Professional phagocytes are recruited for the clearance of obsolete nonprofessional phagocytes in the Drosophila ovary.

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.

Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

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.

Granulocyte dynamics: a key player in the immune priming effects of crickets.

This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the killed Bt-primed group, suggesting a correlation between the heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the killed Bt-primed group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the PBS buffer-primed group exhibited extracellular DNA trap cell death (ETosis), while in the killed Bt-primed group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, the heat-killed Bt-primed group, the heat-killed E. coli-primed group, and the PBS-primed group were re-injected with live Bt 2 and 9 days post priming. Two days later, only the PBS-primed group displayed low survival rates. After injecting live Bt 9 days later, the heat-killed E. coli-primed group surprisingly showed a similarly low survival rate, while the heat-killed Bt-primed group exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health.

Drosophila immune cells transport oxygen through PPO2 protein phase transition.

Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae.

Background: In response to the replace mammal research models with insects in preliminary immunological studies, interest has grown in invertebrate defense systems. The immunological response is regulated by cytokines; however, while their role in mammals is well understood, little is known of their function in insects. A suitable target for studies into insect immunology is Galleria mellonella (Lepidoptera), the wax moth: a common host for human fungal and bacterial pathogens. G. mellonella is also a perfect subject for studies into the presence of cytokine-like proteins. Specific objectives: The main goal of present research was detection in insect immunocompetent cells the 18 mammalian cytokines (IL-1α, IL-1ß, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-ß, GM-CSF, M-CSF, G-CSF), which play important role in immunological response and indication how their level change after fungal infection. Methodology: The changes of cytokine-like proteins level were detected in hemocytes taken from G. mellonella larvae infected with entomopathogenic fungus, C. coronatus. The presence of cytokine-proteins was confirmed with using fluorescence microscopy (in cultured hemocytes) and flow cytometry (in freshly collected hemolymph). The ELISA test was used to detect changes in concentration of examined cytokine-like proteins. Results: Our findings indicated the presence of eighteen cytokine-like molecules in G. mellonella hemocytes during infection with C. coronatus. The hemocytes taken from infected larvae demonstrated higher fluorescence intensity for six cytokine-like proteins (GM-CSF, M-CSF, IL-3, IL-15, IL-1ß and IL-19) compared to untreated controls. ELISA test indicated significantly higher IL-3 and IL-15. M-CSF, IL-1α and IL-19 concentration in the hemolymph after fungal infection, and significantly lower TNF-ß and G-CSF. Conclusions: Our findings confirm that the selected cytokine-like molecules are present in insect hemocytes and that their concentrations change after fungal infection, which might suggest that they play a role in the anti-fungal immunological response.

Leeches Baicalobdella torquata feed on hemolymph but have a low effect on the cellular immune response of amphipod Eulimnogammarus verrucosus from Lake Baikal.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

Expression profile and immunomodulatory roles of methionine-enkephalin and delta opioid receptor in Octopus ocellatus.

In this study, the expressions and distributions of methionine-enkephalin (Met-enk) and δ opioid receptor in the nervous system of Octopus ocellatus, and the immune regulatory mechanisms of Met-enk on O. ocellatus were explored. The distributions and expressions of Met-enk and δ opioid receptor were assessed by immunohistochemistry and enzyme-linked immunosorbent assay. UV-spectrophotometer, microplate reader, and flow cytometer were used to examine the effects of different concentrations of Met-enk on phagocytosis, antioxidant effects, and body surface mucus immunity of O. ocellatus hemocytes. The data were used to study the mechanisms of Met-enk immunity regulation in O. ocellatus. According to the results, the expression levels of Met-enk and δ opioid receptor in O. ocellatus lymphocytes were higher than those in hemocytes. The expression levels of Met-enk in the ganglia of O. ocellatus decreased in the following order: pedal ganglia > cerebral ganglia > visceral ganglia > optic ganglia > stellate ganglia. Moreover, the phagocytic activity of O. ocellatus hemocytes was enhanced with increasing Met-enk concentration. With increasing Met-enk concentration, the expressions of nitric oxide, total nitric oxide synthase, inducible nitric oxide synthase, catalase, hydrogen peroxide, myeloperoxidase, reduced glutathione, α-naphthy acetate esterase, and methionine aminopeptidases decreased in serums of O. ocellatus in the experimental group compared to the blank group. Similarly, the content of reduced glutathione in the hemocytes of O. ocellatus was also lower in the experimental group than in the blank group; however, the expressions of other substances were higher compared to the blank group. Furthermore, α-naphthy acetate esterase, myeloperoxidase, and hydrogen peroxide expressions in mucus immunity trials of the body surface were lower in the experimental group compared to the blank group. These results indicate that the distributions and expressions of Met-enk and δ opioid receptor in the nervous system of O. ocellatus were related to axoplasmic transport and immune regulation mechanisms. Met-enk participates in cellular immunity, humoral immunity, and mucus immunity in the form of neurotransmitters, thereby regulating the immune response of O. ocellatus.

Expression of octopamine/tyramine receptors and immune regulation in Litopenaeus vannamei under acute and chronic thermal stress.

This study investigated the impact of hyperthermal (34 °C) and hypothermal (14 °C) stress on the expression of the octopamine/tyramine receptor (LvOA/TA-R) and immune parameters in Litopenaeus vannamei, which is a species critical to the aquaculture industry. Given the sensitivity of aquatic organisms to climate change, understanding the physiological and immune responses of L. vannamei to temperature variations is essential for developing strategies to mitigate adverse effects. This research focuses on the immune response and expression changes of LvOA/TA-R under acute (0.5, 1, and 2 h) and chronic (24, 72, and 168 h) thermal stress conditions. Our findings reveal that thermal stress induces changes in LvOA/TA-R expression and impacts immune responses. Immune parameters such as total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, lysozyme activity, clearance efficiency, and phagocytosis exhibited a general trend of significant decline under the stress conditions. LvOA/TA-R had a higher expression in haemocyte under hyperthermal stress. The study elucidated that thermal stress modifies the expression of the LvOA/TA-R and diminishes immune functionality in L. vannamei, underscoring the potential influence of climate change on industry.

Apolipoprotein D3 and LOX product play a role in immune-priming of a lepidopteran insect, Spodoptera exigua.

Immune-priming occurs in insects after a prior pathogen exposure. However, its underlying mechanism in insects remains elusive. In the present work, immune-priming was detected in a lepidopteran insect, Spodoptera exigua. Specifically, a prior infection with a heat-killed pathogenic bacterium, Escherichia coli, led to increased survival upon the second infection of different pathogens. Plasma collected from larvae with the prior infection possessed the immune-priming factor(s) that significantly up-regulated cellular and humoral immune responses of naïve larvae. Our study also finds that variations in the timing of plasma collection for priming larvae resulted in distinct impacts on both cellular and humoral responses. However, when the active plasma exhibiting the immune-priming was heat-treated, it lost this priming activity, therefore suggesting that protein factor(s) play a role in this immune-priming. An immunofluorescence assay showed that the hemocytes collected from the immune-primed larvae highly reacted to a polyclonal antibody specific to a vertebrate lipocalin, apolipoprotein D (ApoD). Among 27 ApoD genes (Se-ApoD1 âˆ¼ Se-ApoD27) of S. exigua, Se-ApoD3 was found to be highly induced during the immune-priming, in which it was shown to be expressed in hemocytes and fat body from a fluorescence in situ hybridization analysis. RNA interference of Se-ApoD3 expression significantly impaired the immune-priming of S. exigua larvae. Moreover, the inhibition of eicosanoid biosynthesis suppressed the immune-priming, in which treatment with a lipoxygenase (LOX) inhibitor-and not treatment with a cyclooxygenase inhibitor-suppressed immune-priming. Further, an addition of LOX product such as lipoxin A4 or lipoxin B4 significantly rescued the lost immune-priming activity. Taken together, these results suggest that a complex of ApoD3 and LOX product mediates the immune-priming activity of S. exigua.

Potential threats of microplastics and pathogenic bacteria to the immune system of the mussels Mytilus galloprovincialis.

As one of the main components of marine pollution, microplastics (MPs) inevitably enter the mussel aquaculture environment. At the same time, pathogenic bacteria, especially pathogens such as Vibrio, can cause illness outbreaks, leading to large-scale death of mussels. The potential harm of MPs and pathogenic bacteria to bivalve remains unclear. This study designed two experiments (1) mussels (Mytilus galloprovincialis) were exposed to 100 particles/L or 1,000 particles/L polymethyl methacrylate (PMMA, 17.01 ± 6.74 µm) MPs and 1 × 107 CFU/mL Vibrio parahaemolyticus at the same time (14 days), and (2) mussels were exposed to 100 particles/L or 1,000 particles/L MPs for a long time (30 days) and then exposed to 1 × 107 CFU/mL V. parahaemolyticus to explore the effects of these two stresses on the mussel immune system. The results showed that after the combined exposure of V. parahaemolyticus and MPs, the lysosomal membrane stability of hemocytes decreased, lysozyme activity was inhibited, and hemocytes were induced to produce more lectins and defensins to fight pathogenic invasion. Long-term exposure to MPs caused a large amount of energy consumption in mussels, inhibited most of the functions of humoral immunity, increased the risk of mussel infection with pathogenic bacteria, and negatively affected mussel condition factor, the number of hemocytes, and the number of byssuses. Mussels may allocate more energy to deal with MPs and pathogenic bacterial infections rather than for growth. Above all, MPs exposure can affect mussel immune function or reduce its stress resistance, which in turn has an impact on mollusk farming.

Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

A C-type lectin (CTL2) mediated both humoral and cellular immunity against bacterial infection in Tribolium castaneum.

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Analysis of X-ray irradiation effects on the mortality values and hemolymph immune cell composition of Apis mellifera and its parasite, Varroa destructor.

Varroa destructor is one of the most destructive enemies of the honey bee, Apis mellifera all around the world. Several control methods are known to control V. destructor, but the efficacy of several alternative control methods remains unexplored. Irradiation can be one of these unknown solutions but before practical application, the effectiveness, and the physiological effects of ionizing radiation on the host and the parasite are waiting to be tested. Therefore, the objective of our study was to investigate the effects of different doses (15, 50, 100, and 150 Gy) of high-energy X-ray irradiation through mortality rates and hemocyte composition changes in A. mellifera workers and record the mortality rates of the parasite. The mortality rate was recorded during short-term (12, 24, and 48 h) and long-term periods (3, 6, 12, 18, and 24d). The sensitivity of the host and the parasite in case of the higher doses of radiation tested (50, 100, and 150 Gy) been demonstrated by total mortality of the host and 90 % of its parasite has been observed on the 18th day after the irradiation. V. destructor showed higher sensitivity (1.52-times higher than the adult honey bee workers) at the lowest dose (15 Gy). A. mellifera hemocytes were influenced significantly by radiation dosage and the elapsed time after treatment. The higher radiation doses increased plasmatocyte numbers in parallel with the decrease in prohemocyte numbers. On the contrary, the numbers of granulocytes and oencoytes increased in the treated samples, but the putative effects of the different dosages on the recorded number of these hemocyte types could not be statistically proven. In summary, based on the outcome of our study X-ray irradiation can be deemed an effective tool for controlling phoretic V. destructor. However, further research is needed to understand the physiological response of the affected organisms.

In vitro effects of the harmful benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata on immune responses of the farmed oyster Crassostrea gasar.

Oyster culture is a sustainable solution to food production. However, this activity can be severely impacted by the presence and proliferation of harmful microalgae such as the benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata. This study aimed to evaluate the in vitro effects of P. hoffmannianum and O. cf. ovata on immune system cells (hemocytes) of the native cultured oyster Crassostrea gasar. The direct toxicity of both dinoflagellates was first evaluated assessing hemocyte viability exposed to eight concentrations of each HAB species. No reduction in hemocyte viability was found with the exposure to cell culture or the crude extract of P. hoffmannianum, but O. cf. ovata culture induced hemocyte death in a concentration-dependent manner. Ostreopsis cf. ovata concentration that promoted half of maximal reduction in hemocyte viability (EC50) was 779 cells mL-1. Posteriorly, hemocytes were exposed to both dinoflagellate cells and crude extracts to investigate their effects on hemocyte functional parameters. Despite no direct toxicity of the dinoflagellate cells, P. hoffmannianum extract caused a threefold increase in ROS production and decreased the phagocytosis rate by less than half. Ostreopsis cf. ovata cells and crude extracts also triggered an increase in ROS production (two-fold), but the phagocytosis rate was reduced (by half) only in response to the two lower cell concentrations. These results indicate a harmful potential of both dinoflagellates through a direct toxicity (only for O. cf. ovata) and functional impairment of hemocytes (both species) which could expose C. gasar oyster to opportunistic infections.

Molecular characterization, immune functions and DNA protective effects of peroxiredoxin-1 gene in Antheraea pernyi.

Peroxiredoxins are antioxidant proteins that detoxify peroxynitrite, hydrogen peroxide, and organic hydroperoxides, impacting various physiological processes such as immune responses, apoptosis, cellular homeostasis, and so on. In the present study, we identified and characterized peroxiredoxin 1 from Antheraea pernyi (thereafter designated as ApPrx-1) that encodes a predicted 195 amino acid residue protein with a 21.8 kDa molecular weight. Quantitative real-time PCR analysis revealed that the mRNA level of ApPrx-1 was highest in the hemocyte, fat body, and midgut. Immune-challenged larval fat bodies and hemocytes showed increased ApPrx-1 transcript. Moreover, ApPrx-1 expression was induced in hemocytes and the whole body of A. pernyi following exogenous H2O2 administration. A DNA cleavage assay performed using recombinant ApPrx-1 protein showed that rApPrx-1 protein manifests the ability to protect supercoiled DNA damage from oxidative stress. To test the rApPrx-1 protein antioxidant activity, the ability of the rApPrx-1 protein to remove H2O2 was assessed in vitro using rApPrx-1 protein and DTT, while BSA + DDT served as a control group. The results revealed that ApPrx-1 can efficiently remove H2O2 in vitro. In the loss of function analysis, we found that ApPrx-1 significantly increased the levels of H2O2 in ApPrx-1-depleted larvae compared to the control group. We also found a significantly lower survival rate in the larvae in which ApPrx-1 was knocked down. Interestingly, the antibacterial activity was significantly higher in the ApPrx-1 depleted larvae, compared to the control. Collectively, evidence strongly suggests that ApPrx-1 may regulate physiological activities and provides a reference for further studies to validate the utility of the key genes involved in reliving oxidative stress conditions and regulating the immune responses of insects.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas.

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

The involvement of interferon regulatory factor 8 in regulating the proliferation of haemocytes in oyster Crassostrea gigas.

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (CgIRF8) in regulating haemocyte proliferation of oyster were further investigated. CgIRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of CgIRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of CgIRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgIRF8 protein in haemocytes. After the expression of CgIRF8 was inhibited by the injection of CgIRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of CgGATA and CgSCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein CgCDK2, CgCDC6, CgCDC45 and CgPCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that CgIRF8 was able to activate the CgGATA promoter in HEK293T cells after transfection of CgGATA and CgIRF8. These results collectively indicated that CgIRF8 promoted haemocyte proliferation by regulating the expression of CgGATA and other related genes in the immune response of oyster.