Aquaculture industry suffers significant limitations such as low resistance to diseases and expensive feed. This study investigated the antibacterial and immunostimulatory activities of ZnO-Ulva lactuca nanocomposite (ZnO-Ul NC) in the Procambarus clarkii. Zinc oxide nanoparticles (ZnO NPs) and ZnO-Ul NC were synthetized and characterized by electron microscopies as well as Fourier transform infrared spectroscopy. ZnO NPs and ZnO-Ul NC inhibited the growth of the isolated species Citrobacter freundii and Enterobacter hormaechei. For immunostimulatory evaluation, six crayfish groups (control, U. lactuca, ZnO L, ZnO H, ZnO-Ul L, and ZnO-Ul H) were fed on commercial diet, Ulva lactuca powder, and low or high dose of ZnO NPs or ZnO-Ul NCs, respectively for 90 days. The highest levels of total hemocyte count, granular cells%, phenoloxidase (PO) activity in hemolymph, and NO, superoxide dismutase (SOD), and GSH in hepatopancreas were all reported in the ZnO-Ul groups. The expression of proPO, SOD, and lysozyme exhibited the highest upregulation in the ZnO-Ul H group. Taken together, dietary ZnO-Ul NC significantly improved the non-specific immunity and antioxidant milieu of the crayfish at the genomic and proteomic levels. ZnO-Ul NC is cost effective, easily synthesized, and a promising immunostimulant for Procambarus clarkii that could be used in the aquaculture.

BACKGROUND: In this study, a probiotic mixture (Honeybeeotic) consisting of seven bacterial strains isolated from a unique population of honeybees (Apis mellifera ligustica) was used. That honeybee population was located in the Roti Abbey locality of the Marche Region in Italy, an area isolated from human activities, and genetic contamination from other honeybee populations. The aim was to investigate the effects of this probiotic mixture on the innate immunity and intestinal microbiome of healthy common honeybees in two hives of the same apiary. Hive A received a diet of 50% glucose syrup, while hive B received the same syrup supplemented with the probiotics, both administered daily for 1 month. To determine whether the probiotic altered the immune response, phenoloxidase activity and hemolymph cellular subtype count were investigated. Additionally, metagenomic approaches were used to analyze the effects on gut microbiota composition and function, considering the critical role the gut microbiota plays in modulating host physiology.
RESULTS: The results revealed differences in hemocyte populations between the two hives, as hive A exhibited higher counts of oenocytoids and granulocytes. These findings indicated that the dietary supplementation with the probiotic mixture was safe and well-tolerated. Furthermore, phenoloxidase activity significantly decreased in hive B (1.75 ± 0.19 U/mg) compared to hive A (3.62 ± 0.44 U/mg, p < 0.005), suggesting an improved state of well-being in the honeybees, as they did not require activation of immune defense mechanisms. Regarding the microbiome composition, the probiotic modulated the gut microbiota in hive B compared to the control, retaining core microbiota components while causing both positive and negative variations. Notably, several genes, particularly KEGG genes involved in amino acid metabolism, carbohydrate metabolism, and branched-chain amino acid (BCAA) transport, were more abundant in the probiotic-fed group, suggesting an effective nutritional supplement for the host.
CONCLUSIONS: This study advocated that feeding with this probiotic mixture induces beneficial immunological effects and promoted a balanced gut microbiota with enhanced metabolic activities related to digestion. The use of highly selected probiotics was shown to contribute to the overall well-being of the honeybees, improving their immune response and gut health.

Herein, we focused on the larvicidal effects and potential mechanisms of 5-ethenyl-2,2\'-bithiophene (5 EB), a compound isolated from Echinops ritro L. on Aedes aegypti larvae. Our results show that 5 EB exhibits pronounced larvicidal activity against A. aegypti larvae, with an LC50 = 0.24 mg/L, considerably lesser than that of the traditional insecticide, rotenone. Observations using fluorescence microscopy, electron microscopy, and imaging flow cytometry demonstrated that 5 EB targets the hemocytes of larvae, leading to the disruption of their intracellular membrane systems. This disruption leads to considerable damage to the cellular structure and function, leading to the death of test subjects. Note that additional investigation into the molecular mechanism of 5 EB\'s action was conducted using transcriptomic analysis. Both GO and KEGG enrichment analyses reported that the differentially expressed genes were predominantly associated with membranes, lysosomes, and catalytic activities. To summarize, this study provides new options for developing new, environmentally friendly, plant-based larvicides for mosquito control.

A correlation between the mechanical properties of cells and various diseases has been emerging in recent years. Atomic force microscopy (AFM) has been widely used to measure a single cell\'s apparent Young\'s modulus by treating it as a fully elastic object. More recently, quantitative characterization of the complete viscoelasticity of single cells has become possible. We performed AFM-based nano-indentation experiments on hemocytes isolated from third instar larvae to determine their viscoelasticity and found that live hemocytes, like many other cells, follow a scale-free power-law rheology (PLR) akin to soft glasses. Further, we examined the changes in the rheological response of hemocytes in the presence of pathogenic protein aggregates known to cause neurodegenerative diseases such as Huntington\'s disorder and amyotrophic lateral sclerosis. Our results show that cells lose their fluidity and appear more solid-like in the presence of certain aggregates, in a manner correlated to actin reorganization. More solid-like cells also display reduced intracellular transport through clathrin-mediated endocytosis (CME). However, the cell\'s rheology remains largely unaffected and is similar to that of wild-type (WT) hemocytes, if aggregates do not perturb the actin organization and CME. Moreover, the fluid-like nature was significantly recovered when actin organization was rescued by overexpressing specific actin interacting proteins or chaperones. Our study, for the first time, underscores a direct correlation between parameters governing glassy dynamics, actin organization and CME.

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the β-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood.

Nanoparticles (NPs) are widely used in various fields, including antifouling paints for ships and industrial structures submerged in water. The potential impact of NPs on aquatic organisms, particularly their potential toxicity, is a significant concern, as their negative impact has been relatively poorly studied. In this study, we evaluated the effect of different concentrations of bimetallic Ag-TiO₂ and ZnTi₂O₄-TiO₂ NPs, which could potentially be used in antifouling coatings, on the hemocytes of the Mediterranean mussel Mytilus galloprovincialis. Hemocytes were exposed to NPs at concentrations of 0.1-1 mg/L for 1 and 2 h, and the production of reactive oxygen species (ROS), levels of DNA damage, and number of dead cells were measured. Exposure to Ag-TiO₂ NPs at 1 mg/L concentration for 1 h suppressed ROS production in hemocytes and reduced the relative number of agranulocytes in cell suspensions, without inducing DNA damage or cell death. Exposure to ZnTi2O4-TiO2 NPs did not cause changes in the ratio of granulocytes to agranulocytes in suspensions, nor did it affect other functional parameters of hemocytes. However, after a 2 h exposure period, ZnTi2O4-TiO2 NPs (1 mg/L) significantly reduced the production of ROS by hemocytes. These findings suggest that Ag-TiO2 and ZnTi2O4-TiO2 NPs have low acute toxicity for marine bivalves.

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

Cell death is an important process in the body, as it occurs throughout every tissue during development, disease, and tissue regeneration. Phagocytes are responsible for clearing away dying cells and are typically characterized as either professional or nonprofessional phagocytes. Professional phagocytes, such as macrophages, are found in nearly every part of the body while nonprofessional phagocytes, such as epithelial cells, are found in every tissue type. However, there are organs that are considered \"immune-privileged\" as they have little to no immune surveillance and rely on nonprofessional phagocytes to engulf dying cells. These organs are surrounded by barriers to protect the tissue from viruses, bacteria, and perhaps even immune cells. The Drosophila ovary is considered immune-privileged, however the presence of hemocytes, the macrophages of Drosophila, around the ovary suggests they may have a potential function. Here we analyze hemocyte localization and potential functions in response to starvation-induced cell death in the ovary. Hemocytes were found to accumulate in the oviduct in the vicinity of mature eggs and follicle cell debris. Genetic ablation of hemocytes revealed that the presence of hemocytes affects oogenesis and that they phagocytose ovarian cell debris and in their absence fecundity decreases. Unpaired3, an IL-6 like cytokine, was found to be required for the recruitment of hemocytes to the oviduct to clear away obsolete follicle cells. These findings demonstrate a role for hemocytes in the ovary, providing a more thorough understanding of phagocyte communication and cell clearance in a previously thought immune-privileged organ.

The interactions induced by RIP homotypic interaction motif (RHIM) are essential for the activation of inflammatory signaling and certain cell death pathways. In the present study, a RHIM-containing protein was identified from Pacific oyster Crassostrea gigas, which harbored a RHIM domain and a Death domain (designated CgRHIM-containing protein). The mRNA transcripts of CgRHIM-containing protein were constitutively expressed in all the examined tissues of oysters, with the highest expression level in mantle. The CgRHIM-containing protein was mainly distributed in the cytoplasm of oyster haemocytes. After high temperature stress, the expression levels of CgRel and CgBcl-2 increased significantly, and reached the peak level at 12 h, then decreased gradually. The transcripts of CgRHIM-containing protein, Cgcaspase-8 and Cgcaspase-3 in haemocytes up-regulated at 12 h after high temperature stress. Moreover, the protein abundance of CgRHIM-containing protein increased significantly, and the ubiquitination level of CgRHIM-containing protein in haemocytes showed an increasing trend at first and then decreased. After the expression of CgRHIM-containing protein was knocked down by siRNA, the mRNA expression levels of CgRel and CgBcl-2 decreased significantly at 6 h after high temperature stress, and those of CgFADD-like, Cgcaspase-8 and Cgcaspase-3, as well as the apoptosis rate of haemocytes also decreased significantly at 24 h. These results indicated that CgRHIM-containing protein might regulate haemocyte apoptosis in oysters upon high temperature stress via mediating the expression of Rel, Bcl-2 and caspase-8/3.

Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5\'-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca2+). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops.