Functional analyses of TRAF6 gene in Argopecten scallops.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

Potential roles of IFI44 genes in high resistance to Vibrio in hybrids of Argopecten scallops.

Vibrio bacteria are often fatal to aquatic organisms and selection of Vibrio-resistant strains is warranted for aquaculture animals. In this study, we found that hybrids between bay scallops and Peruvian scallops exhibited significantly higher resistance to Vibrio challenge, but little is available on its mechanism. Interferon induced protein 44 (IFI44), a member of the type I interferon (IFN) family, plays an important role in the IFN immune response in invertebrates, which may also participate in the resistance to Vibrio in scallops. To explore the roles of IFI44 genes in the resistance to Vibrio, they were identified and characterized in the bay scallop (designated as AiIFI44), the Peruvian scallop (designated as ApIFI44), and their reciprocal hybrids (designated as AipIFI44 and ApiIFI44, respectively). Their open reading frame (ORF) sequences were all 1434 bp, encoding 477 amino acids, but with large variations among the four genes. The AipIFI44 and ApiIFI44 exhibited higher similarity with ApIFI44 than with AiIFI44. All four genes have a TLDc structural domain with significant variations in sequences among them. Predicted differences in conformation and posttranslational modifications may lead to altered protein activity. We further demonstrated that the AiIFI44, AipIFI44 and ApiIFI44 expressed in all the tested tissues, with the highest expression in the gills and hepatopancreas. In response to Vibrio anguillarum challenge, the profile of mRNA expression of IFI44 gene differed among the bay scallops and the two hybrids. In the bay scallops, it increased at 6 h but dramatically decreased after 12-48 h. However, the mRNA expression of both AipIFI44 and ApiIFI44 decreased at 6 h but continuously increased thereafter and reached the highest value at 48 h. The results in the present study suggest the immune responds of IFI44 in scallops and it may be related to the higher resistance to Vibrio bacterial in hybrids.

Characterization of TRAF genes and their responses to Vibrio anguillarum challenge in Argopecten scallops.

The tumor necrosis factor receptor-related factor (TRAF) family has been reported to be involved in many immune pathways, such as TNFR, TLR, NLR, and RLR in animals. However, little is known about the roles of TRAF genes in the innate immune of Argopecten scallops. In this study, we first identified five TRAF genes, including TRAF2, TRAF3, TRAF4, TRAF6 and TRAF7, but not TRAF1 and TRAF5, from both the bay scallop A. irradians (Air) and the Peruvian scallop A. purpuratus (Apu). The phylogenetic analysis showed that the TRAF genes in Argopecten scallops (AiTRAF) belong to the branch of molluscan TRAF family, which lacks TRAF1 and TRAF5. Since TRAF6 is a key bridge factor in the tumor necrosis factor superfamily and plays an important role in innate and adaptive immunity, we cloned the ORFs of the TRAF6 gene in both A. irradians and A. purpuratus, as well as in two reciprocal hybrids (Aip for the hybrid Air × Apu and Api for the hybrid Apu × Air). Differences in conformational and post-translational modification resulted from the variation in amino acid sequences may cause differences in activity among them. Analysis of conserved motifs and protein structural domains revealed that AiTRAF contains typical structural domains similar to those of other mollusks and has the same conserved motifs. Tissue expression of TRAF in Argopecten scallops challenged by Vibrio anguillarum was examined by qRT-PCR. The results showed that AiTRAF were higher in gill and hepatopancreas. When challenged by Vibrio anguillarum, the expression of AiTRAF was significantly increased compared with the control group, indicating that AiTRAF may play an important role in the immunity of scallops. In addition, the expression of TRAF was higher in Api and Aip than in Air when challenged by Vibrio anguillarum, suggesting that TRAF may have contributed to the high resistance of Api and Aip to Vibrio anguillarum. The results of this study may provide new insights into the evolution and function of TRAF genes in bivalves and ultimately benefit scallop breeding.

Plipastatin and surfactin coproduction by Bacillus subtilis pB2-L and their effects on microorganisms.

To convert the lipopeptide non-producer strain Bacillus subtilis pB2 into a plipastatin and surfactin coproducer, a gene expression cassette composed of a constitutive promoter (P43), functional gene sfp, and pleiotropic regulatory gene degQ was integrated into the chromosomal amyE locus of strain B. subtilis pB2 by homologous recombination, which generated a plipastatin and surfactin co-producer. Thirteen plipastatins and fifteen surfactins were identified in lipopeptide extracts using analytical techniques, and their effects on microorganisms were described by microscopic, cytoskeleton analysis and flow-cytometry, respectively. Plipastatins isolated from the engineered strain pB2-L exhibited strong antifungal activity (MIC 16 µg ml-1) by disrupting the cell walls, membranes and cytoskeleton of Fusarium oxysporum f. sp. cucumerinum hyphae. Surfactins affected the cell membrane of Staphylococcus aureus (MIC 20 µg ml-1), resulting in nucleic acid leakage and ultimately, cell death. Based on the convenience of genetic manipulation in the engineering strain, this work could be useful for the rational design of lipopeptide synthetases via the recombination of gene fragments to generate arrays of peptide derivatives and thus expand the diversity of microbial-produced lipopeptides.

Natural and anthropogenic controls on environmental change during the Holocene based on a multi-proxy record obtained from subalpine peatland in southern China.

The interactions between past climate, human activity and environmental change in subtropical mountainous areas are poorly understood due to the lack of reliable records in South China. In this study, the evolution of the East Asian summer monsoon (EASM) during the Holocene, and the interactions between regional human activity and environmental change, were studied using multi-proxy records from a subalpine peat core recovered from South China. The chronology of this peat core has been well-constrained by 10 AMS 14C dates of peat stems. A series of proxy indicators, including carbon isotopes (δ13C), loss on ignition (LOI), magnetic susceptibility (MS), the chemical index of alteration (CIA), and geochemical elements from the Shiwangutian (SWGT) peatland were used to reconstruct the palaeohydrological changes during the Holocene. Regional moisture levels showed a generally arid-wet-arid pattern, and three phases of climatic change were detected as follows. 1) Between 11,600 and 9000 cal yr BP, the EASM was weak and a relatively dry climate developed. 2) Between 9000 and 4000 cal yr BP, the prevalence of humid climatic conditions was associated with a strong summer monsoon. 3) After 4000 cal yr BP, the climate shifted to relatively dry conditions. Further comparisons and analysis suggested that solar insolation, migration of the Intertropical Convergence Zone (ITCZ), and El Niño-Southern Oscillation (ENSO) activity played an important role in determining the variations in Holocene EASM intensity. In addition, the increase in both MS and heavy metal concentrations over the last 1000 years is consistent with an increase in the population of Hunan Province. Therefore, it can be inferred that population growth and the associated expansion of cropland and mining led to an increase in soil erosion and metal tool use. These findings suggest that the impact of human activity generally outweighed the natural climatic controls on the environment and landscape in the mountainous region of southern China over the last 1000 years.

Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation.

Effects of fengycin from Bacillus subtilis fmbJ on apoptosis and necrosis in Rhizopus stolonifer.

The lipopeptide antibiotic fengycin, produced by Bacillus subtilis, strongly inhibits growth of filamentous fungi. In this study, we evaluated the effects of fengycin treatment on apoptosis and necrosis in Rhizopus stolonifer by means of cell staining and epifluorescence microscopy. At fengycin concentrations less than 50 µg/ml, treated fungal cells demonstrated a dose-dependent increase in apoptosis-associated markers compared with the untreated control. These markers included chromatin condensation, reactive oxygen species accumulation, mitochondrial membrane potential depolarization, phosphatidylserine externalization, and the occurrence of DNA strand breaks. These results showed that fungal cells were impaired in a number of important functions and entered apoptosis upon treatment with low concentrations of fengycin. In contrast, high concentrations (>50 µg/ml) induced necrosis, indicating that the fungicidal action of fengycin operates via two modes: apoptosis at low concentrations and necrosis at high concentrations. Additionally, the apoptotic effect that we have shown suggests that lower concentrations of fengycin than previously thought may be effective for food preservation.