Spatial Transcriptome Profiling of Mouse Hippocampal Single Cell Microzone in Parkinson's Disease.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and cognitive functions. It is still unknown how gene expression changes in single-cell in different spatial locations of the hippocampus of Parkinson's disease. The purpose of this study was to analyze the gene expression features of single cells in different spatial locations of mouse hippocampus, and to explore the effects of gene expression regulation on learning and memory mechanisms. Here, we obtained 74 single-cell samples from different spatial locations in a mouse hippocampus through microdissection technology, and used single-cell RNA-sequencing and spatial transcriptome sequencing to visualize and quantify the single-cell transcriptome features of tissue sections. The results of differential expression analysis showed that the expression of Sv2b, Neurod6, Grp and Stk32b genes in a hippocampus single cell at different locations was significantly different, and the marker genes of CA1, CA3 and DG subregions were identified. The results of gene function enrichment analysis showed that the up-regulated differentially expressed genes Tubb2a, Eno1, Atp2b1, Plk2, Map4, Pex5l, Fibcd1 and Pdzd2 were mainly involved in neuron to neuron synapse, vesicle-mediated transport in synapse, calcium signaling pathway and neurodegenerative disease pathways, thus affecting learning and memory function. It revealed the transcriptome profile and heterogeneity of spatially located cells in the hippocampus of PD for the first time, and demonstrated that the impaired learning and memory ability of PD was affected by the synergistic effect of CA1 and CA3 subregions neuron genes. These results are crucial for understanding the pathological mechanism of the Parkinson's disease and making precise treatment plans.

A comprehensive characterization of cell-free RNA in spent blastocyst medium and quality prediction for blastocyst.

The rate of pregnancy can be affected by many factors in assisted reproductive technology (ART), and one of which is the quality of embryos. Therefore, selecting the embryos with high potential is crucial for the outcome. Fifteen spent blastocyst medium (SBM) samples were collected from 14 patients who received in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), seven from high-grade embryos and eight from low-grade embryos. Cell-free RNA (cf-RNA) profile of SBM samples were analyzed by RNA sequencing in the present study. It was found that a large amount of cf-RNA were released into SBM, including protein-coding genes (68.9%) and long noncoding RNAs (lncRNAs) (17.26%). Furthermore, a high correlation was observed between blastocyst genes and SBM genes. And the cf-mRNAs of SBM were highly fragmented, and coding sequence (CDS) and untranslated (UTR) regions were released equally. Two hundred and thirty-two differentially expressed genes were identified in high-grade SBM (hSBM) and low-grade SBM (lSBM), which could be potential biomarker in distinguishing the embryos with different quality as an alternative or supplementary approach for subjective morphology criteria. Hence, cf-RNAs sequencing revealed the characterization of circulating transcriptomes of embryos with different quality. Based on the results, the genes related to blastocyst quality were screened, including the genes closely related to translation, immune-signaling pathway, and amino acid metabolism. Overall, the present study showed the types of SBM cf-RNAs, and the integrated analysis of cf-RNAs profiling with morphology grading displayed its potential in predicting blastocyst quality. The present study provided valuable scientific basis for noninvasive embryo selection in ART by RNA-profiling analysis.

Extracellular cell-free RNA profile in human large follicles and small follicles.

Background: Previous studies have shown that a large number of valuable and functional cell-free RNAs (cfRNAs) were found in follicular fluid. However, the species and characteristics of follicular fluid cfRNAs have not been reported. Furthermore, their implications are still barely understood in the evaluation of follicular fluid from follicles of different sizes, which warrants further studies. Objective: This study investigated the landscape and characteristics of follicular fluid cfRNAs, the source of organization, and the potential for distinguishing between follicles of different sizes. Methods: Twenty-four follicular fluid samples were collected from 20 patients who received in vitro fertilization (n = 9) or ICSI (n = 11), including 16 large follicular fluid and 8 small follicular fluid samples. Also, the cfRNA profile of follicular fluid samples was analyzed by RNA sequencing. Results: This result indicated that the concentration of follicular fluid cfRNAs ranged from 0.78 to 8.76 ng/ml, and fragment length was 20-200 nucleotides. The concentration and fragment length of large follicular fluid and small follicular fluid samples were not significantly different (p > 0.05). The technical replica correlation of follicular fluid samples ranged from 0.3 to 0.9, and the correlation of small follicular fluid samples was remarkably (p < 0.001) lower than that of large follicular fluid samples. Moreover, this study found that cfRNAs of the follicular fluid could be divided into 37 Ensembl RNA biotypes, and a large number of mRNAs, circRNAs, and lncRNAs were observed in the follicular fluid. The number of cfRNAs in large follicular fluid was remarkably (p < 0.05) higher than that of small follicular fluid. Furthermore, the follicular fluid contained a large amount of intact mRNA and splice junctions and a large number of tissue-derived RNAs, which are at a balanced state of supply and elimination in the follicular fluid. KEGG pathway analysis showed that differentially expressed cfRNAs were enriched in several pathways, including thyroid hormone synthesis, the cGMP-PKG signaling pathway, and inflammatory mediator regulation of TRP channels. In addition, we further showed that four cfRNAs (TK2, AHDC1, PHF21A, and TTYH1) serve as a potential indicator to distinguish the follicles of different sizes. The ROC curve shows great potential to predict follicular fluid from follicles of different sizes [area under the curve (AUC) > 0.88]. Conclusion: Overall, our study revealed that a large number of cfRNAs could be detected in follicular fluid and could serve as a potential non-invasive biomarker in distinguishing between follicles of different sizes. These results may inform the study of the utility and implementation of cfRNAs in clinical practice.

Prediction of Time-Series Transcriptomic Gene Expression Based on Long Short-Term Memory with Empirical Mode Decomposition.

RNA degradation can significantly affect the results of gene expression profiling, with subsequent analysis failing to faithfully represent the initial gene expression level. It is urgent to have an artificial intelligence approach to better utilize the limited data to obtain meaningful and reliable analysis results in the case of data with missing destination time. In this study, we propose a method based on the signal decomposition technique and deep learning, named Multi-LSTM. It is divided into two main modules: One decomposes the collected gene expression data by an empirical mode decomposition (EMD) algorithm to obtain a series of sub-modules with different frequencies to improve data stability and reduce modeling complexity. The other is based on long short-term memory (LSTM) as the core predictor, aiming to deeply explore the temporal nonlinear relationships embedded in the sub-modules. Finally, the prediction results of sub-modules are reconstructed to obtain the final prediction results of time-series transcriptomic gene expression. The results show that EMD can efficiently reduce the nonlinearity of the original data, which provides reliable theoretical support to reduce the complexity and improve the robustness of LSTM models. Overall, the decomposition-combination prediction framework can effectively predict gene expression levels at unknown time points.

Sensitive and Low-Bias Transcriptome Sequencing Using Agarose PCR.

Transcriptome sequencing has emerged as an important research tool for exploring the mysteries of life at the single-cell level. However, its wide application is limited by the bias associated with the amplification reactions which is essential for library building of trace RNA. In this study, low-melting-point agarose was added to the amplification reactions to take advantage of its molecular crowding effect and polymer cross-linked structure to improve the sensitivity of the reactions and reduce bias. To further evaluate the performance of the method, it was applied to transcriptome sequencing of microregion samples from brain tissue sections of mice with Parkinson's disease at the single cell level. The results showed that agarose PCR had better performance than in-tube PCR. Further application of agarose PCR to transcriptome library sequencing could obtain data closer to that of unamplified. With the addition of low melting point agarose, the sensitivity of the amplification reaction was significantly increased, while homogeneity was increased by approximately 2-fold. Not only that, but this work also provides 11% sensitivity improvement for spatial transcriptomic study on Parkinson's disease-associated gene detection. The agarose PCR provides a new tool for efficient and homogeneous amplification of trace samples and can be widely used for spatial transcriptome library sequencing and studies.

Comparative analysis of circular RNA enrichment methods.

The circRNAs sequencing results vary due to the different enrichment methods and their performance is needed to systematic comparison. This study investigated the effects of different circRNA enrichment methods on sequencing results, including abundance and species of circRNAs, as well as the sensitivity and precision. This experiment was carried out by following four common circRNA enrichment methods: including ribosomal RNA depletion (rRNA-), polyadenylation and poly (A+) RNA depletion followed by RNase R treatment (polyA+RNase R), rRNA-+polyA+RNase R and polyA+RNase R+ rRNA-. The results showed that polyA+RNase R+ rRNA - enrichment method obtained more circRNA number, higher sensitivity and abundance among them; polyA+RNase R method obtained higher precision. The linear RNAs can be thoroughly removed in all enrichment methods except rRNA depletion method. Overall, our results helps researchers to quickly selection a circRNA enrichment of suitable for own study among many enrichment methods, and it provides a benchmark framework for future improvements circRNA enrichment methods.[Figure: see text].

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing. RESULTS: Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified. CONCLUSIONS: The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

Low bias multiple displacement amplification with confinement effect based on agarose gel.

Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30 µm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing.

Bias in RNA-seq Library Preparation: Current Challenges and Solutions.

Although RNA sequencing (RNA-seq) has become the most advanced technology for transcriptome analysis, it also confronts various challenges. As we all know, the workflow of RNA-seq is extremely complicated and it is easy to produce bias. This may damage the quality of RNA-seq dataset and lead to an incorrect interpretation for sequencing result. Thus, our detailed understanding of the source and nature of these biases is essential for the interpretation of RNA-seq data, finding methods to improve the quality of RNA-seq experimental, or development bioinformatics tools to compensate for these biases. Here, we discuss the sources of experimental bias in RNA-seq. And for each type of bias, we discussed the method for improvement, in order to provide some useful suggestions for researcher in RNA-seq experimental.

Effects of brain tissue section processing and storage time on gene expression.

The pre-processing of samples is important factors that affect the results of the RNA-sequencing (RNA-seq) data. However, the effects of frozen sections storage conditions on the integrity of RNA and sequencing results haven't been reported. The study of frozen section protection schemes can provide reliable experimental results for single-cell and spatial transcriptome sequencing. In this study, RNA was isolated to be studied for RNA from brain section at different temperatures (RT: room temperature, -20 °C) and storage time (0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, 7day, 3week, 6month). The stability of reference genes was validated using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the storage at room temperature significantly affected RNA integrity number (RIN), and the RIN value was lower with the prolongation of storage, while the storage at -20 °C exerted less effect on the RIN value. Cresyl violet staining for brain tissue sections had little effect on RNA integrity. 1925, 899 and 3390 differential expression genes (DEGs) were screened at 2 h, 4 h and 8 h at room temperature, respectively. A total of 892, 478 and 619 genes were shown to be differentially expressed at -20 °C for 7d, 3w and 6 m, respectively. Among them, the expression of glycoprotein m6a (Gpm6a), calmodulin 1 (Calm1), calmodulin 1 (Calm2), thymosin, beta 4, X chromosome (Tmsb4x), ribosomal protein S21 (Rps21) and so on were correlated with RNA quality. According to the expression stability of 4 reference genes (Actb: beta-actin; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S ribosomal; Hprt1: hypoxanthine phosphoribosyltransferase 1), 18S is the most stable reference gene in the brain. In conclusion, the storage temperature and time of frozen sections have significant effects on RNA integrity and sequencing results. But there are still some genes that are stable and not affected by worsening of overall RNA integrity ie the decrease of RIN value. In addition, 1% cresyl violet staining can protect RNA.

Encoding Method of Single-cell Spatial Transcriptomics Sequencing.

Despite significant advances in parallel single-cell RNA sequencing revealing astonishing cellular heterogeneity in many tissue types, the spatial information in the tissue context remains missing. Spatial transcriptome sequencing technology is designed to distinguish the gene expression of individual cells in their original location. The technology is important for the identification of tissue function, tracking developmental processes, and pathological and molecular detection. Encoding the position information is the key to spatial transcriptomics because different methods have different encoding efficiencies and application scenarios. In this review, we focus on the latest technologies of single-cell spatial transcriptomics, including technologies based on microwell plates, barcoded bead arrays, microdissection, in situ hybridization, and barcode in situ targeting, as well as mixed separation-based technologies. Moreover, we compare these encoding methods for use as a reference when choosing the appropriate technology.

Plasma RNA sequencing of extracellular RNAs reveals potential biomarkers for non-small cell lung cancer.

BACKGROUND: Lung cancer is one of the most common malignancies, and it has extremely high incidence and mortality rates. Although there have been many studies focused on lung cancer biomarkers, few have reported the extracellular RNA profiles of lung cancer. In this study, we used RNA-seq technology to analyze extracellular RNAs in low volume peripheral blood plasma; we compared the differentially expressed genes from the plasma of non-small cell lung cancer (NSCLC) patients with that of healthy controls. METHODS: We used RNA-seq technology and bioinformatics to analyze the extracellular RNA (exRNA) sequences of 12 human plasma samples (500 µl per sample), 6 from NSCLC patients and 6 from healthy controls. Subsequently, we used gene ontology (GO) enrichment, KEGG analysis and coexpression experiments to compare the differentially expressed genes (DEGs) and identify tumor biomarkers that were highly correlated with NSCLC. These DEGs were further verified by quantitative PCR. RESULTS: Approximately 20 million clean reads were produced for each plasma sample; 50-80% of the reads aligned to the human references, and hundreds of thousands of reads were counted in each plasma sample. In addition, a total of 640 genes (368 upregulated and 272 downregulated) were differentially expressed between NSCLC plasma and normal plasma. Further, we identified 7 key DEGs that are highly correlated with lung tumorigenesis: COX1, COX2, COX3, ND1, ND2, ND4L, and ATP6. CONCLUSION: exRNA-seq from a small amount (400-500 µl) of plasma opens new possibilities for exploring lung cancer biomarkers in the plasma.

Transcriptomic profiling of differentially expressed genes and related pathways in different brain regions in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease. However, the expression pattern and the differential gene in different brain regions and its functions remain unclear although many studies have been reported. In this present study, PD mouse model were build and four brain regions (cerebral cortex: CC, hippocampus: HP, striatum: ST, and cerebellum: CB) were separated for RNA-seq analysis. Results showed that different expressed genes were found between the different brain regions and more differential genes found in ST and HP when compared with control groups. Among them, Lrrk2, Mtor, Gxylt1, C920006o11Rik, Vdac1, Drd4, and Ncan showed the most significant to PD. PDCC vs. PDHP, PDHP vs. PDST and PDCC vs. PDST groups have 334, 722 and 495 differentially expressed genes (DEGs), respectively. Functional analyses results showed that the differential genes mainly related with posttranscriptional regulation of gene expression and protein localization to organelle and so on, which involved in AMPK, PI3K-Akt signaling pathway, and GABA-ergic synapse. Network biology analysis showed LRRK2, DRD2, IGF-1, GNAI1, GNAI3, PRKACA, PPP2R5C, and PIK3R1 play a major role in protein regulation of PD. Therefore, HP and ST play more important roles in the development of PD and it is also suggested the potential target gene for diagnosis and treatment of PD.

Transcriptomic Profiling of Circular RNA in Different Brain Regions of Parkinson's Disease in a Mouse Model.

Parkinson's disease (PD) is the second most common neurodegenerative disease and although many studies have been done on this disease, the underlying mechanisms are still poorly understood and further studies are warranted. Therefore, this study identified circRNA expression profiles in the cerebral cortex (CC), hippocampus (HP), striatum (ST), and cerebellum (CB) regions of the 1-methyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model using RNA sequencing (RNA-seq), and differentially expressed circRNA were validated using reverse transcription quantitative real-time PCR (qRT-PCR). Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and competing endogenous RNA (ceRNA) network analyses were also performed to explore the potential function of circRNAs. The results show that, compared with the control group, 24, 66, 71, and 121 differentially expressed circRNAs (DE-circRNAs) were found in the CC, HP, ST, and CB, respectively. PDST vs. PDCB, PDST vs. PDHP, and PDCB vs. PDHP groups have 578, 110, and 749 DE-circRNAs, respectively. Then, seven DE-cirRNAs were selected for qRT-PCR verification, where the expressions were consistent with the sequencing analysis. The GO and KEGG pathway analyses revealed that these DE-circRNAs participate in several biological functions and signaling pathways, including glutamic synapse, neuron to neuron synapse, cell morphogenesis involved in neuron differentiation, Parkinson's disease, axon guidance, cGMP-PKG signaling pathway, and PI3K-Akt signaling pathway. Furthermore, the KEGG analysis of the target genes predicted by DE-circRNAs indicated that the target genes predicted by mmu_circRNA_0003292, mmu_circRNA_0001320, mmu_circRNA_0005976, and mmu_circRNA_0005388 were involved in the PD-related pathway. Overall, this is the first study on the expression profile of circRNAs in the different brain regions of PD mouse model. These results might facilitate our understanding of the potential roles of circRNAs in the pathogenesis of PD. Moreover, the results also indicate that the mmu_circRNA_0003292-miRNA-132-Nr4a2 pathway might be involved in the regulation of the molecular mechanism of Parkinson's disease.

The fragmentation patterns of maternal plasma cell-free DNA and its applications in non-invasive prenatal testing.

The discovery of cell-free DNA (cfDNA) in maternal plasma has opened up new promises for the development of non-invasive prenatal testing (NIPT). Application of cfDNA in NIPT of fetus diseases and abnormalities is restricted by the low amount of fetal DNA molecules in maternal plasma. Fetus-derived cfDNA in maternal plasma are shorter than maternal DNA, thus leveraging the maternal and fetus-derived cfDNA molecules size difference has become a novel and more accurate method for NIPT. However, multiple biological properties such as size distribution of plasma DNA, proportion of fetal-derived DNA and methylation levels in maternal plasma across different gestational ages still remain largely unknown. Further insights into the size distribution and fragmentation pattern of circulating plasma cfDNA will shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in prenatal diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In the review, we start by summarizing the research techniques for the determination of the fragmentation profiles of cfDNA in maternal plasma. We then summarize the main progress and findings in size profiles of maternal plasma cfDNA and cffDNA. Finally, we discuss the potential diagnostic applications of plasma cfDNA size profiling.

Regulation of bile acid metabolism-related signaling pathways by gut microbiota in diseases.

Over the past decade, there has been increasing attention on the interaction between microbiota and bile acid metabolism. Bile acids are not only involved in the metabolism of nutrients, but are also important in signal transduction for the regulation of host physiological activities. Microbial-regulated bile acid metabolism has been proven to affect many diseases, but there have not been many studies of disease regulation by microbial receptor signaling pathways. This review considers findings of recent research on the core roles of farnesoid X receptor (FXR), G protein-coupled bile acid receptor (TGR5), and vitamin D receptor (VDR) signaling pathways in microbial-host interactions in health and disease. Studying the relationship between these pathways can help us understand the pathogenesis of human diseases, and lead to new solutions for their treatments.

Analysis of genome-wide in cell free DNA methylation: progress and prospect.

DNA methylation is an important epigenetic marker that affects gene expression. Cell-free DNA methylation detection is a promising approach as abnormal distribution of DNA methylation is one of the hallmarks of many cancers and methylation changes occur early during carcinogenesis. This review summarizes the existing literature and reviews on the detection methods based on next generation sequencing for DNA methylation. The review also discusses the feasibility of detecting cfDNA methylation and the latest progress.

Selection of reference genes for miRNA quantitative PCR and its application in miR-34a/Sirtuin-1 mediated energy metabolism in Megalobrama amblycephala.

MiRNAs are small, non-coding RNAs that downregulate gene expression at post-transcriptional levels. They have emerged as important regulators involved in metabolism, immunity, and cancer. Real-time quantitative PCR (RT-qPCR) is an effective and main method for quantifying target miRNA. For robust RT-qPCR method, suitable reference genes play crucial roles in data normalization. Blunt snout bream (Megalobrama amblycephala) is an economically important aquaculture species; however, no reference genes dedicated for qPCR method has been identified for this species so far. The objective of this study was to screen stable reference genes for miRNA RT-qPCR and demonstrated its application in energy metabolism in blunt snout bream. The stabilities of ten potential reference genes (miR-21-1-5p, miR-107a-3p, miR-222a-3p, miR-146a-5p, miR-101a-3p, miR-22a-3p, miR-103-3p, miR-456-3p, miR-221-3p, and U6 (RNU6A)) were evaluated in nine tissues (brain, muscle, liver, skin, spleen, heart, gill, intestine, and eye) under normal condition and in three tissues (liver, intestine, and spleen) under four stresses (heat stress, ammonia stress, bacterial challenge, and glycolipid stress). Using GeNorm, NormFinder, and RefFinder softwares, we discovered that different tissues and stresses are both important variability factors for the expression stability of miRNAs. After verifying miR-34a/Sirtuin-1 expressions in high-carbohydrate diet-induced blunt snout bream, we eventually identified that the most stable reference gene in this species was miR-221-3p, and the best combination of reference genes were miR-221-3p and miR-103-3p.

Dietary fructooligosaccharide can mitigate the negative effects of immunity on Chinese mitten crab fed a high level of plant protein diet.

An 8-week feeding trial was carried out under controlled condition to evaluate the effect of dietary fructooligosaccharide (FOS) on growth performance, whole body composition, antioxidant status and immunity of crabs fed high levels of plant protein diets. Thus, six experimental diets were formulated (designated as F0P50, F0P60, F0P70, F0.2P50, F0.2P60 and F0.2P70), which contain two FOS levels (0 or 0.2%) and three plant protein levels (50, 60, or 70%) according to a 2 × 3 factorial design. The results showed that weight gain increased significantly as dietary plant protein level decreased from 70% to 50%. At 50% plant protein level, the addition of 0.2% FOS can significantly elevate weight gain (WG) (P < 0.05). The highest value in survival rate was observed in crabs fed F0.2P50 and F0.2P60 diet. Crabs fed F0.2P50 diet showed significantly higher crude protein content (P < 0.05) compared with those in other groups, but there were no significant differences in the contents of moisture, crude lipid and ash among all groups (P > 0.05). Catalase (CAT) activity in crabs fed F0.2P50 increased significantly (P < 0.05) compared with crabs fed F0P60, F0P70, F0.2P60 and F0.2P70, but malondialdehyde (MDA) concentrations decreased significantly (P < 0.05). Meanwhile, nitric oxide (NO) concentration, acid phosphatase (ACP) and alkaline phosphatase (AKP) activities of crabs fed 0.2% FOS diets increased significantly (P < 0.05) compared with crabs fed 0% FOS diets. The expressions of prophenoloxidase (propo) was significantly (P < 0.05) affected only by dietary plant protein levels with the highest values observed in 50% plant protein diet, whereas the opposite was true for Myeloid differentiation factor 88 (myd88). The mRNA expressions of mitochondrial manganese superoxide dismutase (mtmnsod), lipopolysaccharide-induced TNF-α factor (litaf) and toll like receptors (tlrs) were significantly affected (P < 0.05) by both FOS and plant protein levels. The cytosolic manganese superoxide dismutase (cytmnsod) mRNA expressions in F0.2P50 and F0.2P60 groups were significantly higher than those in F0P70 and F0.2P70 groups. The results in this study indicated that supplementation with 0.2% FOS can enhance growth performance in crabs fed lower plant protein diets and as well improve immunity in those fed with higher plant protein diets.