Biological Function of Prophage-Related Gene Cluster ΔVpaChn25_RS25055~ΔVpaChn25_0714 of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is the primary foodborne pathogen known to cause gastrointestinal infections in humans. Nevertheless, the molecular mechanisms of V. parahaemolyticus pathogenicity are not fully understood. Prophages carry virulence and antibiotic resistance genes commonly found in Vibrio populations, and they facilitate the spread of virulence and the emergence of pathogenic Vibrio strains. In this study, we characterized three such genes, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055, within the largest prophage gene cluster in V. parahaemolyticus CHN25. The deletion mutants ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 were derived with homologous recombination, and the complementary mutants ΔVpaChn25_0713-com, ΔVpaChn25_0714-com, ΔVpaChn25_RS25055-com, ΔVpaChn25_RS25055-0713-0714-com were also constructed. In the absence of the VpaChn25_RS25055, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055-0713-0714 genes, the mutants showed significant reductions in low-temperature survivability and biofilm formation (p < 0.001). The ΔVpaChn25_0713, ΔVpaChn25_RS25055, and ΔVpaChn25_RS25055-0713-0714 mutants were also significantly defective in swimming motility (p < 0.001). In the Caco-2 model, the above four mutants attenuated the cytotoxic effects of V. parahaemolyticus CHN25 on human intestinal epithelial cells (p < 0.01), especially the ΔVpaChn25_RS25055 and ΔVpaChn25_RS25055-0713-0714 mutants. Transcriptomic analysis showed that 15, 14, 8, and 11 metabolic pathways were changed in the ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 mutants, respectively. We labeled the VpaChn25_RS25055 gene with superfolder green fluorescent protein (sfGFP) and found it localized at both poles of the bacteria cell. In addition, we analyzed the evolutionary origins of the above genes. In summary, the prophage genes VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055 enhance V. parahaemolyticus CHN25's survival in the environment and host. Our work improves the comprehension of the synergy between prophage-associated genes and the evolutionary process of V. parahaemolyticus.

Approaches to Reduce Rice Blast Disease Using Knowledge from Host Resistance and Pathogen Pathogenicity.

Rice is one of the staple foods for the majority of the global population that depends directly or indirectly on it. The yield of this important crop is constantly challenged by various biotic stresses. Rice blast, caused by Magnaporthe oryzae (M. oryzae), is a devastating rice disease causing severe yield losses annually and threatening rice production globally. The development of a resistant variety is one of the most effective and economical approaches to control rice blast. Researchers in the past few decades have witnessed the characterization of several qualitative resistance (R) and quantitative resistance (qR) genes to blast disease as well as several avirulence (Avr) genes from the pathogen. These provide great help for either breeders to develop a resistant variety or pathologists to monitor the dynamics of pathogenic isolates, and ultimately to control the disease. Here, we summarize the current status of the isolation of R, qR and Avr genes in the rice-M. oryzae interaction system, and review the progresses and problems of these genes utilized in practice for reducing rice blast disease. Research perspectives towards better managing blast disease by developing a broad-spectrum and durable blast resistance variety and new fungicides are also discussed.

Identification of Elite R-Gene Combinations against Blast Disease in Geng Rice Varieties.

Rice blast, caused by the Magnaporthe oryzae fungus, is one of the most devastating rice diseases worldwide. Developing resistant varieties by pyramiding different blast resistance (R) genes is an effective approach to control the disease. However, due to complex interactions among R genes and crop genetic backgrounds, different R-gene combinations may have varying effects on resistance. Here, we report the identification of two core R-gene combinations that will benefit the improvement of Geng (Japonica) rice blast resistance. We first evaluated 68 Geng rice cultivars at seedling stage by challenging with 58 M. oryzae isolates. To evaluate panicle blast resistance, we inoculated 190 Geng rice cultivars at boosting stage with five groups of mixed conidial suspensions (MCSs), with each containing 5-6 isolates. More than 60% cultivars displayed moderate or lower levels of susceptibility to panicle blast against the five MCSs. Most cultivars contained two to six R genes detected by the functional markers corresponding to 18 known R genes. Through multinomial logistics regression analysis, we found that Pi-zt, Pita, Pi3/5/I, and Pikh loci contributed significantly to seedling blast resistance, and Pita, Pi3/5/i, Pia, and Pit contributed significantly to panicle blast resistance. For gene combinations, Pita+Pi3/5/i and Pita+Pia yielded more stable pyramiding effects on panicle blast resistance against all five MCSs and were designated as core R-gene combinations. Up to 51.6% Geng cultivars in the Jiangsu area contained Pita, but less than 30% harbored either Pia or Pi3/5/i, leading to less cultivars containing Pita+Pia (15.8%) or Pita+Pi3/5/i (5.8%). Only a few varieties simultaneously contained Pia and Pi3/5/i, implying the opportunity to use hybrid breeding procedures to efficiently generate varieties with either Pita+Pia or Pita+Pi3/5/i. This study provides valuable information for breeders to develop Geng rice cultivars with high resistance to blast, especially panicle blast.

Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

Specific miRNA-G Protein-Coupled Receptor Networks Regulate Sox9a/Sox9b Activities to Promote Gonadal Rejuvenation in Zebrafish.

Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary-gonadal axis, for which the sex gonad serves as the primary source of sex steroid hormones and germ cells. To maintain hormonal stasis and fertility throughout the lifespan, inducing gonadal stem cell renewal is an attractive strategy. The follicle-stimulating hormone/cAMP/MAPK/Sox9 signaling axis and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, yet the regulatory networks are largely unknown. By genome-wide transcriptome mining and gonadal microinjections, we identify two G protein-coupled receptor (GPCR)-regulatory circuits: miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Coinjection of a Sox9a-miR430a mixture promotes spermatogenesis, whereas Sox9b-miR218a mixture increases primordial ovarian follicles. Coimmunoprecipitation and mass spectrometry indicate that the two mixtures differentially modulate Sox9a/Sox9b multiple covalent modifications. We further reveal that miR430a and Sox9a synergistically activate testicular protein kinase C (PKC)/Akt signaling, whereas the miR218a and Sox9b mixture constrains ovary PKC/Akt signaling. pMIR-GFP reporter assay demonstrate that miR430a and miR218a target the 3' untranslated region (UTR) of four GPCR targets (lgr4, grk5l, grk4, and grp157). Knockdown of these GPCR genes or two Sox9 genes alters miR430a and miR218a regulation in the above gonad-specific PKC and Akt signaling pathways. These results establish two specific miRNA-GPCR-Sox9 networks and provide mechanistic insight into gonadal differentiation and rejuvenation. Stem Cells 2019;37:1189-1199.

Correlative light and electron microscopic analyses of mitochondrial distribution in blastomeres of early fish embryos.

Early embryos of vertebrates undergo remarkable dynamic molecular events, such as embryonic gradient, cellular polarity, and asymmetry necessary for cell fate decisions. Correlative light and electron microscopy (CLEM) is a powerful tool to investigate rare or dynamic molecular events and has been developed for relatively small cells in culture and tissues but is not yet available for large cells of early development stage embryos. Here we report the capability of CLEM in blastomeres of medaka fish by using the mitochondria detection system. A short N-terminal signal peptide of the mitochondrial protein Tom20 was linked to green fluorescent protein (GFP), resulting in a fusion protein termed Tom20:GFP. The subcellular location of Tom20:GFP in medaka blastomeres reveals the lack of mitochondrial distribution in pseudopodia as well as inconspicuous redistribution during divisions. Blastomeres, after sample preparation procedures including high-pressure freezing and freeze substitution, are able to preserve fluorescence, antigenicity, and fine structures, which allows for precise correlation between the Tom20:GFP fluorescence and mitochondria on merged light and electron micrographs. Furthermore, nanogold immunostaining for Tom20:GFP and endogenous Tom20 revealed their specific localization on the mitochondrial outer membrane. Our results extend the CLEM approach to early development stage embryos of a vertebrate.

Formation and cultivation of medaka primordial germ cells.

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.

Effects on growth performance and immunity of Monopterus albus after high temperature stress.

To investigate the impact of the effect of high temperature stimulation on Monopterus albus larvae after a certain period of time, five experimental groups were established at different temperatures. Then, the M. albus under high temperature stress was fed at 30°C for 70 days. After that, the growth index of the M. albus was counted and analyzed. In terms of growth index, high temperature stress had significant effects on FCR, FBW, WGR, and SGR of M. albus (p < 0.05). The SR increased after being stimulated by temperature (p < 0.1). The study revealed that liver cells of M. albus were harmed by elevated temperatures of 36°C and 38°C. In the experimental group, the activities of digestive enzymes changed in the same trend, reaching the highest point in the 32°C group and then decreasing, and the AMS activity in the 38°C group was significantly different from that in the 30°C group (p < 0.05). The activities of antioxidase in liver reached the highest at 34°C, which was significantly different from those at 30°C (p < 0.05). In addition, the expression levels of TLR1, C3, TNF-α, and other genes increased in the experimental group, reaching the highest point at 34°C, and the expression level of the IL-1ß gene reached the highest point at 32°C, which was significantly different from that at 30°C (p < 0.05). However, the expression level of the IRAK3 gene decreased in the experimental group and reached its lowest point at 34°C (p < 0.05). The expression level of the HSP90α gene increased with the highest temperature stimulus and reached its highest point at 38°C (p < 0.05). In the α diversity index of intestinal microorganisms in the experimental group, the observed species, Shannon, and Chao1 indexes in the 34°C group were the highest (p < 0.05), and ß diversity analysis revealed that the intestinal microbial community in the experimental group was separated after high temperature stimulation. At the phylum level, the three dominant flora are Proteus, Firmicutes, and Bacteroides. Bacteroides and Macrococcus abundance increased at the genus level, but Vibrio and Aeromonas abundance decreased. To sum up, appropriate high-temperature stress can enhance the immunity and adaptability of M. albus. These results show that the high temperature stimulation of 32°C-34°C is beneficial to the industrial culture of M. albus.

A delayed and unsynchronized ovary development as revealed by transcriptome of brain and pituitary of Coilia nasus.

Coilia nasus is an anadromous fish that has been successfully domesticated in the last decade due to its high economic value. The fish exhibits a delayed ovary development during the reproductive season, despite breeding and selection for five to six offspring. The molecular mechanism of the delayed ovary development is still unknown, so the obstacles have not been removed in the large-scale breeding program. This study aims to investigate the key genes regulating ovarian development by comparing the transcriptomes of ovarian-stage IV and stage II brain/pituitary of Coilia nasus. Ovarian stages were validated by histological sections. A total of 75,097,641 and 66,735,592 high-quality reads were obtained from brain and pituitary transcriptomes, respectively, and alternatively spliced transcripts associated with gonadal development were detected. Compared to ovarian Ⅱ- brain, 515 differentially expressed genes (DEGs) were upregulated and 535 DEGs were downregulated in ovarian Ⅳ- brain, whereas 470 DEGs were upregulated and 483 DEGs were downregulated in ovarian Ⅳ- pituitary compared to ovarian Ⅱ- pituitary. DEGs involved in hormone synthesis and secretion and in the GnRH signaling pathway were screened. Weighted gene co-expression network analysis identified gene co-expression modules that were positively correlated with ovarian phenotypic traits. The hub genes Smad4 and TRPC4 in the modules were co-expressed with DEGs including Kiss1 receptor and JUNB, suggesting that ovarian development is controlled by a hypothalamic-pituitary-gonadal axis. Our results have provided new insights that advance our understanding of the molecular mechanism of C. nasus reproductive functions and will be useful for future breeding.

Effects of Substituting Tenebrio molitor and Elodea nuttallii as Feed on Growth, Flesh Quality and Intestinal Microbiota of Red Swamp Crayfish (Procambarus clarkii).

This study aimed to evaluate the impact of substituting a portion of feed with Tenebrio molitor (TM) and Elodea nuttallii (EN) on crayfish culture. A total of 270 crayfish (5.1 ± 0.4 g) were fed three different diet combinations (A: 100% feed; B: 80% feed + 10% TM + 10% EN; C: 75% feed + 15% TM + 10% EN) for 12 weeks. The findings demonstrated that group C had an important beneficial impact on the growth performance of crayfish. This was evidenced by a rise in digestive enzyme activity (trypsin, lipase, and cellulase) in the intestinal and hepatopancreas, as well as an upregulation in the expression of growth-related genes (ghsr, igfbp7, mhc, mlc1, mef2, and pax7) in the muscle. Furthermore, the assessment of the flesh quality of crayfish muscle in group C was conducted. The findings indicated a significant increase (p < 0.05) in the energy value (moisture, crude protein, and crude lipid) within the muscle. The levels of delicious amino acids (Glu, Ala, Ser, Gly, and Tyr) and polyunsaturated fatty acids (ARA, DHA) were enhanced, resulting in an improved nutritional profile and flavor of the muscle while maintaining the Σn-3/Σn-6 ratio. The remodeling of the intestinal microbiota (abundance of Proteobacteria and ratio of Firmicutes/Bacteroidota bacteria) also revealed improved growth performance. Additional research is necessary to ascertain whether excessive use of TM or EN feed substitution can have negative effects on crayfish culture.