Danli Wang

Cloning and expression analysis of a transformer gene in Daphnia pulex during different reproduction stages.

The full-length cDNA of a transformer gene (Dptra) was cloned from the cladoceran Daphnia pulex using RACE. Dptra expression was assessed by qPCR and whole-mount in situ hybridization in different reproductive stages. The Dptra cDNA, 1652bp in length, has a 1158-bp open reading frame that encodes a 385 amino acid polypeptide containing one Sex determination protein N terminal (SDP_N) superfamily, eight putative phosphorylation sites, and an arginine-serine (RS)-rich domain at the N-terminus. Dptra showed 81%, 53%, 51% and 45% identity to orthologous genes in Daphnia magna, Apis mellifera, Apis cerana and Bombus terrestris, respectively. Phylogenetic analysis based on deduced amino acid sequences revealed that Dptra clustered in the hymenopteran clade and was most closely related to D. magna and A. mellifera. qPCR showed that Dptra expression increased significantly (P<0.05) in different reproductive stages in the following order: male, ephippial female, parthenogenetic female, resting egg and juvenile female. Dptra expression is significantly different between males and females and it is significantly greater in ephippial females and males than in parthenogenetic D. pulex (with summer eggs). Whole-mount in situ hybridization revealed that Dptra was expressed at different levels between males and females. In males, hybridization signals were found in the first antennae, second antennae and thoracic limb, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. This suggests that the Dptra gene plays significant roles in switching modes of reproduction and in sexual differentiation.

Identification and expression analysis of a doublesex1 gene in Daphnia pulex during different reproductive stages

The gene doublesex (dsx) has shown deep conservation in the sex determination in many organisms. Environmental stimuli initiate a switch in the reproductive strategy of Daphnia pulex from asexual to sexual reproduction; however, occasionally, changes in environmental conditions will not lead to this transition. So study genetic responses to environmental stimuli and the molecular basis for the switch of reproductive stages are urgently needed. Therefore, we isolated and sequenced a D. pulex doublesex1 gene (Dpdsx1) and analyzed its expression and location by quantitative polymerase chain reaction (qPCR) and whole-mount in situ hybridization in D. pulex during different stages of reproduction. The predicted amino acid sequence has 335 amino acids that contained one DM domain and one dimerization domain, which is characteristic of insect orthologs of Dsx. Real-time PCR showed that Dpdsx1 expression decreased significantly (P < 0.05) in different reproductive stages in the following order: male, parthenogenetic female, ephippial female, resting egg, and juvenile female. Whole-mount in situ hybridization revealed that Dpdsx1 is expressed in the first antennae, first thoracic limb and compound eye in males, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. Dpdsx1 could not be detected in the gonads of males or ephippial and parthenogenetic females. Taken together, these different reproductive stages’ and sex specific expression patterns are regulated temporally and spatially. We speculate that Dpdsx1 may involve in switching different stages of reproduction and in sexual differentiation in D. pulex.

Pathological analysis of hemolymphs of Charybdis japonica infected with Vibrio alginolyticus

We explored the pathogenic mechanism of Vibrio alginolyticus in the stone crab Charybdis japonica by studying the hemolymph of C. japonica artificially infected by V. alginolyticus. To this end, Wright-Geimsa staining and electron microscopy were used, and phenoloxidase (PO) activity and the immune protection rate of C. japonica injected with immune polysaccharide during infection were analyzed. The results indicated that the total hemocyte and hyaline hemocyte (HH) counts in diseased crabs were significantly lower than those in healthy crabs (P < 0.05), whereas the large granule hemocytes (LGHs) were significantly higher in diseased crabs than in healthy crabs (P < 0.05). The cellular sizes of HHs and LGHs showed an increasing trend after V. alginolyticus infection, while the nuclear/cytoplasmic ratio (NP) of these cells showed a sharp decline after V. alginolyticus infection (P < 0.05). Micro-pathological analysis of hemocytes revealed fewer hemocytes in the hemolymph of diseased crabs and the presence of disintegrated cells. Ultrastructural and micro-pathological analyses showed damage in all types of hemocytes. The mitochondria were damaged and incomplete in structure, parts of the nuclear membrane were anamorphic and parts of the nuclei had shrunk, hematocyte nuclei exhibited heterochromatinization, hemocyte granules were increased in the polysaccharide-treated group infected with V. alginolyticus, and the numbers of mitochondria and rough endoplasmic reticulum were also increased. PO activity in the two Vibrio-infected groups peaked at 6 h and 24 h after infection, respectively, and PO activity increased in the hemolymph of infected crabs but sharply decreased with prolonged infection. Finally, the PO activities in the two Vibrio-infected groups were significantly lower than controls at 120 h post-infection (P < 0.05). Interestingly, PO activity was higher in polysaccharide-treated crabs than non-polysaccharide-challenged infected crabs, resulting in an immunoprotective rate of 69.64% at 7 days post-infection. This phenomenon suggests that polysaccharides could enhance the organisms antibacterial defenses by improving immune-related enzyme activity.

Expression and activation of Daphnia pulex Caspase-3 are involved in regulation of aging

Death-mediating proteases such as Caspases have been implicated in aging. Remarkably, active Caspase-3 can trigger widespread damage and degeneration, playing a key role in causing cell death. In order to explore the relationship between Caspase-3 and aging in Daphnia pulex, we cloned and analyzed the full-length cDNA sequence of its Caspase-3 gene. Both mRNA expression and activity of D. pulex Caspase-3 increased with age. Moreover, different forms of Caspase-3 appeared with aging. The expression of casp3-L was higher and decreased with age, while that of casp3-S was weak and increased with age, consistent with the trend in Caspase-3 activity. Mhc mRNA expression declined over time and was negatively correlated with age and Caspase-3. In situ hybridization results showed that Caspase-3 mRNA was expressed in different growth and reproduction stages, and its expression levels in embryos and larva were lower than that in adult D. pulex. Western blot analysis revealed the presence of Caspase-3 in the form of zymogens with a molecular weight of ~36kDa. Overall, this study explored age-associated gene regulation to provide a basis for the molecular mechanism of D. pulex reproductive conversion.

Expression analysis of a transformer gene in Daphnia pulex after RNAi

In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

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.

Estradiol-17β 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-17β (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.

Erratum to: “Expression analysis of a transformer gene in Daphnia pulex after RNAi”

ACKNOWLEDGMENTS This work was financially supported by grants from the National Natural Science Foundation of China (nos. 31172043, 31572221), the Innovation Team Project of Ningbo, China (2011B81003), the Open Fund of Zhejiang Provincial Top Key Discipline of Aquaculture in Ningbo University (xkzsc1409), the K.C. Wong Magna Fund in Ningbo University, and the Hu Lan Excellent Dr. Fund in Ningbo University.