Identification and structures of P-selectin genes in Bivalvia
Herein, a total of 1, 3, 19, 47, 1, 2, 7 and 10 P-selectin genes were identified in M. philippinaru, B. platifrons, S. constricta, M. mercenaria, P. maximus, P. fucata, C. gigas and C. virginica, respectively, while no P-selectin gene was identified in M. yessoensis (Fig. 1 and Additional file 1: Sequence S1). The number of P-selectin genes accounted for 0.0027%, 0.0089%, 0.0664%, 0.0773%, 0.0025%, 0.0065%, 0.0111%, and 0.0166% of whole-genome protein coding genes in M. philippinaru, B. platifrons, S. constricta, M. mercenaria, P. maximus, P. fucata, C. gigas and C. virginica, respectively. The CAFE output result showed that the P-selectin genes of the Venerida branch had expanded significantly (p < 1e-5) compared with adjacent branches (Fig. 1). This result indicated that P-selectin genes had expanded in the genomes of Venerida species, including S. constricta and M. mercenaria.
SMART analyses revealed that the functional domains of known P-selectin proteins were dominated by complement cofactor protein (CCP) domains; parts of these proteins contained C-type lectin-like (CLECT) domains and epidermal growth factor (EGF) domains (Fig. 2A). P-selectins were classified into type-A and type-B based on the functional domains. In addition to CCP domains, type-A P-selectins contained CLECT domains or EGF domains, while these two domains were not found in type-B P-selectins. The majority of P-selectins in the nine bivalves belonged to type-B. However, 3 type-A P-selectins were identified in S. constricta and 15 type-A P-selectins were identified in M. mercenaria (Fig. 1 and Fig. 2B). The above results indicated that the expansion of P-selectin genes and the presence of type-A P-selectins might be related to specific functions in Venerida.
Comparative phylogeny of P-selectin genes in Bivalvia
Phylogenetic analysis showed that the P-selectin genes could be categorized into subfamilies Ⅰ, Ⅱ, Ⅲ, and Ⅳ (Fig. 3). 34 P-selectin genes, including 3 from S. constricta, 28 from M. mercenaria, 2 from B. platifrons, and 1 from M. philippinaru, were clustered in subfamily Ⅰ; 24 P-selectin genes, including 3 from S. constricta, 5 from M. mercenaria, 1 from B. platifrons, 2 from P. fucata, 1 from P. maximus, 4 from C. gigas, and 8 from C. virginica were clustered in subfamily Ⅱ; 31 P-selectin genes, including 12 from S. constricta, 14 from M. mercenaria, 3 from C. gigas, and 2 from C. virginica, were clustered in subfamily Ⅲ; 2 P-selectin genes from S. constricta were clustered in subfamily Ⅳ (Fig. 3). Interestingly, except for one type-A P-selectin (Sc_P_ selectin_17) belonging to subfamily Ⅲ, all other type-A P-selectins were clustered in subfamily Ⅰ Fig. 3 The phylogenetic tree of P-selectin genes. M. philippinaru, B. platifrons, S. constricta, M. mercenaria, P. maximus, P. fucata, C. gigas and C. virginica genes are distinguished by different colors. Subfamilies Ⅰ, Ⅱ, Ⅲ, and Ⅳ are highlighted in purple, dark blue, dark green and light green, respectively. Branches marked with yellow represent type-A P-selectins and dark blue branches represent type-B P-selectins.
Synteny and duplication of P-selectin genes in S. constricta and M. mercenaria
In total, 19 P-selectin genes were mapped to 6 chromosomes of the S. constricta genome, and 47 P-selectin genes were mapped to 13 chromosomes and 1 scaffold of M. mercenaria (Fig. 4). In S. constricta, 63.15% (12 out of 19) of P-selectin genes were demonstrated to be tandem duplications and were distributed in 5 tandem arrays of 2–3 genes. In M. mercenaria, 65.96% (31 out of 47) of P-selectin genes were found in tandem duplicated regions, which comprised 7 clusters of 2 to 12 genes (Fig. 4). Interestingly, all 3 S. constricta type-A P-selectin genes were located on the same chromosome, and 12 of 15 M. mercenaria type-A P-selectin genes were located in 2 adjacent clusters of the same chromosome. The above results implied that tandem duplications of P-selectin genes were common occurrences among S. constricta and M. mercenaria genomes.
Tissue expression pattern analysis
The spatial expression patterns of P-selectin genes were analyzed using the RNA-seq data of different tissues of S. constricta collected from the public online database. According to the expression patterns of P-selectin genes, the samples from different tissues were clustered into two groups (Fig. 5). One cluster contained three samples from the gills, and another cluster included nine samples from the testes, ovaries and hepatopancreas. Some P-selectin genes, Sc_P_selectin_18, Sc_P_selectin_07, Sc_P_selectin_05, Sc_P_selectin_12, Sc_P_selectin_01, Sc_P_selectin_11, Sc_P_selectin_02 and Sc_P_selectin_10, were relatively highly expressed in S. constricta gills, which was the primary tissue involved in the response to environment stress in bivalves (Fig. 5). The different expression patterns suggested that P-selectin genes might play tissue-specific roles in S. constricta gill.
Expression pattern of S. constricta P-selectin genes under acute salt stress and ammonia stress
To investigate the potential function of S. constricta P-selectins in adapting to environmental stresses, transcriptomes of S. constricta under acute salt stress and ammonia stress were analyzed. Under acute salt stress, 19 P-selectin genes were expressed in S. constricta juveniles, among which 3 genes belonged to type-A P-selectins and 16 genes belonged to type-B P-selectins. Under ammonium stress, 14 P-selectin genes were expressed in the gills of S. constricta, among which 3 and 11 genes belonged to type-A and type-B P-selectins, respectively (Fig. 6).
Under acute salt stress, samples subjected to low salinity were clustered to one branch, while samples subjected to high salinity and optimal salinity were clustered to another branch. Several P-selectins, Sc_P_selectin_03, Sc_P_selectin_19, Sc_P_selectin_02, Sc_P_selectin_06 and Sc_P_selectin_07, were up-regulated under low salt stress. In contrast, Sc_P_selectin_05, Sc_P_selectin_13 and Sc_P_selectin_17 were up-regulated under high salt stress (Fig. 6A). Under ammonia stress, samples subjected to high ammonia nitrogen and control seawater were distinguished based on the expression patterns of P-selectin genes. Sc_P_selectin_13, Sc_P_selectin_15 and Sc_P_selectin_16 were up-regulated under high ammonia nitrogen conditions (Fig. 6A).
The results indicated that P-selectins were responsible for diverse responses to environmental stresses, and P-selectins might play roles when S. constricta face severe environments such as acute variation of salinity and ammonia nitrogen.