A novel microRNA and its pfk target control growth length in the freshwater shrimp Neocaridina heteropoda.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression and play roles in a wide range of physiological processes, including ontogenesis. Herein, we discovered a novel miRNA, novel miR-26, which inhibits translation of the phosphofructokinase (PFK) gene by targeting the 3' untranslated region (UTR) of pfk directly, thereby inhibiting molting and body length growth of the freshwater shrimp Neocaridina heteropoda Lowering expression of pfk by RNA interference (RNAi) led to a longer ecdysis cycle and smaller individuals. This phenotype was mirrored in shrimps injected with novel miR-26 agomirs, but the opposite phenotype occurred in shrimps injected with novel miR-26 antagomirs (i.e. the ecdysis cycle was shortened and body length was increased). After injection of 20-hydroxyecdysone (ecdysone 20E), expression of the novel miR-26 was decreased, while expression of pfk was up-regulated, and the fructose-1,6-diphosphate metabolite of PFK accumulated correspondingly. Furthermore, expression of eIF2 (eukaryotic initiation factor 2) increased under stimulation with fructose-1,6-diphosphate, suggesting that protein synthesis was stimulated during this period. Taken together, our results suggest that the novel miR-26 regulates expression of pfk and thereby mediates the molting and growth of N. heteropoda.

Bursicon homodimers induce innate immune by activating the expression of anti-microbial peptide genes in the shrimp Neocaridina heteropoda.

Bursicon is a neurohormone belonging to the cystine knot protein family. It consists of two subunits (burs α and burs ß) and plays a pivotal role in cuticle tanning and wing expansion in insects. Recent studies show that homologous crustacean bursicon stimulates cuticle thickening and granulation of hemocytes in the crab Callinectes sapidus. Here we investigate whether bursicon homodimers function in immunoprotective defense systems of shrimp. We found that abdominal ganglion was the main neurohemal release site of bursicon in Neocaridina heteropoda. Bacterial infections induced overexpression of burs α (bursicon α) and burs ß (bursicon ß). RNAi of burs α, burs ß or both inhibited the expression of anti-microbial peptide (AMP) genes. Treating shrimp adults with r-bursicon (recombinant bursicon) homodimers led to up-regulation of three AMP genes. Besides, through the induced AMPs, r-bursicon homodimers enhanced the bacteriostasis of shrimp in vivo and in vitro. These findings demonstrate a novel function of bursicon in crustacean that it induces innate immune via up-regulating the expression of genes encoding AMPs.

Molecular cloning and characterization of glycogen synthase in Eriocheir sinensis.

Glycogen plays an important role in glucose and energy homeostasis at cellular and organismal levels. In glycogen synthesis, glycogen synthase (GS) is a rate-limiting enzyme catalysing the addition of α-1,4-linked glucose units from (UDP)3-glucose to a nascent glycogen chain using glycogenin (GN) as a primer. While studies on mammalian liver GS (GYS2) are numerous, enzymes from crustaceans, which also use glycogen and glucose as their main energy source, have received less attention. In the present study, we amplified full-length GS cDNA from Eriocheir sinensis. Tissue expression profiling revealed the highest expression of GS in the hepatopancreas. During moulting, GS expression and activity declined, and glycogen levels in the hepatopancreas were reduced. Recombinant GS was expressed in Escherichia coli Rosetta (DE3), and induction at 37°C or 16°C yielded EsGS in insoluble inclusion bodies (EsGS-I) or in soluble form (EsGS-S), respectively. Enzyme activity was measured in a cell-free system containing glucose-6-phosphate (G6P), and both forms possessed glycosyltransferase activity, but refolded EsGS-I was more active. Enzyme activity of both GS and EsGS-I in the hepatopancreas was optimum at 25°C, which is coincident with the optimum growth temperature of Chinese mitten crab, and higher (37°C) or lower (16°C) temperatures resulted in lower enzyme activity. Taken together, the results suggest that GS may be important for maintaining normal physiological functions such as growth and reproduction.