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Hainan Province is located in the southernmost part of China. Natural conditions, such as high temperature, drought, and typhoons, have become important factors restricting the development of the red swamp crayfish (Procambarus clarkii) industry. This study aimed to examine the effects of drought stress on growth, ovarian development, antioxidant capacity, sex hormone content, and reproductive gene expression of P. clarkii. An annual survey was conducted on the ovarian development of female P. clarkii in Hainan Province, China, from January 2023 to January 2024. Under the influence of drought and prolonged light, the ovaries of P. clarkii entered the stage of rapid development in May, and the maturity was the highest in September, reaching a maximum of 2.88. In this study, female P. clarkii with a body weight of 31.09 ± 1.20 g were randomly divided into 5 groups with 6 replicates per group for drought stress experiment at 0 d (control group), 10 d, 20 d, 30 d, and 40 d, respectively. Compared with the control group, the maturity of P. clarkii was significantly improved after 10 d, 30 d, and 40 d of stress. The levels of GSH-Px, SOD, and T-AOC in hepatopancreas and hemolymph were significantly decreased after 30 d of drought, and MDA level was significantly increased after 10 d, 20 d, and 30 d of drought treatment. The contents of E, PROG, 17A-OHP, and E2 in the hemolymph of female P. clarkii were significantly decreased after more than 30 d of drought stress. Cyclin B mRNA transcription level decreased significantly after 20 d of drought. The expression levels of vgr and cdc2 significantly increased after 30 d and 40 d of drought, while foxL2 significantly decreased and pcna significantly increased after 40 d of drought. Based on the above results, 30 d drought was the most effective in promoting the ovarian development of female P. clarkii.

Rhodopseudomonas palustris (RP) are known anaerobic bacteria with probiotic properties containing several bioactive compounds and enzymes that benefit aquatic animals. However, studies on the use of RP on aquatic animal species are limited. This study investigated the effects of dietary supplementation with RP formulation on the growth, non-specific immunity, and disease resistance of juvenile ivory shells (Babylonia areolata). The experiment was conducted for 8 weeks, with B. areolata fed a control diet (RP0) and four diets containing four different RP formulations, with doses of 1 (RP1), 5 (RP2), 10 (RP3), and 20 (RP4) g/kg, respectively. Higher levels of R. palustris in the formulation led to increased final weight, weight gain, and specific growth rate in B. areolata. The crude protein content was significantly higher in the RP4 group compared to the RP0 group. However, there was no significant difference in the crude lipid content. Higher levels of R. palustris in the RP4 formulation group increased the trypsin and lipase activities. Dietary supplementation with RP significantly increased the total antioxidant capacity, superoxide dismutase, and catalase activities and decreased the malondialdehyde content in B. areolata. Acid phosphatase and alkaline phosphatase activities were significantly increased in the RP4 group compared to the RP0 group. Dietary RP significantly increased the expression levels of antioxidant-related (superoxide dismutase, Cu/Zn-superoxide dismutase, glutathione S-transferase A-like, ferritin) and immune-related (acid phosphatase, cytochrome c) genes. Higher levels of R. palustris in the formulations RP3 and RP4 increased the survival rate of B. areolata challenged with Vibrio parahaemolyticus. These findings indicate that R. palustris preparation could improve growth performance, muscle composition, and digestive capacity and may act as an immune booster for preventing disease in B. areolata.

Vitamin C (VC) plays an essential role in the physiological function and normal growth of aquatic animals. However, the effects and requirements of VC on juvenile ivory shell (Babylonia areolata) are still unknown. This study aimed to investigate the effects of VC on growth, muscle composition, non-specific immunity, and resistance to ammonia stress of B. areolata and determine its requirement for VC. Six experimental diets (Diet 1, Diet 2, Diet 3 Diet 4 Diet 5, and Diet 6) with different levels of VC (0, 100, 200, 400, 800, and 1200 mg/kg diet, respectively) were fed to juvenile B. areolate for 3 months. The weight gain rate (WGR) and specific growth rate (SGR) of B. areolata increased in parallel with the VC content, and those of the 800 mg/kg group were significantly higher than those of the control group. There was no significant difference in the survival rate (SR), flesh shell ratio (FSR), viscerosomatic index (VSI), soft tissue index (STI), and muscle composition among the experimental groups. VC (800 mg/kg) significantly increased the total antioxidant capacity (T-AOC) of the hepatopancreas. In addition, VC (400–800 mg/kg) significantly increased the activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) of the hepatopancreas and significantly decreased the content of malondialdehyde (MDA). VC significantly induced the expression levels of functional genes, such as SOD, GST, CYP450, ferritin, mucin-5AC, and CYC in the hepatopancreas and increased the survival rate of B. areolata under ammonia stress. These results indicate that supplementation with dietary VC could increase the growth, antioxidant capacity, immunity, and resistance against the stress caused by ammonia. The optimal dietary VC requirement for juvenile B. areolata was 400–800 mg/kg of VC.

This study was to investigate the effects of Alpinia officinarum Hance stems and leaves extract (AOE) on the growth, non-specific immunity, intestinal microflora of shrimp (Litopenaeus vannamei). A total of 540 juvenile L. vannamei (2.69 ± 0.03 g) were randomly divided into six groups, and feed experiment with diet supplement of 0 g/kg (CG), 0.1 g/kg (AO-1), 0.2 g/kg (AO-2), 0.3 g/kg (AO-3), 0.5 g/kg (AO-4) and 0.7 g/kg(AO-5) AOE was carried out for 8 weeks, respectively. Compared with the control group (CG), the WGR and SGR of the AO-1, AO-3, and AO-4 groups were significantly increased. Crude protein content in the muscle of L. vannamei in AO-2 was significantly increased, while the water content was significantly decreased. The crude lipid content of each extract group was significantly decreased. Compared with the control group, activities of GSH-Px, SOD, and T-AOC in the cell-free hemolymph of AO-3 group were significantly enhanced, and the MDA content of each extract group was significantly decreased. Activities of CAT, SOD, and T-AOC in the hepatopancreas of AO-2 group were significantly increased. Gene expression levels of lvcat, lvgsh-px, and lvmn-sod in AO-3 group were obviously higher than those in the control group, while lvpropo expression was dramatically higher in AO-2 and AO-3 groups. Moreover, AOE addition in feed changed the composition and species abundance of intestinal microflora of L. vannamei at phylum and genus levels. Through α diversity analysis, the biodiversity of the flora was improved, while β diversity analysis changed the similarity of the flora between the control and addition groups. Therefore, addition of 0.2–0.3 g/kg AOE could improve the growth, immunity and optimize the composition of intestinal microorganisms of L. vannamei.

In recent years, the problems associated with continuous cropping (CC) that cause soil degradation have become increasingly serious. As a key soil quality property, dissolved organic matter (DOM) affects the circulation of carbon and nutrients and the composition of bacterial communities in soil. However, research on the changes in the molecular composition of DOM after CC is limited. In this study, the soil chemical properties, DOM chemical diversity, bacterial community structure, and their interactions are explored in the soil samples from different CC years (CC1Y, CC3Y, CC5Y, and CC7Y) of tobacco. With increasing CC year of tobacco, most of the soil chemical properties, such as total carbon, total nitrogen and organic matter, decreased significantly, while dissolved organic carbon first decreased and then increased. Likewise, the trends of DOM composition differed with changing duration of CC, such as the tannin compounds decreased from 18.13 to 13.95%, aliphatic/proteins increased from 2.73 to 8.85%. After 7 years of CC, the soil preferentially produced compounds with either high H/C ratios (H/C > 1.5), including carbohydrates, lipids, and aliphatic/proteins, or low O/C ratios (O/C

The current study was conducted to determine the effects of dietary Radix bupleuri root extract (RBE) on the growth performance, muscle composition, immune responses, and microcystin-LR (MC-LR) stress resistance of juvenile red claw crayfish (Cherax quadricarinatus). Crayfish (0.28 ± 0.02 g) were fed experimental diets containing graded RBE levels as 0 (Diet 1), 0.2 (Diet 2), 0.4 (Diet 3), 0.6 (Diet 4), 0.8 (Diet 5) and 1.6 (Diet 6) g/kg RBE for 8 weeks. The results showed that specific growth rate (SGR) and weight gain rate (WGR) were significantly increased in the Diet 4 and Diet 5 groups. The content of muscle crude protein was significantly increased in the Diet 4 and Diet 5 groups, and the crude lipid content was significantly decreased in the Diet 5 group. Dietary RBE did not alter the structure of the hepatopancreas. Total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activities in hepatopancreas and plasma were significantly increased in Diet 4 and Diet 5 groups compared to the control group, while the malondialdehyde (MDA) content in hepatopancreas and plasma was significantly decreased in the Diet 5 group. Plasma alkaline phosphatase (AKP) activity was significantly higher in the Diet 3, Diet 4, Diet 5 and Diet 6 groups. Hepatopancreas and plasma PO activities were significantly increased in Diet 4 and Diet 5 groups. The mRNA levels of extracellular copper/zinc superoxide dismutase (ecCu, Zn-SOD), hemocyanin, C-type lysozyme (C-LZM), GPx, and selenium-dependent glutathione peroxidase (Se-GPx) were significantly up-regulated in the hepatopancreas of crayfish fed Diet 5 than those of the control group. After MC-LR challenge, the survival rates of crayfish in the Diet 4 and Diet 5 groups were significantly higher than the control group. The findings suggest that 0.6–0.8 g/kg of RBE can improve growth and muscle composition and enhance immunity and microcystin-LR resistance.

Microcystin-LR (MC-LR), the most prevalent cyanobacterial toxin, poses a serious threat to the survival of aquatic animals. Herein, crayfish (Cherax quadricarinatus) were treated with MC-LR at 33.06 μg kg−1 for 48 h, and the effects of MC-LR on behavior, histomorphology, oxidative stress, non-specific immunity, and gene expression were investigated to elucidate the molecular mechanisms underlying antioxidation and detoxification in crayfish. MC-LR caused vacuolation and disruption of crayfish hepatopancreatic cells, although no significant effect was observed on the structure of intestinal cells. Moreover, MC-LR increased the activity of antioxidant enzymes (SOD, CAT, GPx, GST) and up-regulated the expression of genes encoding antioxidant enzymes (ecCu,Zn-SOD, SOD, Se-GPx, GPx3P, GST). In addition, GSH was found depleted in the hepatopancreas and plasma. MC-LR activated the immune system, enhanced immunoenzyme activity (ACP, AKP) and induced the expression of immunity-related genes (C-LZM, proPO, hemocyanin). Furthermore, caspase and HSP70 genes were induced in the hepatopancreas of crayfish under MC-LR stress. Taken together, MC-LR was shown to cause a positive response in crayfish, and our findings suggested that the antioxidant activity and immune system play an important role in protecting crayfish from MC-LR stress by regulating gene expression and activities of antioxidant and immune enzymes.

Abstract Background Circular RNAs (circRNAs) are related to the pathogenesis and progression of triple‐negative breast cancer (TNBC). The aim of this study was to investigate the role and mechanism of hsa_circ_0001925 in TNBC progression. Methods Hsa_circ_0001925, microRNA (miR)‐1299 and Yin Yang 1 (YY1) levels were examined in TNBC via reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) and western blot. Cell counting kit‐8 (CCK‐8), colony formation, 5‐ethynyl‐2′‐deoxyuridine (EdU) staining, flow cytometry, wound healing assay and tube formation assay were conducted to estimate the effects of hsa_circ_0001925 on malignant phenotypes of TNBC tumors. Several protein levels were measured with western blot. The regulatory relationship between miR‐1299 and hsa_circ_0001925 or YY1 was validated using a dual‐luciferase reporter and RNA immunoprecipitation (RIP) assays. Xenograft assay was used to estimate the effect of hsa_circ_0001925 in TNBC in vivo. Results Hsa_circ_0001925 and YY1 levels were upregulated, while miR‐1299 abundance was downregulated in TNBC tissues and cells. Hsa_circ_0001925 silencing constrained cell proliferation, migration and angiogenesis whereas it promoted apoptosis in vitro, and hsa_circ_0001925 silencing significantly curbed xenograft tumor growth in vivo. Hsa_circ_0001925 acted as a miRNA sponge for miR‐1299. Hsa_circ_0001925 decreased YY1 expression by sponging miR‐1299. MiR‐1299 downregulation alleviated the effects of hsa_circ_0001925 knockdown on BC progression. MiR‐1299 interacted with the 3′ untranslated region (3′ UTR) of YY1, and YY1 overexpression partly reversed the effects of miR‐1299 overexpression on BC progression. Conclusion Our findings showed that hsa_circ_0001925 mediated TNBC progression via regulating miR‐1299/YY1 axis, providing a potential target for BC treatment.

Abstract Background Strawberries are an important economic fruit crop world-wide. In strawberry cultivation, continuous cropping (CC) can seriously threaten yield and quality. However, our understanding of the gene expression changes in response to CC and during subsequent defense processes is limited. In this study, we analyzed the impact of CC on the transcriptome of strawberry roots using RNA-Seq technology to elucidate the effect of CC and the subsequent molecular changes. Results We found that CC significantly affects the growth of strawberry plants. The transcriptome analysis identified 136 differentially expressed genes (DEGs), including 49 up-regulated and 87 down-regulated DEGs. A Gene Ontology (GO) analysis indicated that the up-regulated DEGs were mainly assigned to defense-related GO terms, and most down-regulated DEGs were assigned to nutrient-related GO terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the responsive DEGs were classified in a large number of important biological pathways, such as phenylalanine metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, glutathione metabolism and plant-pathogen interaction. We also found that four WRKY transcription factors and three peroxidase genes involved in plant defense pathways were up-regulated in the roots of strawberry plants subjected to CC. Conclusion Several unigenes involved in plant defense processes, such as CNGCs, WRKY transcription factors, PR1, and peroxidase genes with highly variable expression levels between non-CC and CC treatments may be involved in the regulation of CC in strawberry. These results indicate that strawberry roots reallocate development resources to defense mechanisms in response to CC. This study will further deepen our understanding of the fundamental regulatory mechanisms of strawberry resource reallocation in response to CC.

Red claw crayfish (Cherax quadricarinatus) is a valuable aquatic species with fast growth, big size, delicious meat, and strong resistance to diseases worldwide. Microcystin-LR (MC-LR) is the most common and potently toxic and is known to infect aquatic animals. The present study was performed at the transcriptome level to investigate the mechanism of C. quadricarinatus in responses to MC-LR by RNA sequencing (RNA-seq) analysis. A total of 57,153,501, 51,955,723, and 56,316,927 clean reads were retrieved from the three libraries of C. quadricarinatus (0 h, 6 h, and 48 h). A total of 37,616 unigenes were identified and compared with NR, Swiss-Prot, Pfam, COG, GO, and KEGG databases. A total of 4980 and 4585 differentially expressed genes (DEGs) were obtained at 6 h and 48 h compared with control (0 h), respectively. Most of these DEGs were up-regulated in 6 h, but most were down-regulated in 48 h, indicating that gene expression levels were inhibited by MC-LR in 48 h. Based on the GO and KEGG pathways analyses of DEGs, we obtained various pathways related to apoptosis, immunity, and detoxification. A total of 234 DEGs were related to the immune system, including 19 immune metabolic pathways, such as the Toll-like receptor signaling pathway, Toll and Imd signaling pathway, interleukin-17 (IL-17) signaling pathway, etc. Besides, detoxification-associated and apoptosis-associated pathways were also identified, including drug metabolism-cytochrome P450 and glutathione metabolism, apoptosis and p53 signaling pathway, etc. Twelve DGEs involved in immune response and MC-LR detoxification were discovered and validated by quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the C. quadricarinatus might counteract the toxicity of MC-LR at the transcriptomic level by increasing expression levels of immune- and detoxification-related genes. In summary, this study first reported the response mechanism of the C. quadricarinatus with injected MC-LR at the transcriptional level. The identification of differential expression genes and pathways can help us to understand further the response mechanism of C. quadricarinatus under MC-LR stress.

Alpinia oxyphylla is a homology of medicine and food. This study aims to investigate the dominant chemical composition and explore the antioxidant properties of the ethanol extract of the leaves and stems of A. oxyphylla (AOE) on juvenile shrimp, Litopenaeus vannamei. An in vitro test showed that AOE and its dominant chemical composition procyanidin B-2 (1) and epicatechin (2) presented DPPH and ABTS radical scavenging activities. A shrimp feeding supplement experiment revealed that shrimp growth parameters and muscle composition were improved significantly when fed with a 200 mg/Kg AOE additive. Meanwhile, the activities of antioxidant enzymes (CAT, GSH-Px, SOD, and T-AOC) in serum and the liver and the expression of related genes (LvMn-SOD, LvCAT, LvproPo, and LvGSH-Px) were enhanced with various degrees in different AOE additive groups while the content of MDA was significantly decreased. Moreover, the antioxidative effect of AOE additive groups on shrimp was also observed in an acute ammonia nitrogen stress test.

Objective: To investigate the correlation of fibronectin 1 (FN1) expression with prognosis and tumor-infiltrating immune cells in breast cancer (BRCA).Methods: FN1 mRNA and protein expressions were analyzed through Tumor Immune Estimation Resource (TIMER), Gene Set Cancer Analysis (GSCA), Human Protein Atlas (HPA) databases, and immunohistochemical analysis. The clinicopathological characteristics and genetic factors affecting the FN1 mRNA expression were assessed by various public databases. Then, we analyzed the prognostic value of FN1 in BRCA by Kaplan-Meier plotter, receiver operating characteristic, and Cox regression analyses. Further, the UCSC Xena database was used to retrieve TCGA-BRCA expression profiles for functional enrichment analysis and immune cell infiltration analysis. The potential drugs for the BRCA patients with high- FN1 expression were identified using the connectivity map analysis.Results: FN1 was upregulated in BRCA tissues compared with normal tissues. High FN1 mRNA expression was correlated with poor clinical outcomes and had good performance in predicting the survival status of BRCA patients. Further, Cox regression analysis showed that FN1 was an independent prognostic factor for predicting the overall survival of patients with BRCA. Moreover, hypermethylation of FN1 contributed to a better prognosis for BRCA patients. Functional enrichment analyses revealed the ECM-receptor interaction pathway and focal adhesion as the common pathways. Moreover, FN1 showed a significant association with tumor-infiltrating immune cells and immune checkpoint inhibitors. Several drugs such as telmisartan, malotilate, and seocalcitol may have therapeutic effects in BRCA patients with high FN1 expression.Conclusion: FN1 might serve as a novel prognostic biomarker and a novel therapeutic target for BRCA. Besides, the association of FN1 with immune cells and immune checkpoint inhibitors may provide assistance for BRCA treatment.

Graded levels of supplemental copper (Cu) (0, 10, 25, 40, 55 and 110 mg/kg) were fed to juvenile tiger shrimp (Penaeus monodon) reared in 5‰ salinity water for 8 weeks. According to the results, shrimps fed a diet supplemented with 40 mg/kg Cu had the highest weight gain (WG). Shrimps fed a diet with 110 mg/kg supplemental Cu had the lowest total haemocyte count (THC) and the highest reactive oxygen species (ROS) production and apoptotic ratio in haemocytes, suggesting that excessive levels of dietary Cu caused oxidative stress and induced haemocyte apoptosis. In plasma, the activities of copper, zinc-superoxide dismutase (Cu, Zn-SOD) and total superoxide dismutase (T-SOD) were higher in shrimps fed diets with 40 and 55 mg/kg supplemental Cu than those fed diets with ≤ 25 and 110 mg/kg supplemental Cu. Hepatic Cu, Zn-SOD activity increased in shrimps fed diets with 25–55 mg/kg supplemental Cu, while hepatic T-SOD activity increased in shrimps fed diets with 40 and 55 mg/kg supplemental Cu. In addition, diets with 25–55 mg/kg supplemental Cu increased the plasma total antioxidant capacity (T-AOC), while diets with 10–55 mg/kg supplemental Cu increased the hepatic T-AOC. Hepatic malondialdehyde (MDA) content was lowest in shrimps fed diets with 25 and 40 mg/kg supplemental Cu and highest in shrimps fed a diet with 110 mg/kg. Based on the growth and antioxidant activities, the optimum requirement of Cu for the antioxidant status of P. monodon was 30.7–61.9 mg/kg.

Air exposure exerts unavoidable environmental stress during the transport of crayfish. This study evaluated survival, histological structure, non-specific immunity and gene expression in red claw crayfish (Cherax quadricarinatus) under air exposure. Survival experiment showed that crayfish under air exposure started dying after 24 h, and survival rate decreased to 62.2% and 20.0% after 36 h and 48 h respectively. In the second experiment, crayfish divided into six groups with different air exposure time (0 h, 4 h, 8 h, 12 h, 24 h and 36 h). Crayfish maintained in the normal culture environment were used as control (0 h), whereas other five groups were placed in tanks without water. Histopathological study found that air exposure resulted in swelling and vacuolation of hepatocytes and enterocytes. Total antioxidant capacity (T-AOC), activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and alkaline phosphatase (AKP) increased at 4–8 h short-term air exposure, and then decreased. T-AOC in hepatopancreas was suppressed significantly at 36 h exposure. Malondialdehyde (MDA) levels in both hepatopancreas and hemolymph raised after 4 h and then reduced to the initial level. Expression of antioxidant genes (SOD, GPx and Trx 1), immune-related genes (C-LZM, CRU-2, and SERPIN3), anti-stress related genes (HSP70 and HSP90) were induced after short-term air exposure, and then down-regulated. Expression of SERP, HSP21 and NFKBIA maintained high levels during 36 h air exposure, indicating that these genes play vital roles in the defense mechanism against air exposure. In brief, our study demonstrates that air exposure causes pathological changes in hepatopancreas and intestines, and altered antioxidant and immune status in C. quadricarinatus. The suppressed antioxidant status, leading to tissue oxidative damage, is one of the main reason for death of crayfish under long-term air exposure.

The objective of the present study was to investigate the effects of different doses of dietary Eucommia ulmoides leaf extract (ELE) on juvenile red claw crayfish (Cherax quadricarinatus). A total number of 720 red claw crayfish (initial body weight of 0.24 ± 0.01 g) were randomly assigned to six groups and fed diets containing 0 (Diet 1), 0.5 (Diet 2), 1 (Diet 3), 2 (Diet 4), 4 (Diet 5) and 10 (Diet 6) g dry weight (dw) ELE kg (dw)−1 diets for eight weeks and challenged with microcystin-LR stress. The results indicated that dietary supplementation with 1–2 g dw ELE kg (dw)−1 diet could significantly improve the weight gain rate (WGR) and specific growth rate (SGR) of crayfish. Muscle crude protein contents of crayfish fed Diet 2, Diet 3, and Diet 4 were significantly higher than those of the control group. Compared with the control group, dietary ELE could increase total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) activities and decrease malondialdehyde (MDA) level of crayfish. Dietary ELE significantly increased the relative expression levels of SOD, thioredoxin 1 (TRX1), GPx, selenium-dependent glutathione peroxidase (Se-GPx), cytochrome P450 (CYP450), anti-lipopolysaccharide factor (ALF) and C-type lysozyme (C-LZM) mRNA of crayfish compared with the control group during the feeding experiment. When subjected to MC-LR stress for 48 h, the mRNA expression levels of SOD, GPx, Se-GPx, glutathione-s-transferase 1 (GST1), ALF, hemocyanin (HEM), and C-LZM in the hepatopancreas could be improved to varying degrees compared with the Diet 1. Supplementation of 1–2 g dw ELE kg (dw)−1 diet could improve the survival rate (SR) of crayfish under MC-LR stress. These results indicated that dietary ELE (1–2 g dw ELE kg (dw)−1 diet) could improve the growth performance, muscle protein, and non-specific immune response and increase the SR of crayfish under MC-LR stress by regulating the mRNA expression levels of the immune- and antioxidant-related genes.

Morphology and immune functions of Penaeus monodon haemocytes were analysed by flow cytometry (FCM). Basing on the relative size and granularity, there are three types of haemocyte identified by FCM, including hyaline cells (HC), semigranular cells (SGC) and granular cells (GC). GC and SGC were significantly larger than HC. GC contained more granules in the cytoplasm than the two other types. SGC were the most abundant cells, while HC were the least. The mean total apoptotic cell ratio was 5.09 % in the fresh shrimp haemocytes. SGC contributed most to the phagocytic activity, and HC contributed least, probably due to the great difference of percentage. GC had most mass of mitochondria and lysosome, and HC contained least. Defined by different characteristic of lysosomal mass, three subsets of haemocytes were observed. GC were most active in non-induced and zymosan-induced reactive oxygen species (ROS) production, while HC showed lowest oxidative activity. Both non-induced and zymosan-induced esterase activity (EA) in GC were higher than those in SGC and HC. The present flow cytometric analysis clearly demonstrated the variation in morphologic and immunological characterisation among the three haemocyte subpopulations. The facts that SGC account for most of the number of circulating haemocyte, while GC contain most organelles in relation to energy supply and immune defence, and have highest immune activites, suggesting that GC and SGC are the main haemocyte types involved in the cellular defence of P. monodon.