Cladocerans have been widely used as bioassay organisms to evaluate the impact of toxic substances. In this study, the activities of selected immunological enzymes and MDA level were investigated to characterize Daphnia similoides’ immune response to ammonia stress. The activity of ACP and CAT showed no significant change during the test. The activity of AKP was significantly increased in the 0.19 and 0.38 mg/l NH3-N exposure groups after 6 h. After 24 h, a significant depression occurred in the high concentrations. After 24 h, significant decreases in SOD activity were observed at the concentrations of 0.38, 0.57 and 0.76 mg/l NH3-N. After 24 h, a significant change in MDA was also observed in 0.57 and 0.76 mg/l. These results suggest that changes in whole-body immune parameters of Daphnia similoides are effective to predict sub-lethal ammonia toxicity and useful as an early warning tool in monitoring studies of natural environments.
To explore the role of the Doublesex gene (Dsx1) in the switching between reproductive modes in Daphnia pulex Leydig, 1860, we performed gene silencing using RNA interference (RNAi). We also investigated the expression of gfp (Green Fluorescent Protein) by quantitative-PCR, and the expression of Dsx1 mRNA after RNAi by real-time PCR. Dsx1 expression was significantly greater in males than in females, and was down-regulated in experimental groups compared with the control group. The decrease was more significant in females (35% of controls) than males (47% of controls). The gene fragment of Dsx1 amplified by PCR was ligated to the pEASY-Blunt vector to obtain the recombinant plasmid expressed recombinant protein induced by isopropyl-β-D-thiogalactoside (IPTG). After having been purified by Ni-column affinity chromatography, the recombinant protein as antigen was used to immune rabbits. The antiserum was successfully purified by the protein A method to obtain the Dsx1 polyclonal antibody, an IgG concentration of 2.65 mg/ml, titer 240 000. Western blotting showed that RNAi treatment significantly reduced DSX1 protein levels. Whole-mount immunofluorescence analysis revealed that the fluorescence intensity of appendages was significantly lower following RNAi treatment than the negative control group, and comparable with the blank control group, further confirming the decrease in Dsx1 expression after RNAi treatment. The number of offspring in all experimental groups was measured, and was significantly greater in the RNAi-treated group compared with the control group. These results provide a foundation for further work on the molecular basis of switching between the two modes of reproduction in cladocerans. The findings also further increase our understanding of the regulatory effects of Dsx1 in the differing sexes in these crustaceans.
Daphnia pulex is a freshwater microcrustacean that is known for its cyclical parthenogenesis. In D. pulex, parthenogenetic reproduction switches to sexual reproduction when the living conditions worsen. However, this transformation also occurs over age under favourable living conditions. Thus, there might be a relationship between aging and reproductive conversion. We performed Illumina RNA sequencing, generating 51 712 680 and 59 854 588 raw reads from 1 day-old D. pulex and 25 day-old D. pulex, respectively. From these reads, 60 776 transcripts were assembled and 36 569 (60.15%) unigenes were annotated. A number of significantly differentially expressed genes associated with growth, aging, and reproduction were identified and Quantitative real-time PCR for six genes confirmed the transcriptome data. RNA interference (RNAi) of the caspase-3 gene (casp3) that is a key gene for growth, development, aging, and reproduction, was conducted, which achieved an 80% reduction in casp3 mRNA expression. Meanwhile, the mRNA expression of upstream genes jnk and akt, and sir2 (encoding a substrate of casp3) were also detected. The change of jnk mRNA expression before and after RNAi was not significant () and the mRNA levels of sir2 decreased, while akt increased after casp3 RNAi (). The results indicated the interrelationships of some genes in the senescence pathway and helped to identify the molecular mechanism of the aging progress in D. pulex. Overall, the difference in mRNA expression profile during aging of D. pulex forms a basis for further studies aimed at understanding the role of the transcriptional level in regulating aging and reproductive transformation.