Identification and Evolution Analysis of the Genes Involved in the 20-Hydroxyecdysone Metabolism in the Mud Crab, Scylla paramamosain: A Preliminary Study

Abstract

Background: 20-Hydroxyecdysone (20E) is the most ubiquitous ecdysteroid (Ecd) and plays critical roles during the life cycle of arthropods. To elucidate the metabolism pathway of 20E in the economically important species, Scylla paramamosain, we conducted a comprehensive exploration of the genes involved in the 20E metabolism pathway.

Methods: A comprehensive exploration of genes involved in the 20E metabolism pathway was conducted, including gene annotation, local blast using the Drosophila ortholog as query, and TreeFam ortholog genes identification. Bioinformatics and expression profiling of the identified genes were performed to assess their roles in the 20E metabolism of green mud crabs.

Results: This experiment indicated that, except for CYP306a1 and CYP314a1, all other ortholog genes involved in the Drosophila 20E metabolism can be found in the mud crab, suggesting that the function of these two genes might be replaced by other CYP genes or the "active" Ecd in mud crabs was not the 20E. All genes had the typical features of each gene family, clustered with the specific clade in the phylogenetic trees. In addition, all the identified genes had the highest expression level in the Y-organ, and sex-biased gene expression was observed in these genes.

Conclusions: This study provided some valuable insights into the metabolism and diversity of ecdysteroids in crustaceans.

Keywords: CYP18a1; CYP302a1; CYP307a1; Neverland; Scylla paramamosain; Y-organ.

Figure 1

Tertiary structural modelling of 20E metabolism proteins.

Figure 2

Multiple sequence alignment analysis. Sequences with 100% similarity are highlighted with a black background, sequences with over 75% similarity are highlighted with a dark gray background, and sequences with over 50% similarity are highlighted with a light gray background. (A) Multiplex protein sequences alignment of Nvds. (B) Multiplex protein sequences alignment of CYP315a1. (C) Multiplex protein sequences alignment of CYP307a1. (D) Multiplex protein sequences alignment of CYP302a1. (E) Multiplex protein sequences alignment of CYP18a1.

Figure 2

Multiple sequence alignment analysis. Sequences with 100% similarity are highlighted with a black background, sequences with over 75% similarity are highlighted with a dark gray background, and sequences with over 50% similarity are highlighted with a light gray background. (A) Multiplex protein sequences alignment of Nvds. (B) Multiplex protein sequences alignment of CYP315a1. (C) Multiplex protein sequences alignment of CYP307a1. (D) Multiplex protein sequences alignment of CYP302a1. (E) Multiplex protein sequences alignment of CYP18a1.

Figure 2

Multiple sequence alignment analysis. Sequences with 100% similarity are highlighted with a black background, sequences with over 75% similarity are highlighted with a dark gray background, and sequences with over 50% similarity are highlighted with a light gray background. (A) Multiplex protein sequences alignment of Nvds. (B) Multiplex protein sequences alignment of CYP315a1. (C) Multiplex protein sequences alignment of CYP307a1. (D) Multiplex protein sequences alignment of CYP302a1. (E) Multiplex protein sequences alignment of CYP18a1.

Figure 2

Multiple sequence alignment analysis. Sequences with 100% similarity are highlighted with a black background, sequences with over 75% similarity are highlighted with a dark gray background, and sequences with over 50% similarity are highlighted with a light gray background. (A) Multiplex protein sequences alignment of Nvds. (B) Multiplex protein sequences alignment of CYP315a1. (C) Multiplex protein sequences alignment of CYP307a1. (D) Multiplex protein sequences alignment of CYP302a1. (E) Multiplex protein sequences alignment of CYP18a1.

Figure 2

Multiple sequence alignment analysis. Sequences with 100% similarity are highlighted with a black background, sequences with over 75% similarity are highlighted with a dark gray background, and sequences with over 50% similarity are highlighted with a light gray background. (A) Multiplex protein sequences alignment of Nvds. (B) Multiplex protein sequences alignment of CYP315a1. (C) Multiplex protein sequences alignment of CYP307a1. (D) Multiplex protein sequences alignment of CYP302a1. (E) Multiplex protein sequences alignment of CYP18a1.

Figure 3

Evolutionary tree. (A) The phylogenetic tree of Neverland in different species. The tree is constructed using the Neighbor-Joining (NJ) method. The size of the circle on the branch represents the bootstrap support rate. (B) The phylogenetic tree of P450 family genes involved in 20E metabolism. The trees is constructed using the Neighbor-Joining (NJ) method. The size of the circle on the branch represents the bootstrap support rate.

Figure 3

Evolutionary tree. (A) The phylogenetic tree of Neverland in different species. The tree is constructed using the Neighbor-Joining (NJ) method. The size of the circle on the branch represents the bootstrap support rate. (B) The phylogenetic tree of P450 family genes involved in 20E metabolism. The trees is constructed using the Neighbor-Joining (NJ) method. The size of the circle on the branch represents the bootstrap support rate.

Figure 4

The results of the relative expression of genes in different tissues. Note: Hep: hepatopancreas; YO: Y-organ; Mu: muscle; Gill: gill; Cu: cuticle; TG: thoracic ganglion; He: hemocytes; OV: ovary; Te: testes. “*” indicates significant difference between the same or related tissues in different gender (p < 0.05); “**” indicates extremely significant difference between the same or related tissues in different gender (p < 0.01); “ns” indicate no significant difference. (A–E) Different lowercase or uppercase letters indicate significant differences among the different tissues in the same gender.