Dietary effects of vitamin C on antioxidant capacity, intestinal microbiota and the resistance of pathogenic bacteria in cultured Silver pomfret (Pampus argenteus).

As most teleosts are unable to synthesize vitamin C, supplemental diets containing vitamin C diets play a crucial role in fish health. The aim of this study was to investigate the effect of dietary vitamin C on the intestinal enzyme activity and intestinal microbiota of silver pomfre (Pampus argenteus). Four experimental diets were supplemented with basic diets containing 300 mg of vitamin C/kg (group tjl3), 600 mg of vitamin C/kg (group tjl6), and 1200 mg of vitamin C/kg (group tjl12), as well as vitamin C-free supplemental basic diet (group tjl0), respectively. The four diets were fed to juvenile P. argenteus (average initial weight: 4.68 ± 0.93 g) for 6 weeks. The results showed that the activity of SOD (superoxide dismutase) and CAT (catalase) increased significantly while that of MDA (malondialdehyde) decreased significantly in group tjl3 compared to vitamin group tjl0. At the genus level, groups tjl0, tjl6, and tjl12 contained the same dominant microbial community, Stenotrophomonas, Photobacterium, and Vibrio, whereas group tjl3 was dominated by Stenotrophomonas, Delftia, and Bacteroides. Among the fish fed with a basic diet containing 300 mg of vitamin C/kg, the intestines exhibited a notable abundance of probiotic bacteria, including lactic acid bacteria (Lactobacillus) and Bacillus. The abundance of Aeromonas in groups tjl3 and tjl6 was lower than that of the vitamin C-free supplemental basic diet group, whereas Aeromonas was not detected in group tjl12. In addition, a causative agent of the disease outbreak in cultured P. argenteus, Photobacterium damselae subsp. Damselae (PDD) was the dominant microbiota community in groups tjl0, tjl6 and tjl12, whereas the abundance of PDD in group tjl3 was the lowest among the diets. Taken together, the diets supplied with vitamin C could influence the composition microbial community of P. argenteus. The low level of vitamin C (300 mg of vitamin C/kg per basic diet) supplementation could not only improve the antioxidant capacity but also resist the invasion of pathogenic bacteria.

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.

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.

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 organism's antibacterial defenses by improving immune-related enzyme activity.