Dkkl1 gene plays an important role in mammalian testicular development and spermatogenesis, but the research of Dkkl1 gene in turtles is still limited. Therefore, in order to explore its potential function and mechanism of action in turtles, we cloned a cDNA fragment of Dkkl1 gene from Chinese soft-shelled turtle (Pelodiscus sinensis), and analyzed its sequence characteristics, expression pattern and response to exogenous hormone treatment. The cDNA of Dkkl1 gene was 823 bp in length, with 3' UTR of 67 bp, 5' UTR of 90 bp, open reading frame of 666 bp, and encoded 222 amino acids in total. Dkkl1 protein is a kind of alkaline protein with poor stability and high hydrophilicity. The secondary and tertiary structures of Dkkl1 protein are mainly dominated by α-helix and irregular coil. Amino acid sequence homology comparison showed high similarity with Chinemys reevesii Dkkl1 protein (81%), but low homology with Dermochelys coriacea Dkkl1 protein (70%). RT-PCR and RT-qPCR analyses reveal that Dkkl1 mRNA was significantly highly expressed in spermathecae of 3-winter-age P. sinensis adults (P<0.001), while it was hardly expressed in the rest of somatic tissues. Moreover, the expression of Dkkl1 gene in P. sinensis spermathecae gradually increased with age and peaked at 3-winter-age. In addition, both 17β-estradiol and 17α-methyltestosterone treatments significantly inhibited the expression of Dkkl1 gene in adult P. sinensis spermathecae (P<0.05). The results suggest that the Dkkl1 gene might play an important role in the process of testicular development and spermatogenesis in P. sinensis.
To explore the early embryonic development process of bullfrogs (Rana catesbeiana) and supplement their biological information and imaging data, we obtained the fertilized eggs from artificially bred bullfrogs by natural oviposition to investigate their morphological changes and biological characteristics. The results show that the size of fertilized eggs was (1.41±0.31) mm. At temperature of (24.5±0.4) ℃, the early embryonic development process could be divided into 7 phases and 24 stages. It took 177 h 38 min for the fertilized eggs to reach the operculum closure stage, and the total accumulated temperature was 4 249.71 h·℃. The embryonic development process showed a pattern of a fast speed at the first several stages (From fertilized egg to 64-cell stage) and then a slowdown at the latter stages. When being stimulated by external stimuli, the embryo had an annular contraction response by bending the head and tail. Later, as the embryo hatched until the completion of gill closure, they become tadpoles.
Yangtze sturgeon (Acipenser dabryanus) is an endemic fish in China and has been listed as a critically endangered (CR) species by the International Union for Conservation of Nature (IUCN). In order to provide a reference basis for healthy breeding and disease analysis of juvenile Yangtze sturgeon, we analyzed the composition, diversity and dynamic changes of microbial flora between its surface mucus, intestinal content and culture water by using Illumina high-throughput sequencing technology. The results show the diversity of microorganisms in culture water was higher than that in mucus and intestinal content. The microbial community structure of mucus and intestinal content were more similar, and could be clearly distinguished from the microbial community of culture water. On phylum level, mucus and intestinal content were mainly composed of Actinobacteria, Proteobacteria and Firmicutes, while the culture water was mainly composed of Proteobacteria, Bacteroidetes and Firmicutes. Seven potential pathogenic genera were found, mainly in culture water and mucus which might lead to the occurrence of diseases under certain circumstances. BugBase phenotype prediction analysis reveals that both Gram negative and Gram positive bacteria were present in the mucus, intestinal content and culture water of juvenile Yangtze sturgeon. The dominant microbiota of intestinal content was Gram positive bacteria, accounting for over 60%. The dominant microbial communities in mucus and culture water were both Gram negative bacteria, accounting for 60% and 70%, respectively. In terms of oxygen demand, mucus and intestinal content were mainly composed of aerobic bacteria, while culture water was mainly composed of aerobic and facultative anaerobic bacteria.
Ammonia (NH4+-N), nitrate (NO3−-N) and nitrite (NO2−-N) are the main contaminants in industrial aquaculture systems. High nitrogen concentration in water is likely to cause aquaculture water pollution and endanger the safety of aquatic animals. Aerobic denitrifying bacteria are widely used to remove nitrogen-containing aquaculture wastewater. In order to obtain a strain that can safely and efficiently treat wastewater with high ammonia nitrogen concentration, we studied the aerobic denitrifying bacteria WM28 with high ammonia nitrogen resistance screened from aquaculture ponds. The strain was identified through morphological observation, physiological and biochemical tests and 16S rRNA gene sequencing. The environmental and biological safety of the strains were evaluated through antibiotic tests and zebrafish (Danio rerio) toxicity tests. The growth and denitrification performance were measured in three single nitrogen source simulated wastewater, and the denitrification capacity was tested in high concentration ammonia nitrogen simulated wastewater. WM28 was identified as Rhodococcus ruber with high antibiotic sensitivity and good biosafety. The removal rates were 100%, 76.3% and 66.99% after 48 h incubation in single NH4+-N, NO3−-N and NO2−-N media, respectively. Their removal rates of NH4+-N reached 100% at high concentrations of 100–500 mg·L−1 NH4+-N in simulated wastewater experiment after 48 h. 700 mg·L−1 NH4+-N was removed by more than 88% after 116 h. At 120th hour, the initial NH4+-N concentration of 1 000 mg·L−1 was still capable of denitrification with a removal rate of 74.38%, which indicates that strain WM28 has great tolerance to high ammonia nitrogen. In summary, strain WM28 is a safe and efficient aerobic denitrifying bacteria with high ammonia tolerance, and has promising application prospects in the treatment of aquaculture and industrial wastewater.
Development of sea cucumber larvae needs to go through stages of auricularia, doliolaria, pentactula and juvenile. High mortality rate from metamorphosis and development of planktonic larvae to attachment stage of seedlings is a common problem in tropical sea cucumbers breeding. However, the gene regulation mechanism underlying the metamorphosis development of tropical sea cucumber is still unclear. In this study, we chose larval Holothuria leucospilota from four periods of early auricularia (A), mid auricularia (B), late auricularia (C) and doliolaria (D) as samples for high-throughput transcriptome sequencing to investigate the molecular mechanism underlying its metamorphosis development. The results show that a total of 83.6 GB Raw reads were generated, and 93 528 Unigenes were obtained by splicing. Pairwise comparisons between adjacent groups of the four sequencing libraries show that the number of genes with significant differential expression in A_vs_B, B_vs_C and C_vs_D were 17 732, 11 757 and 11 319, respectively. GO function enrichment shows that differential genes were mainly enriched in GO functions related to cell growth, such as molecular function and catalytic activity. In addition, KEGG pathways were analyzed, and the results show that the differential genes were significantly enriched in pathways related to cell differentiation, proliferation and apoptosis, such as PI3K-Akt, cell cycle and cancer pathways. Among them, the enrichment frequency of the pathway in cancer significantly increased during the metamorphosis process, which indicates a key role in the transformation of larval growth and development mode. The screened differentially expressed genes and predicted functional information can lay a foundation for the research on the regulatory mechanism of growth and development, artificial breeding and molecular improvement application of H. leucospilota.
