Hemocyte Mortality Research Articles

Exposure to nanopollutants (nZnO) enhances the negative effects of hypoxia and delays recovery of the mussels’ immune system

Aquatic environments face escalating challenges from multiple stressors like hypoxia and nanoparticle exposure, with impact of these combined stressors on mussel immunity being poorly understood. We investigated the individual and combined effects of short-term and long-term hypoxia and exposure to zinc oxide nanoparticles (nZnO) on immune system of the mussels (Mytilus edulis). Hemocyte functional traits (mortality, adhesion capacity, phagocytosis, lysosomal abundance, and oxidative burst), and transcript levels of immune-related genes involved in pathogen recognition (the Toll-like receptors, the complement system components, and the adaptor proteins MyD88) were assessed. Short-term hypoxia minimally affected hemocyte parameters, while prolonged exposure led to immunosuppression, impacting hemocyte abundance, viability, phagocytosis, and defensin gene expression. Under normoxia, nZnO stimulated immune responses of mussel hemocytes. However, combined nZnO and hypoxia induced more pronounced and rapid immunosuppression than hypoxia alone, indicating a synergistic interaction. nZnO exposure hindered immune parameter recovery during post-hypoxic reoxygenation, suggesting persistent impact. Opposing trends were observed in pathogen-sensing and pathogen-elimination mechanisms, with a positive correlation between pathogen-recognition system activation and hemocyte mortality. These findings underscore a complex relationship and potential conflict between pathogen-recognition ability, immune function, and cell survival in mussel hemocytes under hypoxia and nanopollutant stress, and emphasize the importance of considering multiple stressors in assessing the vulnerability and adaptability of mussel immune system under complex environmental conditions of anthropogenically modified coastal ecosystems.

Antioxidant Systematic Alteration Was Responsible for Injuries Inflicted on the Marine Blue Mussel Mytilus edulis Following Strontium Exposure.

The ionic properties of strontium (Sr), a significant artificial radionuclide in the marine environment, were estimated using a stable nuclide-substituting experimental system under controlled laboratory conditions. The bio-accumulation of Sr and its impacts, as well as any possible hidden mechanisms, were evaluated based on the physiological alterations of the sentinel blue mussel Mytilus edulis. The mussels were exposed to a series of stress-inducing concentrations, with the highest solubility being 0.2 g/L. No acute lethality was observed during the experiment, but sublethal damage was evident. Sr accumulated in a tissue-specific way, and hemolymph was the target, with the highest accumulating concentration being 64.46 µg/g wet weight (ww). At the molecular level, increases in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and changes in ROS components (H2O2, O2-, and -OH) and antioxidant system activity indicated that the redox equilibrium state in hemocytes was disturbed. Furthermore, the rise in the hemocyte micronucleus (MN) rate (4‱ in the high-concentration group) implied DNA damage. At the cellular level, the structures of hemocytes were damaged, especially with respect to lysosomes, which play a crucial role in phagocytosis. Lysosomal membrane stability (LMS) was also affected, and both acid phosphatase (ACP) and alkaline phosphatase (AKP) activities were reduced, resulting in a significant decline in phagocytosis. The hemolymph population structure at the organ level was disturbed, with large changes in hemocyte number and mortality rate, along with changes in component ratios. These toxic effects were evaluated by employing the adverse outcome pathway (AOP) framework. The results suggested that the disruption of intracellular redox homeostasis is a possible explanation for Sr-induced toxicity in M. edulis.

Effects of reproductive condition on haemocyte parameters in the Pacific abalone Haliotis discus hannai during the spawning period

ABSTRACT The assessment of morphology and immunology of specimens at different maturity stages is essential for the determination of spawning strategy and reducing the risk of mortality. In this paper, female and male Pacific abalones (Haliotis discus hannai) at different statuses (pre-spawning, spawning and post-spawning) were collected to estimate the effects of spawning on the immune response. The haemocyte parameters, including total haemocyte counts (THC), haemocyte mortality, and reactive oxygen species production (ROS) were analysed using flow cytometry. THC decreased after spawning, which was lowest 3 days post-spawning. Haemocyte mortality was higher during spawning, and was highest 3 days post-spawning. Abalone also reduced their metabolic activity after spawning, reflected by an increase in ROS in both females and males. Moreover, our results show that female abalone reach the spawning period with a higher degree of activities of haemocytes and immune responses than male. The results confirmed that variability in abalone haemocytes during the spawning period might related to individual metabolic conditions.

Effects of cadmium exposure on haemocyte immune function of clam Ruditapes philippinarum at different temperatures

Haemocytes are crucial for the immune defence of mollusks. It is important to explore the immune performance of haemocytes of mollusks under the stress of heavy metals with global warming. In order to study the effects of cadmium (Cd) exposure and temperature stress on the haemocyte immune function of clam Ruditapes philippinarum, clams were exposed to different Cd concentrations (0.05, 0.10 and 0.25 mg/L) at 20 °C, 25 °C and 30 °C respectively. Haemocyte mortality, reactive oxygen species (ROS) and superoxide dismutase (SOD) activity were measured at day 1, day 3, day 5 and day 7. The results showed that the changes of the three indexes were not obvious when exposed to 0.05 mg/L of Cd at 20 °C, while significant differences were observed with the increase of temperature, Cd concentration and exposure time. Under a condition of relative high temperature coupling with high concentration of Cd, the clams were significantly influenced, showing an obvious synergistic effect. Selected indexes reflect the clam's response to the combined stress of temperature and Cd. Moreover, R. philippinarum might be an ideal biological index species to the Cd pollution.

Size-dependent effects of plastic particles on antioxidant and immune responses of the thick-shelled mussel Mytilus coruscus

Micro-/nano-plastic particles (MNPs) are present in the ocean with potential detrimental impacts on marine ecosystems. Bivalves are often used as marine bioindicators and are ideal to evaluate the threat posed by various-sized MNPs. We exposed the mussel Mytilus coruscus to MNPs with different particle sizes (70 and 500 nm, 5, 10 and 100 μm) for 3, 72 h and 30 days. The antioxidant responses in digestive gland and the hemolymph were then evaluated. The time of exposure played a strong modulating role in the biological response. A 3-hour exposure had no significant impact on the digestive gland. After 72 h, an increase in oxidative stress was observed in the digestive gland, including increased hydrogen peroxide (H2O2) level, catalase (CAT), glutathione peroxidase (GPx) activities and malondialdehyde (MDA) production. After a 30-day exposure, the oxidative stress decreased while lipid peroxidation increased. A 30-day exposure increased hemocyte mortality (HM) and reactive oxygen species (ROS) levels in the hemolymph, while phagocytosis (PA), lysosome content (LC), mitochondrial number (MN) and mitochondrial membrane potential (MMP) significantly decreased. Longer-term exposure to MNPs caused oxidative stress in the digestive gland as well as impaired viability and immunity of hemocytes. Particle size also influenced the response with smaller particles having more severe effects. A depuration for 7 days was enough to reverse the negative effects observed on the digestive gland and hemolymph. This study provides new insights on the effects of small-sized MNPs, especially nanoplastic particles (NPs), on aquatic organisms, and provides a solid theoretical knowledge background for future studies on toxic effects of MNPs.

Adverse outcome pathways as a tool for optimization of the biomarker-based assessment of pollutant toxicity: A case study of cadmium in the blue mussels Mytilus edulis

Technogenic metals like cadmium (Cd) are discharged into the environment posing significant ecological risks to wildlife and contributing to a decline in biodiversity. Recent developments in toxicity testing and environmental risk assessment have shifted the focus towards prioritizing biological effects. To ensure effective environmental risk assessment, it is essential to develop sensitive sub-organismal biomarkers correlated with ecologically relevant organismal-level responses. Our study proposes a novel approach to enhance the evaluation of biomarker-based toxicity based on adverse outcome pathways (AOP) concept. We exposed the blue mussels M. edulis to Cd concentrations ranging from 200 to 600 µg l−1 (10–30 % of LC50) for 14 days, and assessed a comprehensive panel of 26 biomarkers related to molecular initiation events (Cd uptake and binding), key cellular events (oxidative stress, metabolic disturbances, and cytotoxicity), and adverse organismal outcomes (survival, energy metabolism, and reef-building capacity) in the putative AOP of Cd. Tissue Cd burdens increased linearly with exposure concentrations in the mussels. Although the condition index of the mussels remained unaffected, their biofiltration activity was suppressed at the highest Cd concentration. The negative effects of Cd exposure on the mussels' feeding were linked to reduced energetic scope for growth, impaired byssus production, increased hemocyte mortality, and altered functional traits of hemocytes. Additionally, Cd exposure induced oxidative stress, as evidenced by lower total antioxidant capacity and thiol pool, along with increased lipid peroxidation, leading to enhanced autophagy. Through Random Forest Analysis, we identified a minimal set of sensitive Cd effect biomarkers, including hemocyte mortality (contributing ∼0.32 to classification accuracy), hemocyte acid phosphatase activity, nitric oxide and metallothionein levels in the digestive gland, and whole-organism clearance rate and scope for growth (each contributing ∼0.02–0.08 to classification accuracy). These biomarkers collectively achieved a high accuracy of 90–100 % in distinguishing between control and Cd-exposed groups. Increasing the number of biomarkers did not improve classification accuracy. These findings suggest that using a smaller number of optimized biomarkers based on Adverse Outcome Pathways (AOP) can enhance the assessment of contaminant-induced negative effects while reducing testing costs in environmental risk assessments.

Combined effects of microfibers and polychlorinated biphenyls on the immune function of hemocytes in the mussel Mytilus coruscus

Numerous studies have shown that microplastics can interact with other pollutants in the environment to produce synergistic effects, leading to more serious impacts. To date, there is little consensus on the combined effects of microfibers (MFs) and polychlorinated biphenyls (PCBs, Aroclor 1254), two legacy and alarming environmental pollutants. There is an urgent need to assess the impact of combined exposures on bivalve immune defences. In this study, we assessed the immune response of the mussels (Mytilus coruscus) hemocyte to MFs and PCBs alone and in combination by using flow cytometry. M. coruscus were exposed to MFs (1000 pieces/L) and PCBs (PCBs) (100 ng/L and 1000 ng/L) alone or in combination for 14 consecutive days and recovered for 7 days. The hemocyte of M. coruscus was collected on day 7, 14 and 21. MF exposure alone had no effect on the hemocyte. The total hemocyte count (THC), esterase (EA), lysosomal contents (LC), mitochondrial number (MN) and mitochondrial membrane potential (MMP) of mussels showed a decreasing trend with increasing PCB concentrations, both individually and in combination; The decreases in EA, MN and MMP were associated with the induction of reactive oxygen species (ROS). Hemocyte mortality (HM) was associated with a decrease in THC. Combined exposure to MFs and PCBs would exacerbate the effects on hemocyte immunity. These new findings improve our understanding of the toxic effects of MFs and organic chemical pollutants, and demonstrate the potential mechanism of PCBs to bivalves through changes in hemolymph immunity-related indicators.

Comparative immune responses of blue mussel and zebra mussel haemocytes to simultaneous chemical and bacterial exposure

Biomonitoring at the scale of the aquatic continuum and based on biomarkers, requires various representative species and a knowledge of their sensitivity to contaminants. Mussel immunomarkers are established tools for evaluating immunotoxic stress, but little is known about the consequences of an immune activation by local microorganisms on their response to pollution. This study aims to compare the sensitivity of cellular immunomarkers in two mussel species from different environments, the marine mussel Mytilus edulis (blue mussel) and the freshwater mussel Dreissena polymorpha (zebra mussel), to chemical stressors combined with bacterial challenge. Haemocytes were exposed ex vivo to the contaminants (bisphenol A, caffeine, copper chloride, oestradiol, ionomycin) for 4 h. The chemical exposures were coupled with simultaneous bacterial challenges (Vibrio splendidus and Pseudomonas fluorescens) to trigger activation of the immune response. Cellular mortality, phagocytosis efficiency and phagocytosis avidity were then measured by flow cytometry. The two mussel species had different basal levels since D. polymorpha showed higher cell mortality than M. edulis (23.9 ± 11% and 5.5 ± 3% dead cells respectively), and lower phagocytosis efficiency (52.6 ± 12% and 62.2 ± 9%), but similar phagocytosis avidity (17.4 ± 5 and 13.4 ± 4 internalised beads). Both bacterial strains led to an increase in cellular mortality (+8.4% dead cells in D. polymorpha, +4.9% in M. edulis), as well an activation of phagocytosis (+9.2% of efficient cells in D. polymorpha, +6.2% efficient cells and +3 internalised beads per cell in M. edulis). All chemicals triggered an increase in haemocyte mortality and/or phagocytotic modulations, except for bisphenol A. The two species differed in the amplitude of their response. The addition of a bacterial challenge significantly altered cell responses to chemicals with synergetic and antagonistic variations compared to a single exposure, depending on the compound used and the mussel species. This work highlights the species-specific sensitivity of mussel immunomarkers to contaminants, with or without bacterial challenge, and the necessity of considering the presence of in natura non-pathogenic microorganisms for future in situ applications of immunomarkers.

Effect of size continuum from nanoplastics to microplastics on marine mussel Mytilus edulis: Comparison in vitro/in vivo exposure scenarios

For several decades, plastic has been a global threat in terms of pollution. Plastic polymers, when introduce in the aquatic environment, are exposed to fragmentation processes into microplastics (MPs) and nanoplastics (NPs) which could potentially interact with living organisms. The objective of this work was to study the effects of plastic particles representative of those found in the environment, on the marine mussels Mytilus edulis, under two exposure scenarios: in vivo and in vitro. Whole mussels or cultured hemocytes were exposed for 24 h to NPs and MPs generated from macro-sized plastics collected in the field, but also to reference NPs, at concentrations found in the environment: 0.08, 10 μg and 100 μg·L−1. Results showed that immune response was only activated when mussels were exposed in vivo. However, cytotoxicity (hemocyte mortality) and genotoxicity (DNA damage) parameters were induced after both types of exposure, but in a dose-dependent manner after in vitro hemocyte exposure to all tested plastic conditions. These results indicate that in vitro approaches could be considered as potential predictors of in vivo exposures.

Transcriptome analysis reveals fluid shear stress (FSS) and atherosclerosis pathway as a candidate molecular mechanism of short-term low salinity stress tolerance in abalone

BackgroundTranscriptome sequencing is an effective tool to reveal the essential genes and pathways underlying countless biotic and abiotic stress adaptation mechanisms. Although severely challenged by diverse environmental conditions, the Pacific abalone Haliotis discus hannai remains a high-value aquaculture mollusk and a Chinese predominantly cultured abalone species. Salinity is one of such environmental factors whose fluctuation could significantly affect the abalone’s cellular and molecular immune responses and result in high mortality and reduced growth rate during prolonged exposure. Meanwhile, hybrids have shown superiority in tolerating diverse environmental stresses over their purebred counterparts and have gained admiration in the Chinese abalone aquaculture industry.The objective of this study was to investigate the molecular and cellular mechanisms of low salinity adaptation in abalone. Therefore, this study used transcriptome analysis of the gill tissues and flow cytometric analysis of hemolymph of H. discus hannai (DD) and interspecific hybrid H. discus hannai ♀ x H. fulgens ♂ (DF) during low salinity exposure. Also, the survival and growth rate of the species under various salinities were assessed.ResultsThe transcriptome data revealed that the differentially expressed genes (DEGs) were significantly enriched on the fluid shear stress and atherosclerosis (FSS) pathway. Meanwhile, the expression profiles of some essential genes involved in this pathway suggest that abalone significantly up-regulated calmodulin-4 (CaM-4) and heat-shock protein90 (HSP90), and significantly down-regulated tumor necrosis factor (TNF), bone morphogenetic protein-4 (BMP-4), and nuclear factor kappa B (NF-kB).Also, the hybrid DF showed significantly higher and sustained expression of CaM and HSP90, significantly higher phagocytosis, significantly lower hemocyte mortality, and significantly higher survival at low salinity, suggesting a more active molecular and hemocyte-mediated immune response and a more efficient capacity to tolerate low salinity than DD.ConclusionsOur study argues that the abalone CaM gene might be necessary to maintain ion equilibrium while HSP90 can offset the adverse changes caused by low salinity, thereby preventing damage to gill epithelial cells (ECs).The data reveal a potential molecular mechanism by which abalone responds to low salinity and confirms that hybridization could be a method for breeding more stress-resilient aquatic species.

Identification and Functional Characterization of Toxoneuron nigriceps Ovarian Proteins Involved in the Early Suppression of Host Immune Response.

Simple SummaryThe endoparasitoid of the tobacco budworm Heliothis virescens, Toxoneuron nigriceps, has several strategies to survive, including venom and calyx fluid. This latter contains a Polydnavirus and Ovarian Proteins (OPs). They are injected into the host body together with the egg. Although much research has focused on venom protein components, little is known about OPs. OPs can disrupt the cellular immune response of the host, acting on host hemocytes, the immune cells. In this study we investigated the action of HPLC fractions derived from OPs. Two fractions caused multiple and significant changes in hemocytes, including cellular oxidative stress and actin cytoskeleton disruption, which might explain the high incidence of hemocyte death and loss of function. Furthermore, using a transcriptome and proteomic approach, we identify the proteins of the two fractions that may be involved in the observed host hemocyte alterations. Our results will help to better understand the OP components and their involvement in parasitization strategies.The endophagous parasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) of the larval stages of the tobacco budworm Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) injects the egg, the venom, the calyx fluid, which includes a Polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian Proteins (OPs) into the host body during oviposition. The host metabolism and immune system are disrupted prematurely shortly after parasitization by the combined action of the TnBV, venom, and OPs. OPs are involved in the early suppression of host immune response, before TnBV infects and expresses its genes in the host tissues. In this work, we evaluated the effect of HPLC fractions deriving from in toto OPs. Two fractions caused a reduction in hemocyte viability and were subsequently tested to detect changes in hemocyte morphology and functionality. The two fractions provoked severe oxidative stress and actin cytoskeleton disruption, which might explain the high rate of hemocyte mortality, loss of hemocyte functioning, and hence the host’s reduced hemocyte encapsulation ability. Moreover, through a transcriptome and proteomic approach we identify the proteins of the two fractions: eight proteins were identified that might be involved in the observed host hemocyte changes. Our findings will contribute to a better understanding of the secreted ovarian components and their role in parasitoid wasp strategy for evading host immune responses.

Effects of spawning stress on the immune capacity of blood cockle Tegillarca granosa occurring on the south coast of Korea

Spawning in marine bivalves is a great energy-demanding process, and it often results in lethal and sublethal stresses during the post-spawning period, including depressed immune capacity. The blood cockle Tegillarca granosa (Linnaeus, 1758) distributes widely in silty-mud tidal flats on the south coast of Korea, and they spawn in late summer. To understand the impacts of spawning on immune parameters, we analyzed the total hemocyte count (THC), hemocyte mortality, phagocytosis capacity, and reactive oxygen species (ROS) production of T. granosa in pre-, and post-spawning condition using a flow cytometer. Histology indicated that the blood cockles occurring on the south coast of Korea ripe and ready to spawn in July, and they spawned in August and September. The THC in the blood cockle hemolymph declined from pre-spawning (1.2 × 108 cell mL−1) to post-spawning (0.9 × 108 cell mL−1), possibly due to the spawning stress and the massive infiltration of hemocytes in the gonad to phagocytose and resorb the residual gametes during the post-spawning period. The hemocyte mortality increased linearly from August (4.1%) to November (9.1%), as the histology revealed that the blood cockle completed spawning, and they resorbed the relict gametes. The granulocyte phagocytosis capacity declined dramatically from July (12.7%) to September (6.0%), when the cockles were engaged in active spawning. The flow cytometry revealed that the production of reactive oxygen species (ROS) from the granulocytes and the erythrocytes type II increased linearly from August (0.8–0.9 × 105 A U.) to December (2.1–2.8 × 105 A U.), which may cause stresses at a cellular level during this period. As the data indicated, spawning is a stressful activity inducing depressed immunological capacities in the blood cockles.

Shift in functional and morphological parameters of the Pacific oyster hemocytes after exposure to hypoxia

Bivalve mollusks are widely spread in shallow areas where they are subjected to fluctuations of dissolved oxygen concentration. The rapidly occurring hypoxia can represent a significant impact on bivalve immunity and functional status of the organism. The Pacific oyster (Crassostrea gigas) is an intensively cultivated introduced species in the Black Sea, which is poorly studied as a region for aquaculture. In this work the effect of 72 h hypoxia (2.1 mg O2L−1) on hemocyte functional parameters as well as immune markers of C. gigas were investigated. Hemolymph cellular composition, hemocyte proliferation level, mortality, and mitochondrial membrane potential, phagocytic capacity and spontaneous reactive oxygen species (ROS) production were evaluated using flow cytometry and microscopy. Exposure to hypoxia significantly affected hemolymph cellular composition by increasing the number of agranulocytes. Granulocyte and hyalinocyte number did not change. After 72 h exposure period changes in hemocyte morphology and massive cell aggregation were observed on slides. Cellular and nuclear diameter of agranulocytes decreased upon exposure to hypoxia The non-stimulated production of ROS significantly decreased in all hemocyte types. Phagocytic activity, hemocyte proliferation and mortality level were not significantly influenced by oxygen deficiency. The results of the present work indicate a pronounced negative impact of 72 h hypoxia on physiological parameters of hemocytes, which, however, was not associated with a decrease of their immune capacity.

Hemocyte Responses of the Oyster Crassostrea hongkongensis Exposed to Diel-Cycling Hypoxia and Salinity Change

Marine hypoxia caused by nutrient enrichment in coastal waters has become a global problem for decades, especially diel-cycling hypoxia that occurs frequently in the summer season. On the contrary, sudden rainstorms, and freshwater discharge make salinity in estuarine and coastal ecosystems variable, which often occurs with hypoxia. We found mass mortality of the Hong Kong oyster Crassostrea hongkongensis in the field where hypoxia and salinity fluctuation co-occur in the summer season during the past several years. To investigate the effects of diel-cycling hypoxia and salinity changes on the hemocyte immune function of C. hongkongensis, oysters were exposed to a combined effect of two dissolved oxygen (DO) concentrations (24 h normal oxygen 6 mg/L, 12 h normal oxygen 6 mg/L, and 12 h hypoxia 2 mg/L) and three salinities (10, 25, and 35‰) for 14 days. Subsequently, all treatments were restored to constant normal oxygen (6 mg/L) and salinity under 25‰ for 3 days to study the recovery of hemocyte immune function from the combined stress. Hemocyte parameters were analyzed by flow cytometry, including hemocyte mortality (HM), total hemocyte count (THC), phagocytosis (PHA), esterase (EST) activity, reactive oxygen species (ROS), lysosomal content (LYSO), and mitochondrial number (MN). The experimental results showed that diel-cycling hypoxia and salinity changes have obvious interactive effects on various immune parameters. In detail, diel-cycling hypoxia and decreases in salinity led to increased HM, and low salinity caused heavier impacts. In addition, low salinity, and diel-cycling hypoxia also led to decreases in LYSO, EST, and THC, while the decrease of PHA only occurs in the early stage. On the contrary, ROS production increased significantly under low salinity and hypoxic conditions. After 3-day recovery, THC, PHA, EST, LYSO, and MN were basically restored to normal, while HM and ROS were still significantly affected by diel-cycling hypoxia and salinity change, indicating that the combined stress of diel-cycling hypoxia and salinity changes had latent effects on the immune function of C. hongkongensis. Our results highlight that diel-cycling hypoxia and salinity change may impair the health and survival of the Hong Kong oyster C. hongkongensis and may be the key factors for the mass mortality of this oyster in the field.

Immune responses to ZnO nanoparticles are modulated by season and environmental temperature in the blue mussels Mytilus edulis

Increased production and release of ZnO nanoparticles (nZnO) can cause toxic effects on marine ecosystems and aquatic organisms. However, nZnO toxicity and its modulation by common environmental stressors such as temperature are not yet fully understood. We examined the responses of immune cells (hemocytes) of the blue mussels (Mytilus edulis) exposed to different concentrations (0, 10, 100 μg l−1) of nZnO or dissolved zinc combined with two temperatures (ambient (10 °C in winter and 15 °C in summer) and warming (+5 °C above ambient temperature)) in winter and summer for 21 days. In winter mussels, exposure to nZnO induced a strong transcriptomic response in multiple immune and inflammation-related genes, stimulated phagocytosis and hemocyte mortality yet suppressed adhesion capacity of hemocytes. In summer mussels, the immune cell responses to nZnO were blunted. The transcriptional responses of hemocytes to dissolved Zn were qualitatively similar but weaker than the responses to nZnO. In the absence of the toxic stress, +5 °C warming lead to dysregulation of the transcription of key immune-related genes in the summer but not the winter mussels. Seasonal warm acclimatization and additional warming in summer suppressed the nZnO-induced transcriptional upregulation of antimicrobial peptides, Toll-like receptors and the complement system. These findings demonstrate that nZnO act as an immunogen in M. edulis and indicate that +5 °C warming might have detrimental effect on innate immunity of the temperate mussel populations in summer when exposure to pathogens is especially high.Capsule: ZnO nanoparticles act as an immunotoxicant inducing a strong immune response in the mussels which is dysregulated by warming in summer but not in winter.

Effects of the toxic dinoflagellates Prorocentrum lima and Ostreopsis cf. ovata on immune responses of cultured oysters Crassostrea gasar.

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.

Effect of common salt (NaCl) on reproductive tract dimension, haemolymph haemocyte count and biochemical parameters of Giant African Land snail (A. marginata) during dry season

A study was conducted on the effect of common salt (NaCl) on reproductive tract dimension, haemolymph haemocyte count and biochemical parameters of Giant African Land snail (GALs), Archachatina marginata during the dry season. Forty (40) snails with a weight range of 150 g–180 g were used for this study. Four (4) treatment with each consisting of ten (10) replicates were assigned as follows: T1 (Control: concentrate +0 g of salt (NaCl)), T2 (concentrate +0.05 g of salt (NaCl)), T3 (concentrate+0.15 g of salt (NaCl) and T4 (concentrate + 0.25 of salt (NaCl)). The experiment lasted 13 weeks. Haemolymph was collected at the end of 13 weeks to assess haemocyte count, total protein, albumin, globulin, alanine transaminase (ALT) and aspartate transaminase (AST). The haemocyte count was determined using a Neubeur haemocytometer, while haemolymph biochemical parameters were determined using spectrophotometric methods with the appropriate kit. The snails were also dissected for complete isolation of the reproductive tract for morphometric measurements. Key organs concerned were: Ovo-testis, albumen, little hermaphrodite duct and hepatopancreas. The weight and length of each of these organs were taken together with complete tract weight. Ovo-testis from each treatment was also harvested for histology. A mortality record was taken as the experiment progresses. The result showed that serum biochemical parameters were not significantly (P > 0.05) affected by a various inclusion level of salt used. But it had a significant effect on ovotesis length, albumen weight, little hermaphrodite weight and reproductive tract weight. However, no significant effect was seen in ovotestis weight, albumen length, little hermaphrodite length and hepatopancreas weight and length. It was also discovered that common salt (NaCl) significantly affected (P < 0.01) haemocyte count and mortality record of GALS. Snails fed 0.25 g NaCl had the highest haemocyte count, followed by those fed 0.15 g, while snails fed 0 g and 0.05 g recorded the least value. It was also discovered that 0 g NaCl inclusion into the diet of snail (A. marginata) recorded the highest mortality (60 %) followed by 0.05 g NaCl inclusion level which gave 30 % mortality. However, the inclusion level of 0.15 g and 0.25 g NaCl into snail feed recorded no mortality. Histological evaluation of the ovotestis revealed that snail given 0.25 g inclusion into the concentrate diet had active acini with spermatogonium and well-formed oocyte, followed by those group with 0.15 g with active acini with several ova. It is obvious from this study that NaCl had an immune-modulatory influence on GAS during the dry season via mitigation of mortality without negatively affecting the haemolymph biochemical parameter. Inclusion used in this study also boost reproductive apparatus functions which are usually not active during this period. It is therefore recommended that both 0.15 g and 0.25 g/kg be included into the diet of A. marginata during the dry season to mitigate challenges that confront snail farming during the period to boost production and reduce economic losses due to mortality during this season.

In vitro Evaluation of Programmed Cell Death in the Immune System of Pacific Oyster Crassostrea gigas by the Effect of Marine Toxins.

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.

Role of Ovarian Proteins Secreted by Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) in the Early Suppression of Host Immune Response.

Simple SummaryToxoneuron nigriceps is an endoparasitoid of the tobacco budworm Heliothis virescens. Parasitoid strategies to survive involve different regulating factors that are injected into the host body together with the egg: the venom and the calyx fluid, containing a Polydnavirus (PDV) and Ovarian Proteins (OPs). The combination of these factors increases the success of parasitism. Although many studies have been reported on venom protein components and the knowledge on PDVs is increasing, little is known on OPs. These secretions are able to interfere early with the host cellular immune response, acting specifically on host haemocytes, cells involved in immune response. Our results show that OPs induce several alterations on haemocytes, including cellular oxidative stress condition and modifications of actin cytoskeleton, so inducing both a loss of haemocyte functionality and cell death. Overall, in synergy with PDV and venom, OPs positively contribute to the evasion of the host immune response by T. nigriceps.Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae). During oviposition, T. nigriceps injects into the host body, along with the egg, the venom, the calyx fluid, which contains a Polydnavirus (T. nigriceps BracoVirus: TnBV), and the Ovarian Proteins (OPs). Although viral gene expression in the host reaches detectable levels after a few hours, a precocious disruption of the host metabolism and immune system is observed right after parasitization. This alteration appears to be induced by female secretions including TnBV venom and OPs. OPs, originating from the ovarian calyx cells, are involved in the induction of precocious symptoms in the host immune system alteration. It is known that OPs in braconid and ichneumonid wasps can interfere with the cellular immune response before Polydnavirus infects and expresses its genes in the host tissues. Here we show that T. nigriceps OPs induce several alterations on host haemocytes that trigger cell death. The OP injection induces an extensive oxidative stress and a disorganization of actin cytoskeleton and these alterations can explain the high-level of haemocyte mortality, the loss of haemocyte functionality, and so the reduction in encapsulation ability by the host.

Влияние суточной гипоксии на функциональные показатели гемоцитов Anadara kagoshimensis (Tokunaga, 1906)

Марикультурные хозяйства традиционно расположены в прибрежных участках, где моллюски могут подвергаться воздействию гипоксии. Культивирование в условиях дефицита кислорода приводит к снижению темпов роста, вспышкам заболеваний на фермах и массовой гибели моллюсков. Методом проточной цитометрии исследовано влияние краткосрочной гипоксии на функциональные показатели гемоцитов анадары (Anadara kagoshimensis). Контрольную группу содержали при 6,7–6,8 мг O2·л−1, опытную — при 0,4–0,5 мг O2·л−1. Экспозиция — 24 часа. Содержание кислорода в воде снижали, продувая её газообразным азотом. В гемолимфе моллюска на основании относительного размера и относительной гранулярности идентифицировано две группы гемоцитов: гранулоциты (эритроциты) и агранулоциты (амёбоциты). Эритроциты — преобладающий тип клеток в гемолимфе A. kagoshimensis: их доля составила более 90 % от общего числа клеток. Суточная гипоксия не привела к изменениям клеточного состава гемолимфы анадары. Способность к продукции активных форм кислорода и уровень смертности гемоцитов моллюсков экспериментальной группы также остались на уровне контрольных значений. Результаты проведённого исследования свидетельствуют о толерантности анадары к условиям острого дефицита кислорода.