Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Identification and characterization of a symbiotic agglutination-related C-type lectin from the hydrothermal vent shrimp Rimicaris exoculata.

Rimicaris exoculata (Decapoda: Bresiliidae) is one of the dominant species of hydrothermal vent communities, which inside its gill chamber harbors ectosymbioses with taxonomic invariability while compositional flexibility. Several studies have revealed that the establishment of symbiosis can be initiated and selected by innate immunity-related pattern recognition receptors (PRRs), such as C-type lectins (CTLs). In this research, a CTL was identified in R. exoculata (termed RCTL), which showed high expression at both mRNA and protein levels in the scaphognathite, an organ where the ectosymbionts are attached outside its setae. Linear correlationships were observed between the relative quantities of two major symbionts and the expression of RCTL based on analyzing different shrimp individuals. The recombinant protein of RCTL could recognize and agglutinate the cultivable γ-proteobacterium of Escherichia coli in a Ca2+-dependent manner, obeying a dose-dependent and time-cumulative pattern. Unlike conventional crustacean CTLs, the involvement of RCTL could not affect the bacterial growth, which is a key issue for the successful establishment of symbiosis. These results implied that RCTL might play a critical role in symbiotic recognition and attachment to R. exoculata. It also provides insights to understand how R. exoculata adapted to such a chemosynthesis-based environment.