The intestinal bacterial community over seasons and its relationship with physiological status of Yesso scallop Patinopecten yessoensis.

Emerging evidence indicates that the intestinal bacterial communities associated with eukaryotes play critical roles in the physiological activities and health of their hosts. Yesso scallop Patinopecten yessoensis, one of the cold-water aquaculture species in the North Yellow Sea of China, has suffered from massive mortality in recent years. In the present study, P. yessoensis were collected from Zhangzi Island, Dalian from March 2021 to January 2022 to investigate the intestinal bacterial community and physiological indices. 16S rRNA gene sequencing data revealed that the diversity of intestinal bacteria changed significantly over seasons, with the highest Chao1 (237.42) and Shannon (6.13) indices detected in January and the lowest Chao1 (115.44) and Shannon (2.73) indices detected in July. Tenericutes, Proteobacteria and Firmicutes were dominant phyla in the intestinal bacteria of P. yessoensis, among which Firmicutes and Proteobacteria significantly enriched in August and January, respectively. Mycoplasma was the most abundant genus during the sampling period, which exhibited the highest abundance in October (75.26%) and lowest abundance in August (13.15%). The functional profiles of intestinal bacteria also exhibited seasonal variation, with the pathways related to pentose phosphate and deoxyribonucleotides biosynthesis enriched in August while the glycogen biosynthesis pathway enriched in October. Redundancy analysis showed that seawater pH, dissolved inorganic nitrogen and silicate were major environmental factors driving the temporal succession of scallop intestinal bacteria. Correlation clustering analysis suggested that the relative abundances of Endozoicomonas and Vibrio in the intestine were positively correlated with superoxide dismutase activity in hepatopancreas while negatively correlated with malondialdehyde content in hepatopancreas and glycogen content in adductor muscle. All the results revealed that the intestine harbored a lower bacterial diversity and a higher abundance of Vibrio in August, compared to January, which were closely related to the oxidative stress status of scallop in summer. These findings will advance our understanding of the relationship between seasonal alteration in the intestinal bacteria and the physiological status of scallops.

Water stratification alters phytoplankton assemblages in scallop farming waters of the North Yellow Sea in China.

As evaluation indicators of the primary productivity, the phytoplankton biomass and community structure are of great significance to the fishery industry, which can be driven by ocean currents, nutrients and water stratification. In the present study, the characteristics of phytoplankton assemblages in different water layers of a typical Yesso scallop farming area in Zhangzi Island, the North Yellow Sea were investigated from March 2021 to January 2022. According to the vertical distribution of temperature, water stratification was observed from June to August (stratification period), and disappeared in March, October and the following January with vertical homogeneity (mixing period). 18S rRNA gene sequencing results revealed that Pyrrophyta was the most dominant phylum during the sampling period, with high gene proportions in the stratification (63.36%) and mixing periods (77.35%). The gene proportion of Bacillariophyta in the stratification period was 5.44%, which was significantly lower than that in the mixing period of 8.93% (p < 0.05). Moreover, Pseudo-nitzschia, a toxin-producing taxon affiliated with Bacillariophyta, exhibited a significantly higher proportion in the stratification period than in the mixing period. During the stratification period, a number of toxin-producing taxa such as Pseudo-nitzschia and Karlodinium were enriched in the bottom layer, which was 1.29-fold and 1.37-fold of that in the surface layer, respectively. Redundancy analysis showed that phosphate and water temperature were major environmental factors driving the vertical distribution of phytoplankton assemblages. The phosphate (0.11 µM) and silicate (2.09 µM) concentrations in the surface layer approached the minimum threshold for phytoplankton growth, and the stoichiometric limitation of phosphate was detected in the surface and middle layers. Collectively, these results indicated that the decreased proportion ratio of Bacillariophyta to Pyrrophyta and unfavorable community composition of Bacillariophyta for scallops were observed during summer, which might result from the phosphate limitation driven by water stratification. The results will further our understanding of the dynamics of phytoplankton communities under the background of intensifying ocean stratification and provide ecological guidance for mollusc mariculture.

Increased abundances of potential pathogenic bacteria and expressions of inflammatory cytokines in the intestine of oyster Crassostrea gigas after high temperature stress.

High temperature stress is a significant threat to the health of oysters, but the effects on their intestinal performances are not well understood. In this study, the effects of high temperature stress on the intestinal histology, immune response and associated microbiota were investigated in Crassostrea gigas after rearing at 20, 25 and 28 °C for 21 days. With the increase of temperature, shortened and shed microvilli as well as increased goblet cells were observed in the intestines of oysters. The transcripts of cytokines CgIL17-5, CgTNF-2 and CgTGF-ß and apoptosis-related gene CgCaspase-3 in intestine increased with the increasing temperature. Further, the diversity and composition of the oyster intestinal microbiota changed after high temperature stress. The 16S rRNA gene copy number per ng of DNA in the T25 (5.16 × 105) and T28 (1.63 × 105) groups were higher than that in the control group (8.62 × 104). The Chao 1 index in the T25 (238.00) and T28 (240.17) groups was lower than that in the control group (279.00). The Shannon index decreased progressively with the increasing temperature, with the value in the T28 group (4.44) significantly lower than that in the control group (5.40) (p < 0.05). The abundances of potential pathogenic bacteria such as Acinetobacter, Pseudomonas, Vibrio and Endozoicomonas increased while that of probiotic bacteria Bacillus decreased after high temperature exposure. Functional prediction indicated that the pathways associated with bacterial proliferation were enriched at 25 °C, while those involved in protein synthesis were blocked at 28 °C. Collectively, these results suggested that high temperature stress led to an increased abundances of potential pathogenic bacteria and expressions of inflammatory cytokines in the intestine, which may consequently affect the functional integrity of the intestinal barrier in oysters.

Effects of high temperature stress on the intestinal histology and microbiota in Yesso scallop Patinopecten yessoensis.

High temperature stress posed by global warming is considered as one of the greatest threats to marine ectotherms by altering their behavior and physiological functions. The intestine and its associated microbiota constitute the first defensive line for the animals against environmental stresses, but their responses to high temperature stress in mollusks are largely unknown. In the present study, the changes of intestinal histology and microbiota were investigated in Yesso scallop Patinopecten yessoensis, a cold-water bivalve species, after high temperature stress. The shrinkage of intestinal lumen, shortening of intestinal villi and increased goblet cells were observed in the intestines of scallops exposed to seawater temperatures of 20 °C (T20 group) and 23 °C (T23 group), compared to the control group (15 °C). High-throughput sequencing of 16S rRNA gene showed that the composition of intestinal microbiota rather than the alpha diversity indices changed significantly after high temperature stress. At the phylum level, the relative abundances of Proteobacteria and Firmicutes decreased progressively with increasing temperature, while that of Bacteroidetes increased by 1.18-fold in the T20 group and 0.95-fold in the T23 group. At the genus level, Tenacibaculum and Mycoplasma were significantly enriched after high temperature stress, and Mycoplasma exhibited highest abundance of 39.43% in the T23 group. Functional prediction revealed that the pathways related to amino acid biosynthesis were blocked after high temperature stress, while that of phospholipases showed the opposite trend. According to the results of network analysis, the network connectivity decreased with increasing temperature, while the percentages of negative correlations in the two high temperature groups were higher than that in the control group. Collectively, the intestinal histology and microbial community of P. yessoensis changed significantly after high temperature stress, which would hinder the nutrient absorption and promote the proliferation of pathogenic microorganisms in the intestine of scallops. Our results will provide novel insights into the occurrence mechanism of mass summer mortality in marine mollusks.

Identification of a Bidirectional Promoter from Trichoderma reesei and Its Application in Dual Gene Expression.

The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes' expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems.