motifs of Manila clam TLR proteins. The motifs, numbers 1–10, are displayed in different colored boxes.

3.3. Phylogenetic analysis and classification of tlr genes family

In this study, we identified nine types of TLR genes and classified the genes into TLR1 (1–10), TLR2 (1–10), TLR2–2 (1–5), TLR3 (1–3), TLR4 (1–9), TLR5, TLR6 (1–5), TLR7 (1–2), and TLR13 (1–4), and T1 (1–22) genes with a function similar to that of TLR from R. philippinarum. Clustering analysis of different types of TLRs showed that the results were broadly divided into six branches. Except for the T1 class genes, other TLR type genes were not clustered into one branch according to the gene annotation classification (Fig. 4). To analysis the evolutionary relationships of TLR genes, the phylogenetic tree was constructed using the amino acid sequences from 8 species (Fig. 5). In general, those TLR genes were mainly clustered into 8 branches. Interestingly, the distribution of TLR genes is relatively dense in the red branch, the light yellow branch and the green branch, and most of them are T1, TLR2, TLR4, and TLR13 genes. The results showed that the TLR genes of R. philippinarum are relatively closely related to C. gigas and M. yessoensis, and the TLR genes of L. gigantea and P. canaliculata are relatively closely related. In general, the TLR genes of mollusks are relatively closely related.

Fig. 4.

According to the clustering results of different types of TLR genes, different color branches represent groups with a higher degree of similarity.

Fig. 5.

Phylogenetic analysis of TLR proteins from selected organisms. A phylogenetic tree was constructed by MEGA10 with the Neighbor Joining method and bootstrap of 1000 replications. Details of clusters are shown with different color. Symbols with different colors represent different species.

3.4. Chromosomal location of tlr gene family

TLR genes are widely distributed on the chromosomes of R. philippinarum (Fig. 6). Those genes are mainly distributed in chromosomes 1, 3, 6, 8, 10, and 19. We found that TLR genes were mostly concentrated in the middle of chromosomes. When there are few TLR genes, most of them are concentrated at the ends of the chromosome. There is no TLR gene distribution on chromosomes 7 and 12. The detailed chromosomal locations of TLR genes and the lengths of chromosomes are shown in Table 1.

Fig. 6.

The chromosome distribution of TLR genes. The chromosome numbers are indicated at the top of each chromosome. The figure shows the chromosome of the TLR gene.

3.5. Expression profiles of tlr genes in manila clam at different developmental stages, different tissues and challenged by V. anguillarum

The results showed that with the growth of larvae, there was no significant difference in the expression of TLR genes in the fertilized egg (FE)-Pediveliger (P), and the TLR gene was significantly increased in the stage of single pipe Juvenile(S), and Juvenile (J) stage (Fig. 7A). The expression levels of TLR genes were similar in mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO), and labial palp (L) of Manila clams, and only low expression in adductor muscle (AM) tissue (Fig. 7B). Through heatmap analysis of the expression levels of TLR genes in susceptible individuals, resistant individuals and control clams after V. anguillarum stress, we found that the expression of TLR4, TLR5, TLR7, and TLR13 genes in the resistant group was higher than that in the susceptible group and the control group (Fig. 7C, D).

Fig. 7.

The hierarchical clustering of expression profiles of TLR genes based on the RNA-seq data. The horizontal axis shows the developmental stages, different tissues, and V. anguillarum stimulation. The vertical axis represents different genes name. (A) Expression profile of TLR in fertilized egg (FE), 1st polar body (PB1), 2st polar body (PB2), 2-cell (TC), 8-cell (EC), blastula (B), Gastrula (G), Trochophora (T), Patterm D larva (D), Umbo veliger (U), Pediveliger (P), single water Juvenile (S), Juvenile (J). (B) Expression profile of TLR in different tissues, adductor muscle (AM), mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO), and labial palp (L). (C) Expression profile of TLR under V. anguillarum challenge. VaR: Resistant individuals after V. anguillarum challenge. VaS: susceptible individuals after V. anguillarum challenge. Con control group. The color scale represents relative expression levels. (D) Different folds of TLR gene expression in different comparison combinations between the resistant group (VaR), the susceptible group (VaS) and the control group (Con) after V. anguillarum stress.

4. Discussion

In recent years, there are many studies on TLR genes and most of which focus on vertebrates [1,17]. In invertebrate, TLR has been reported in crustacean Macrobrachium rosenbergii [10], and mollusks Chlamys nobilis and C. farreri [19,41]. Recently, it was reported that four types of TLR genes from R. philippinarum were analyzed based on transcriptome data in Manila clam after the challenge of V. anguillarum [31]. In the present study, we identified nine types of TLR genes using the Hidden Markov Model (HMM) based on the R. philippinarum genome. We also found a protein toll gene with similar function to TLR Named T1 (1–22) (they all contain conserved TLR domains). Genomics based classification of TLR gene families has been reported and there are different classifications of TLR genes [30, 42].

TLRs can be classified based on the number of cysteine clusters in their ectodomains: multiple cysteine cluster TLRs (mccTLRs) and single cysteine cluster TLRs (sccTLRs) [1]. TLRs are functionally partitioned into two categories: those that are localized to host cell membranes and primarily recognize microbial cell membrane components (TLR1, 2, 4, 5, 6, and 10) and those that are localized to endosomes and recognize nucleic acids (TLR3, 7, 8, and 9) [43]. The general TLR protein structure includes a hydrophobic tandem leucine-rich repeat (LRR), a short transmembrane domain, an intracellular Toll/interleukin-1 receptor (TIR), these structures are the key structures of TLR-acting pathways and downstream pathways for immune function [11]. The intracellular TIR domains of TLR specifically interact with the post-receptor adaptor proteins such as MyD88, Tube, and Pelle, and subsequently induce a series of intracellular signaling cascades in Toll signaling pathway to eventually realize a variety of innate immune responses [10]. Studies have shown that interleukin-10(Il10), TGF-beta-activated kinase 1 and MAP3K7-binding protein (TAB), TIR domain-containing adapter molecule 1-like (TICAM1), and Interleukin-1 receptor-associated kinase (Irak) showed high connectivity to TLR genes [17]. TLRs were originally discovered in Drosophila, and in recent years, TLR study has been expanded in humans, indicating that functional defects of TLRs can seriously lead to human diseases [1, 44]. Increasing evidences have suggested that the animals from fly to human share a canonical TLR signaling pathway and many molecules in this pathway are rather conserved [22,45]. In some invertebrates, TLR genes have been found to have markedly expanded and contracted during evolution, such as Anthocidaris crassispina [46], and Patinopecten yessoensis [47]. Interestingly, in our results, all TLR genes are not clustered entirely according to the annotated results (Fig. 4). In the process of TLR gene screening and analysis, we conducted blast comparison through NCBI database and found that each type of TLR gene can compare different types of TLR genes in molluscs, and the similarity of this alignment is consistent, which may be related to gene amplification and contraction.

It was reported that a conserved TLR signaling pathway exists in mollusks, the investigation on TLR and its signaling pathway in C. farreri provide the critical clues to understand the molecular and functional evolution of the TLR signaling pathway [19]. Our results also show that the Manila clam TLR gene has a similar evolutionary relationship with many other mollusks (Fig. 5). The number of exons, and introns in different TLR genes was similar in R. philippinarum. Those structural features are consistent with the phylogenetic relationship (Fig. 7A).

Similar to previous studies, the metabolic, immune and stress related genes in R. philippinarum are highly expressed in digestive glands, gonads and gills [30,48,49]. In V. anguillarum challenged clams, the TLR gene expression level in the resistant individuals are higher than susceptible individuals (Fig. 7C,D), indicating the TLR genes play important roles in the immune response and defense process [31]. The heatmap showed that the expression of TLR1 gene in resistant individuals was relatively lower than that in susceptible group, but overall higher than that in control group without V. anguillarum stress. TLR4, TLR5, TLR7, TLR13 genes are generally highly expressed in individuals with resistance to V. anguillarum infection. Diversity of TLR genes has also been reported in teleosts, comparative genomics studies have attributed this trend of divergence in teleost lineage to enhanced molecular evolutionary rates of nucleotide and protein coding sequences and higher rate of gain and loss of cis-regulatory elements that consequently alters morphology or function [50,51]. For the different antibacterial effects of TLR family members, the study of mice deficient in each TLR have demonstrated that each TLR has a distinct function in terms of PAMP recognition and immune responses [11]. It was reported that TLR and downstream factors, such as MyD88, TRAF6, IκB and NF-κB, were promoted by stimulation with LPS (a component of most gram-negative bacteria) in C. farreri [19,31]. In this study, our results have similar expression trends to TLR genes in C. farreri [19]. In Cyclina sinensis, the TLR4 and TLR13 were significantly increased in hemocytes (P< 0.01) upon challenge with V. anguillarum [19,52], indicating that the TLR4 and TLR13 genes of C. sinensis are functionally similar to those of TLR4 and TLR13 in R. philippinarum. Our results suggest that TLR4, TLR5, TLR7, and TLR13 genes in R. philippinarum play important roles in the process of resistance to V. anguillarum. This may be related to the domains of individual TLR genes. TLR was a typical transmembrane protein, including signal peptide, LRRs, transmembrane domain and TIR domain [27]. An LRR, a protein structural motif which forms an α/β horseshoe, is documented as versatile binding and recognition motif [27,53]. Insertions and deletions of TLR-LRRs are proved to be involved in PAMP recognition in human [27,54]. Drosophila does not have insertions or deletions in the convex surface of Toll-LRRs and DmTolls do not directly interact with microbial PAMPs [26,27,55,56]. The different types of TLR domain components in clams are different, which may be one of the reasons why they are differentially expressed in resistant individuals and susceptible individuals under the stress of V. anguillarum.

5. Conclusion

In this study, a genome-wide analysis of the TLR family was performed in R. philippinarum genome and a total of 71 TLR genes were identified. Based on gene structure and phylogenetic relationship, the TLRs in R. philippinarum were classified into different types (T1, TLR1, TLR2, TLR2–2, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR13). The results suggest that TLR4, TLR5, TLR7, and TLR13 genes play important roles in the process of immune defense and resistance to V. anguillarum in R. philippinarum. This study provides a valuable insight into the immune function of TLR genes in R. philippinarum and serves as a useful resource for further functional characterization of TLR.

Data availability statement

The raw sequences for R. philippinarum have been deposited in the NCBI PRJNA738278 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA738278). The RNA-seq datasets are available in the NCBI Sequence Read Archive (SRA) with accession numbers SRR14870452, SRR14870451, SRR14870442, SRR14870441, SRR14870440, SRR14870439, SRR14870438, SRR14870437, and SRR14870436.

Author contributions

ZY conducted the experiment, analyzed the data and wrote the draft manuscript. ZL and YL involved in bioinformatics analysis. HN and XY conceived the study and revised the manuscript.

Conflict of Interest

We declare that we have no financial and personal relationships with other people 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 “Genome-wide investigation and expression analysis of TLR gene family reveals its immune role in Vibrio tolerance challenge of Manila clam”.

Data Availability

• Data will be made available on request.