Expression and functional analysis of the Akt gene from Daphnia pulex.

Daphnia pulex is a nutrient-rich freshwater crustacean with two different reproduction methods. Akt is a serine/threonine protein kinase that plays an important role in cell growth, survival, and lifespan regulation. To explore the function of Akt in the growth and aging process of Daphnia pulex, we cloned the cDNA sequence of the open reading frame (ORF) of the akt gene based on the bioinformatic analysis of the transcriptome data of D. pulex, and analyzed the structural features of the Akt protein. Gene silencing was performed using RNA interference (RNAi), and the expression of the Akt gene and protein before and after interference were analyzed using qPCR and western blotting. The results showed that the expression of akt in D. pulex at different ages showed a "W" pattern, being significantly higher at 20 days than at 10 days and 15 days (P < .05). The expression trend of Akt protein and mRNA were similar, with lower expression at a younger age (1-5 day), after which expression gradually increased from 10 days age, and showed no significant change after 25 days, which might be caused by a lag of protein translation. RNAi reduced the expression of the Akt gene and protein by at least 76%, and the survival rate and reproductive capacity of D. pulex were significantly lower in the RNAi group compared with those in the control group. This study provides a better understanding of the function of the akt gene in D. pulex.

Ovarian transcriptome analysis of Mactra chinensis provides insights into genes expressed during the intermediate and ripening stages.

Enhancing the production of aquatic animals is very important for fishery management and aquaculture applications. Ovaries have important functions in producing oocytes and hormones. The Chinese clam (Mactra chinensis) is a nutritious saltwater shellfish. Significant biochemical changes take place during the sexual maturation of M. chinensis; however, the genetic mechanisms of this process are unclear. Transcriptome sequencing can determine gene expression changes as development occurrs. In the present study, transcriptome sequencing was used to produce a comprehensive transcript dataset for the ovarian development of M. chinensis. The different ovarian developmental stages were determined using hematoxylin-eosin staining. There was identification of 54,172 unigenes at the intermediate stage and 63,081 at the ripening stage, and 80,141 all-unigenes were assembled to determine the molecular mechanism of ovarian development in M. chinensis. Quantitative real-time PCR for nine mRNAs confirmed the RNA-seq data. Functional annotation of the transcripts indicated there were important pathways in ovarian development, such as those involving the vitellogenin gene. Six pathways associated with ovarian development were identified: estrogen signaling pathway, GnRH signaling pathway, progesterone-mediated oocyte maturation, ovarian steroidogenesis, steroid hormone biosynthesis, and steroid biosynthesis. Significant upregulation of protein kinase alpha (PKA) and calmodulin (CAM) in four of the pathways indicates that PKA and CAM are active in M. chinensis ovarian development during maturation. Results of the present study provide the first comprehensive transcriptomic resource for M. chinensis ovaries, which will increase understanding of the molecular mechanisms underlying sexual maturation and promote molecular nutritional studies of M. chinensis.

Cloning and expression of the lifespan-associated protein Sir2 from Daphnia pulex.

The activity of NAD+-dependent deacetylase Sir2, originally discovered in yeast, is known to be essential for effective longevity. The relationship between Sir2 and lifespan in Daphnia pulex was investigated by cloning and analysis the full-length 1901 bp cDNA. The Sir2 protein includes several zinc finger active sites and two readily hydrolysable low-complexity SD-rich regions. The three-domain structure of Sir2 has a distinct crevice that plays an important regulatory role in the binding of NAD+. D. pulex Sir2 shares 90% amino acid sequence identity with Sir2 from D. pulicaria, 89% with D. magna Sir2, 40% with Mus musculus Sir2, and 39% with the Homo sapiens protein. Expression of Sir2 mRNA was measured at 1, 10, 15, 20,25, 30 and 35 days by real-time PCR (p < .05), and was lowest at 1 day and highest at 20 days (p < .05), after which expression decreased with age. In situ hybridisation showed that Sir2 mRNA was expressed in the chest, the first and second antennae, and the gonadal gland in D. pulex. A Sir2 gene fragment was amplified by PCR, ligated into the pEASY-Blunt vector, and recombinant Sir2 was expressed in Escherichia coli and purified by Ni affinity chromatography. The recombinant protein was used as antigen to immunise rabbits, and antiserum was successfully purified using the protein A method, yielding a Sir2 polyclonal antibody. Western blotting showed that Sir2 is expressed at a 69 kDa protein in D. pulex, in accordance with the predicted value. Sir2 protein abundance increased gradually with age, but remained unchanged after 25 days.

Estradiol-17ß and testosterone levels during the annual reproductive cycle of in Mytilus coruscus.

The aim of the study was to examine seasonal variations in the estradiol-17ß (E2) and testosterone (T) levels in the gonad of Mytilus coruscus in relation to the reproductive cycle. M. coruscus individuals were obtained from March 2012 to February 2013 and gonadal tissue was studied by histological analysis and transmission electron microscopy. Histological data revealed that gametogenesis occurs from autumn to winter (i.e., from September to January) when the water temperature is lower than the rest of the year, and spawning occurs in spring (i.e., March onwards). In this study, we used enzyme-linked immunosorbent assay (ELISA) to measure the estradiol (E2) and testosterone (T) concentrations in Mytilus coruscus. The E2 level ranged from 109.59 to 412.31 pg/g wet weight and from 81.45 to 356.05 pg/g wet weight throughout the year. Furthermore, there was a positive correlation between the E2 level and mean oocyte diameter during the study period, indicating that E2 plays a role in sexual maturation in M. coruscus females and males. Thus, E2 and T may be endogenous modulators in sex determination and in the development and maturation of M. coruscus.

Analysis of the microRNA transcriptome of Daphnia pulex during aging.

Daphnia pulex is an important food organism that exhibits a particular mode of reproduction known as cyclical parthenogenesis (asexual) and sexual reproduction. Regulation of the aging process by microRNAs (miRNAs) is a research hotspot in miRNA studies. To investigate a possible role of miRNAs in regulating aging and senescence, we used Illumina HiSeq to sequence two miRNA libraries from 1-day-old (1d) and 25-day-old (25d) D. pulex specimens. In total, we obtained 11,218,097 clean reads and 28,569 unique miRNAs from 1d specimens and 11,819,106 clean reads and 44,709 unique miRNAs from 25d specimens. Bioinformatic analyses was used to identify 1335 differentially expressed miRNAs from known miRNAs, including 127 miRNAs that exhibited statistically significant differences (P < 0.01); 92 miRNAs were upregulated and 35 were downregulated. Quantitative real-time (qRT)-PCR experiments were performed for nine miRNAs from five samples (1d, 5d, 10d, 15d, 20d and 25d) during the aging process, and the sequencing and qRT-PCR data were found to be consistent. Ninety-four miRNAs were predicted to correspond to 2014 target genes in known miRNAs with 4032 target gene sites. Sixteen pathways changed significantly (P < 0.05) at different developmental stages, revealing many important principles of the miRNA regulatory aging network of D. pulex. Overall, the difference in miRNA expression profile during aging of D. pulex forms a basis for further studies aimed at understanding the role of miRNAs in regulating aging, reproductive transformation, senescence, and longevity.