Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans.

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Natural compound Byakangelicin suppresses breast tumor growth and motility by regulating SHP-1/JAK2/STAT3 signal pathway.

Byakangelicin mostly obtained from the root of Angelica dahurica and has protective effect on liver injury and fibrosis. In addition, Byakangelicin, as a traditional medicine, is also used to treat colds, headache and toothache. Recent studies have shown that Byakangelicin exhibits anti-tumor function; however, the role of Byakangelicin in breast tumor progression and related mechanism has not yet been elucidated. Our study aims to investigate the role of Byakangelicin in breast tumor progression and the underlying mechanism. To measure the effect of Byakangelicin on JAK2/STAT3 signaling, a dual luciferase reporter assay and a Western blot assay were performed. CCK8, colony formation, apoptosis and cell invasion assays were used to examine the inhibitory potential of Byakangelicin on breast cancer cells. Additionally, SHP-1 was silenced by specific siRNA duplex and the function of SHP-1 on Byakangelicin-mediated inhibition of JAK2/STAT3 signaling was evaluated. Byakangelicin treatment significantly inhibited STAT3 transcriptional activity. In addition, Byakangelicin treatment blocked JAK2/STAT3 signaling in a dose-dependent manner. Byakangelicin-treated tumor cells showed a dramatically reduced proliferation, colony formation and invasion ability. Moreover, Byakangelicin remarkedly induced breast cancer cell apoptosis. Furthermore, Byakangelicin regulated the expression of SHP1.In conclusion, our current study indicated that Byakangelicin, a natural compound, inhibits SHP-1/JAK2/STAT3 signaling and thus blocks tumor growth and motility.

Trophic interactions in soil micro-food webs drive ecosystem multifunctionality along tree species richness.

Rapid biodiversity losses under global climate change threaten forest ecosystem functions. However, our understanding of the patterns and drivers of multiple ecosystem functions across biodiversity gradients remains equivocal. To address this important knowledge gap, we measured simultaneous responses of multiple ecosystem functions (nutrient cycling, soil carbon stocks, organic matter decomposition, plant productivity) to a tree species richness gradient of 1, 4, 8, 16, and 32 species in a young subtropical forest. We found that tree species richness had negligible effects on nutrient cycling, organic matter decomposition, and plant productivity, but soil carbon stocks and ecosystem multifunctionality significantly increased with tree species richness. Linear mixed-effect models showed that soil organisms, particularly arbuscular mycorrhizal fungi (AMF) and soil nematodes, elicited the greatest relative effects on ecosystem multifunctionality. Structural equation models revealed indirect effects of tree species richness on ecosystem multifunctionality mediated by trophic interactions in soil micro-food webs. Specifically, we found a significant negative effect of gram-positive bacteria on soil nematode abundance (a top-down effect), and a significant positive effect of AMF biomass on soil nematode abundance (a bottom-up effect). Overall, our study emphasizes the significance of a multitrophic perspective in elucidating biodiversity-multifunctionality relationships and highlights the conservation of functioning soil micro-food webs to maintain multiple ecosystem functions.

Soil nematode abundances drive agroecosystem multifunctionality under short-term elevated CO2 and O3.

The response of soil biotas to climate change has the potential to regulate multiple ecosystem functions. However, it is still challenging to accurately predict how multiple climate change factors will affect multiple ecosystem functions. Here, we assessed the short-term responses of agroecosystem multifunctionality to a factorial combination of elevated CO2 (+200 ppm) and O3 (+40 ppb) and identified the key soil biotas (i.e., bacteria, fungi, protists, and nematodes) concerning the changes in the multiple ecosystem functions for two rice varieties (Japonica, Nanjing 5055 vs. Wuyujing 3). We provided strong evidence that combined treatment rather than individual treatments of short-term elevated CO2 and O3 significantly increased the agroecosystem multifunctionality index by 32.3% in the Wuyujing 3 variety, but not in the Nanjing 5055 variety. Soil biotas exhibited an important role in regulating multifunctionality under short-term elevated CO2 and O3 , with soil nematode abundances better explaining the changes in ecosystem multifunctionality than soil biota diversity. Furthermore, the higher trophic groups of nematodes, omnivores-predators served as the principal predictor of agroecosystem multifunctionality. These results provide unprecedented new evidence that short-term elevated CO2 and O3 can potentially affect agroecosystem multifunctionality through soil nematode abundances, especially omnivores-predators. Our study demonstrates that high trophic groups were specifically beneficial for regulating multiple ecosystem functions and highlights the importance of soil nematode communities for the maintenance of agroecosystem functions and health under climate change in the future.

Elevated O3 Exerts Stronger Effects than Elevated CO2 on the Functional Guilds of Fungi, but Collectively Increase the Structural Complexity of Fungi in a Paddy Soil.

Global climate change is characterized by altered global atmospheric composition, including elevated CO2 and O3, with important consequences on soil fungal communities. However, the function and community composition of soil fungi in response to elevated CO2 together with elevated O3 in paddy soils remain largely unknown. Here we used twelve open-top chamber facilities (OTCs) to evaluate the interactive effect of CO2 (+ 200 ppm) and O3 (+ 40 ppb) on the diversity, gene abundance, community structure, and functional composition of soil fungi during the growing seasons of two rice cultivars (Japonica, Wuyujing 3 vs. Nangeng 5055) in a Chinese paddy soil. Elevated CO2 and O3 showed no individual or combined effect on the gene abundance or relative abundance of soil fungi, but increased structural complexity of soil fungal communities, indicating that elevated CO2 and/or O3 promoted the competition of species-species interactions. When averaged both cultivars, elevated CO2 showed no individual effect on the diversity or abundance of functional guilds of soil fungi. By contrast, elevated O3 significantly reduced the relative abundance and diversity of symbiotrophic fungi by an average of 47.2% and 39.1%, respectively. Notably, elevated O3 exerts stronger effects on the functional processes of fungal communities than elevated CO2. The structural equation model revealed that elevated CO2 and/or O3 indirectly affected the functional composition of soil fungi through community structure and diversity of soil fungi. Root C/N and soil environmental parameters were identified as the top direct predictors for the community structure of soil fungi. Furthermore, significant correlations were identified between saprotrophic fungi and root biomass, symbiotrophic fungi and root carbon, the pathotroph-symbiotroph and soil pH, as well as pathotroph-saprotroph-symbiotroph and soil microbial biomass carbon. These results suggest that climatic factors substantially affected the functional processes of soil fungal, and threatened soil function and food production, highlighting the detrimental impacts of high O3 on the function composition of soil biota.

Simplifying network complexity of soil bacterial community exposed to short-term carbon dioxide and ozone enrichment in a paddy soil.

Global atmospheric changes are characterized by increases in carbon dioxide (CO2) and ozone (O3) concentrations, with important consequences for the soil microbial community. However, the influences of CO2 and O3 enrichment on the biomass, diversity, composition, and functioning of the soil bacterial community remain unclear. We investigated the effects of short-term factorial combinations of CO2 (by 200 ppm) and O3 (by 40 ppb) enrichment on the dynamics of soil bacterial community in paddy soils with two rice varieties (Japonica, Nangeng 5055 (NG5055) vs. Wuyujing 3 (WYJ3)) in an open top chamber facility. When averaged both varieties, CO2 and O3 enrichment showed no individual or combined effect on the abundance or diversity of soil bacterial community. Similarly, CO2 enrichment did not exert any significant effect on the relative abundance of bacterial phyla. However, O3 enrichment significantly reduced the relative abundance of Myxococcota phylum by a mean of 37.5%, which negatively correlated to root N content. Compared to ambient conditions, soil bacterial community composition was separated by CO2 enrichment in NG5055, and by both CO2 and O3 enrichment in WYJ3, with root N content identified as the most influential factor. These results indicated that root N was the top direct predictor for the community composition of soil bacteria. The COG (cluster of orthologous groups) protein of cell motility was significantly reduced by 5.8% under CO2 enrichment, and the COG protein of cytoskeleton was significantly decreased by 14.7% under O3 enrichment. Furthermore, the co-occurrence network analysis indicated that both CO2 and O3 enrichment decreased the network complexity of the soil bacterial community. Overall, our results highlight that continuous CO2 and O3 enrichment would potentially damage the health of paddy soils through adverse impacts on the associations and functional composition of soil microbial communities.

Contribution of tree species to the co-occurrence network of the leaf phyllosphere and soil bacterial community in the subtropical forests.

The underlying mechanisms of the interactions between bacterial communities and tree species are still unknown, primarily attributed to a focus on the soil system while ignoring the leaf phyllosphere, which is a complex and diverse ecosystem that supports microbial diversity in the forest ecosystem. To gain insights into the mechanisms, the effects of seven common subtropical tree species, involving Pinus massoniana Lamb., Mytilaria laosensis Lecomte., Ilex chinensis Sims., Michelia macclurei Dandy., Liquidambar formosana Hance., Quercus acutissima Carruth., and Betula luminifera H.Winkler on the bacterial communities were investigated in the leaf phyllosphere and soil systems. We found that the bacterial community was dominated by Proteobacteria in the leaf phyllosphere (63.2-84.7%), and was dominated by Proteobacteria (34.3-45.0%) and Acidobacteria (32.5-40.6%) in soil. Mycorrhizal types and leaf phenology had no apparent effects on the bacterial abundance in the bacterial diversity in the leaf phyllosphere and soil. The bacterial community composition was greatly influenced by tree species in the leaf phyllosphere rather than in soil, with soil parameters (soil pH and C/N) and litter N identified as the most important factors. Ectomycorrhizal trees exerted positive effects on the complexity of the bacterial community at the expense of decreasing the robustness of the soil bacterial community, potentially threatening ecosystem stability. Evergreen trees decreased the network robustness of bacterial community by 21.9% higher than this of deciduous trees in the leaf phyllosphere. Similarly, evergreen trees decreased soil bacterial abundance by 50.8% and network robustness by 8.0% compared to deciduous trees, indicating the adverse impacts of leaf phenology on the bacterial stability in both leaf and soil. Overall, our results highlight the need for studies of leaf-associated bacteria to comprehensively understand the potential effects of tree species on microbial diversity and stability in subtropical forests.

Mitochondrial ATPase inhibitor factor 1, MjATPIF1, is beneficial for WSSV replication in kuruma shrimp (Marsupenaeus japonicus).

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.

Thymosins participate in antibacterial immunity of kuruma shrimp, Marsupenaeus japonicus.

Thymosins ß are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp.

Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp.

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjß-arrestin2. Further studies found that Mjß-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus.

ß-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity.

The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that ß-arrestins negatively regulate Toll signaling in two different ways. ß-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of ß-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. ß-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that ß-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

Soil ecosystem represents the largest contributor to global nitrous oxide (N2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N-18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N2 O production from nitrifier-induced denitrification, a potential significant source of N2 O production in agricultural soils.

Leucine-rich repeats containing protein functions in the antibacterial immune reaction in stomach of kuruma shrimp Marsupenaeus japonicus.

Leucine rich repeat (LRR) motif exists in many immune receptors of animals and plants. Most LRR containing (LRRC) proteins are involved in protein-ligand and protein-protein interaction, but the exact functions of most LRRC proteins were not well-studied. In this study, an LRRC protein was identified from kuruma shrimp Marsupenaeus japonicus, and named as MjLRRC1. MjLRRC1 was consistently expressed in different tissues of normal shrimp with higher expression in gills and stomach. At the transcriptional level, there were no significant changes of MjLRRC1 after injection of Vibrio anguillarum or Staphylococcus aureus in gills and hepatopancreas. While in V. anguillarum oral infection, MjLRRC1 was upregulated in stomach but not in intestine. The recombinant MjLRRC1 protein could bind to Gram-positive and Gram-negative bacteria, bacterial cell wall components including peptidoglycan, lipoteichoic acid, and lipopolysaccharide. MjLRRC1 regulated the expression of some antimicrobial peptide (AMP) genes and participated in bacteria clearance of stomach. All these results suggested that MjLRRC1 might play important roles in antibacterial immune response of kuruma shrimp.

Myeloid leukemia factor functions in anti-WSSV immune reaction of kuruma shrimp, Marsupenaeus japonicus.

Myeloid leukemia factor (MLF) plays an important role in development, cell cycle, myeloid differentiation, and regulates the RUNX transcription factors. However, the function of MLF in immunity is still unclear. In this study, an MLF was identified and characterized in kuruma shrimp Marsupenaeus japonicus, and named as MjMLF. The full-length cDNA of MjMLF contained 1111 nucleotides, which had an opening reading frame of 816 bp encoding a protein of 272 amino acids with an MLF1-interacting protein domain. MjMLF could be ubiquitously detected in different tissues of shrimp at the transcriptional level. The expression pattern analysis showed that MjMLF could be upregulated in shrimp hemocytes and hepatopancreas after white spot syndrome virus challenge. The RNA interference and protein injection assay showed that MjMLF could inhibit WSSV replication in vivo. Flow cytometry assay showed that MjMLF could induce hemocytes apoptosis which functioned in the shrimp antiviral reaction. All the results suggested that MjMLF played an important role in the antiviral immune reaction of kuruma shrimp. The research indicated that MjMLF might function as a novel regulator to inhibit WSSV replication in shrimp.

Scavenger receptor C promotes bacterial clearance in kuruma shrimp Marsupenaeus japonicus by enhancing hemocyte phagocytosis and AMP expression.

Scavenger receptors (SRs) comprise a large family of structurally diverse glycoproteins located on the cell membrane and function as pattern-recognition receptors (PRRs) participating in innate immunity in different species. Class C scavenger receptor (SRC) has been only identified in invertebrates and its biological functions still need to be researched. In this study, we characterized the anti-bacterial function of a SRC from kuruma shrimp Marsupenaeus japonicus (MjSRC). The mRNA level of MjSRC was up-regulated significantly in hemocytes of kuruma shrimp challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant extracellular domains (MAM and CCP domains) of MjSRC have the ability of binding different bacteria and glycans in vitro. After knockdown of MjSRC, the bacterial clearance ability and phagocytic rate of hemocyte decreased significantly in vivo. Meanwhile, overexpression of MjSRC in shrimp enhanced the clearance ability and phagocytic rate of hemocytes. Further study found that MjSRC could regulate the expression of several antimicrobial peptides (AMPs). All these results indicate that MjSRC plays important roles in antibacterial immunity in kuruma shrimp by enhancing hemocyte phagocytosis and AMP expression.

Long-Term Nickel Contamination Increases the Occurrence of Antibiotic Resistance Genes in Agricultural Soils.

Heavy metal contamination is assumed to be a selection pressure on antibiotic resistance, but to our knowledge, evidence of the heavy metal-induced changes of antibiotic resistance is lacking on a long-term basis. Using quantitative PCR array and Illumina sequencing, we investigated the changes of a wide spectrum of soil antibiotic resistance genes (ARGs) following 4-5 year nickel exposure (0-800 mg kg-1) in two long-term experimental sites. A total of 149 unique ARGs were detected, with multidrug and ß-lactam resistance as the most prevailing ARG types. The frequencies and abundance of ARGs tended to increase along the gradient of increasing nickel concentrations, with the highest values recorded in the treatments amended with 400 mg nickel kg-1 soil. The abundance of mobile genetic elements (MGEs) was significantly associated with ARGs, suggesting that nickel exposure might enhance the potential for horizontal transfer of ARGs. Network analysis demonstrated significant associations between ARGs and MGEs, with the integrase intI1 gene having the most frequent interactions with other co-occurring ARGs. The changes of ARGs were mainly driven by nickel bioavailability and MGEs as revealed by structural equation models. Taken together, long-term nickel exposure significantly increased the diversity, abundance, and horizontal transfer potential of soil ARGs.

Effects of the Nitrification Inhibitor 3,4-Dimethylpyrazole Phosphate on Nitrification and Nitrifiers in Two Contrasting Agricultural Soils.

UNLABELLED: The nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) is a powerful tool that can be used to promote nitrogen (N) use efficiency and reduce N losses from agricultural systems by slowing nitrification. Mounting evidence has confirmed the functional importance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in nitrification and N2O production; however, their responses to DMPP amendment and the microbial mechanisms underlying the variable efficiencies of DMPP across different soils remain largely unknown. Here we compared the impacts of DMPP on nitrification and the dynamics of ammonia oxidizers between an acidic pasture soil and an alkaline vegetable soil using a (15)N tracing and (13)CO2-DNA-stable-isotope probing (SIP) technique. The results showed that DMPP significantly inhibited nitrification and N2O production in the vegetable soil only, and the transient inhibition was coupled with a significant decrease in AOB abundance. No significant effects on the community structure of ammonia oxidizers or the abundances of total bacteria and denitrifiers were observed in either soil. The (15)N tracing experiment revealed that autotrophic nitrification was the predominant form of nitrification in both soils. The (13)CO2-DNA-SIP results indicated the involvement of AOB in active nitrification in both soils, but DMPP inhibited the assimilation of (13)CO2 into AOB only in the vegetable soil. Our findings provide evidence that DMPP could effectively inhibit nitrification through impeding the abundance and metabolic activity of AOB in the alkaline vegetable soil but not in the acidic pasture soil, possibly due to the low AOB abundance or the adsorption of DMPP by organic matter. IMPORTANCE: The combination of the (15)N tracing model and (13)CO2-DNA-SIP technique provides important evidence that the nitrification inhibitor DMPP could effectively inhibit nitrification and nitrous oxide emission in an alkaline soil through influencing the abundance and metabolic activity of AOB. In contrast, DMPP amendment has no significant effect on nitrification or nitrifiers in an acidic soil, potentially owing to the low abundance of AOB and the possible adsorption of DMPP by organic matter. Our findings have direct implications for improved agricultural practices through utilizing the nitrification inhibitor DMPP in appropriate situations, and they emphasize the importance of microbial communities to the efficacy of DMPP.

Involvement of a LysM and putative peptidoglycan-binding domain-containing protein in the antibacterial immune response of kuruma shrimp Marsupenaeus japonicus.

Lysin motif (LysM) is a peptidoglycan and chitin-binding motif with multiple functions in bacteria, plants, and animals. In this study, a novel LysM and putative peptidoglycan-binding domain-containing protein was cloned from kuruma shrimp (Marsupenaeus japonicus) and named as MjLPBP. The cDNA of MjLPBP contained 1010 nucleotides with an open reading frame of 834 nucleotides encoding a protein of 277 amino acid residues. The deduced protein contained a Lysin motif and a transmembrane region, with a calculated molecular mass of 31.54 kDa and isoelectric point of 8.61. MjLPBP was ubiquitously distributed in different tissues of shrimp at the mRNA level. Time course expression assay showed that MjLPBP was upregulated in hemocytes of shrimp challenged with Vibrio anguillarum or Staphylococcus aureus. MjLPBP was also upregulated in hepatopancreas after white spot syndrome virus and bacteria challenge. The recombinant protein of MjLPBP could bind to some Gram-positive and Gram-negative bacteria and yeast. Further study found that rMjLPBP bound to bacterial cell wall components, including peptidoglycans, lipoteichoic acid, lipopolysaccharide, and chitin. The induction of several antimicrobial peptide genes and phagocytosis-related gene, such as anti-lipopolysaccharide factors and myosin, was depressed after knockdown of MjLPBP. MjLPBP could facilitate V. anguillarum clearance in vivo. All the results indicated that MjLPBP might play an important role in the innate immunity of shrimp.

Prohibitin Interacts with envelope proteins of white spot syndrome virus and prevents infection in the red swamp crayfish, Procambarus clarkii.

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.

A fibrinogen-related protein (FREP) is involved in the antibacterial immunity of Marsupenaeus japonicus.

Fibrinogen-related proteins (FREPs) in invertebrates have important functions in innate immunity. In this study, the cDNA of FREP was identified from the kuruma shrimp Marsupenaeus japonicus (MjFREP2). The full-length cDNA of MjFREP2 is 1138 bp with an open reading frame of 954 bp that encodes a 317-amino acid protein comprising a signal peptide and a fibrinogen-like domain. MjFREP2 could be detected in hemocytes, heart, hepatopancreas, gills, stomach, and intestines. MjFREP2 could also be upregulated in hemocytes after Vibrio anguillarum and Staphylococcus aureus challenge. Agglutination and binding assay results revealed that the recombinant MjFREP2 bound to bacteria and polysaccharides. Immunocytochemical analysis results showed that MjFREP2 proteins were mainly distributed in the cytoplasm of hemocytes from unchallenged shrimp and transported to the membrane or secreted out of the cell after V. anguillarum or S. aureus challenge. The secreted MjFREP2 bound to the bacteria presented in shrimp hemolymph. The overexpression of MjFREP2 could enhance bacterial clearance by inducing the phagocytosis of hemocytes. This ability was impaired by knockdown of MjFREP2 with RNA interference. The cumulative mortality of MjFREP2-silenced shrimp was significantly higher than that of the control shrimp. These results suggested that MjFREP2 has an important function in the antibacterial immunity of M. japonicus.