Background: Phosphorus (P) is one of the most essential macronutrients on the planet, and microorganisms (including bacteria and archaea) play a key role in P cycling in all living things and ecosystems. However, our comprehensive understanding of key P cycling genes (PCGs) and microorganisms (PCMs) as well as their ecological functions remains elusive even with the rapid advancement of metagenome sequencing technologies. One of major challenges is a lack of a comprehensive and accurately annotated P cycling functional gene database. Results: In this study, we constructed a well-curated P cycling database (PCycDB) covering 139 gene families and 10 P metabolic processes, including several previously ignored PCGs such as pafA encoding phosphate-insensitive phosphatase, ptxABCD (phosphite-related genes), and novel aepXVWPS genes for 2-aminoethylphosphonate transporters. We achieved an annotation accuracy, positive predictive value (PPV), sensitivity, specificity, and negative predictive value (NPV) of 99.8%, 96.1%, 99.9%, 99.8%, and 99.9%, respectively, for simulated gene datasets. Compared to other orthology databases, PCycDB is more accurate, more comprehensive, and faster to profile the PCGs. We used PCycDB to analyze P cycling microbial communities from representative natural and engineered environments and showed that PCycDB could apply to different environments. Conclusions: We demonstrate that PCycDB is a powerful tool for advancing our understanding of microbially driven P cycling in the environment with high coverage, high accuracy, and rapid analysis of metagenome sequencing data. The PCycDB is available at https://github.com/ZengJiaxiong/Phosphorus-cycling-database.

The outbreak of diseases ordinarily results from the disruption of the balance and harmony between hosts and pathogens. Devoid of adaptive immunity, shrimp rely largely on the innate immune system to protect themselves from pathogenic infection. Two nuclear factor-κB (NF-κB) pathways, the Toll and immune deficiency (IMD) pathways, are generally regarded as the major regulators of the immune response in shrimp, which have been extensively studied over the years. Bacterial infection can be recognized by Toll and IMD pathways, which activate two NF-κB transcription factors, Dorsal and Relish, respectively, to eventually lead to boosting the expression of various antimicrobial peptides (AMPs). In response to white-spot-syndrome-virus (WSSV) infection, these two pathways appear to be subverted and hijacked to favor viral survival. In this review, the recent progress in elucidating microbial recognition, signal transduction, and effector regulation within both shrimp Toll and IMD pathways will be discussed. We will also highlight and discuss the similarities and differences between shrimps and their Drosophila or mammalian counterparts. Understanding the interplay between pathogens and shrimp NF-κB pathways may provide new opportunities for disease-prevention strategies in the future.

C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu ⁹⁹ -Pro ¹⁰⁰ -Asn ¹⁰¹ ) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei . Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

Intestinal microbiota is an integral component of the host and plays important roles in host health. The pacific white shrimp is one of the most profitable aquaculture species commercialized in the world market with the largest production in shrimp consumption. Many studies revealed that the intestinal microbiota shifted significantly during host development in other aquaculture animals. In the present study, 22 shrimp samples were collected every 15 days from larval stage (15 day post-hatching, dph) to adult stage (75 dph) to investigate the intestinal microbiota at different culture stages by targeting the V4 region of 16S rRNA gene, and the microbial function prediction was conducted by PICRUSt. The operational taxonomic unit (OTU) was assigned at 97% sequence identity. A total of 2,496 OTUs were obtained, ranging from 585 to 1,239 in each sample. Forty-three phyla were identified due to the classifiable sequence. The most abundant phyla were Proteobacteria, Cyanobacteria, Tenericutes, Fusobacteria, Firmicutes, Verrucomicrobia, Bacteroidetes, Planctomycetes, Actinobacteria and Chloroflexi. OTUs belonged to 289 genera and the most abundant genera were Candidatus_Xiphinematobacter, Propionigenium, Synechococcus, Shewanella and Cetobacterium. Fifty-nine OTUs were detected in all samples, which were considered as the major microbes in intestine of shrimp. The intestinal microbiota was enriched with functional potentials that were related to transporters, ABC transporters, DNA repair and recombination proteins, two component system, secretion system, bacterial motility proteins, purine metabolism and ribosome. All the results showed that the intestinal microbial composition, diversity and functions varied significantly at different culture stages, which indicated that shrimp intestinal microbiota depended on culture stages. These findings provided new evidence on intestinal microorganism microecology and greatly enhanced our understanding of stage-specific community in the shrimp intestinal ecosystem.

Background: Recently, increasing evidence supports that some complex diseases are not attributed to a given pathogen, but dysbiosis in the host intestinal microbiota (IM). The full intestinal ecosystem alterations, rather than a single pathogen, are associated with white feces syndrome (WFS), a globally severe non-infectious shrimp disease, while no experimental evidence to explore the causality. Herein, we conducted comprehensive metagenomic and metabolomic analysis, and intestinal microbiota transplantation (IMT) to investigate the causal relationship between IM dysbiosis and WFS. Results: Compared to the Control shrimp, we found dramatically decreased microbial richness and diversity in WFS shrimp. Ten genera, such as Vibrio, Candidatus Bacilloplasma, Photobacterium, and Aeromonas, were overrepresented in WFS, whereas 11 genera, including Shewanella, Chitinibacter, and Rhodobacter were enriched in control. The divergent changes in these populations might contribute the observation that a decline of pathways conferring lipoic acid metabolism and mineral absorption in WFS. Meanwhile, some sorts of metabolites, especially lipids and organic acids, were found to be related to the IM alteration in WFS. Integrated with multiomics and IMT, we demonstrated that significant alterations in the community composition, functional potentials, and metabolites of IM were closely linked to shrimp WFS. The distinguished metabolites which were attributed to the IM dysbiosis were validated by feed-supplementary challenge. Both homogenous selection and heterogeneous selection process were less pronounced in WFS microbial community assembly. Notably, IMT shrimp from WFS donors eventually developed WFS clinical signs, while the dysbiotic IM can be recharacterized in recipient shrimp. Conclusions: Collectively, our findings offer solid evidence of the causality between IM dysbiosis and shrimp WFS, which exemplify the ‘microecological Koch’s postulates’ (an intestinal microbiota dysbiosis, a disease) in disease etiology, and inspire our cogitation on etiology from an ecological perspective.

The function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-κB transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins to inhibit viral infection. Taken together, we therefore identify that the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs. The TLR pathway mediated antiviral immune response is well identified in mammals, yet, Toll pathway governing this protection in invertebrates remains unknown. In the present study, we uncover that a shrimp Toll4 from a total of nine Tolls in L. vannamei confers resistance to WSSV thought inducing the NF-κB transcription factor Dorsal to inspire the production of some antimicrobial peptides (AMPs) with antiviral activity. The anti-LPS-factor (ALF) and lysozyme (LYZ) family are identified as the Toll4-Dorsal pathway targeted genes with the ability to interact with viral structural proteins in response to WSSV infection. These results suggest that the Toll receptor induces the expression of AMPs with antiviral activity could be a general antiviral mechanism in invertebrates and Toll pathway established antiviral defense could be conserved during evolution.