Highly Sensitive and Specific Responses of Oyster Hemocytes to Copper Exposure: Single-Cell Transcriptomic Analysis of Different Cell Populations.

Oyster hemocytes are the primary vehicles transporting and detoxifying metals and are regarded as important cells for the occurrence of colored oysters due to copper (Cu) contamination. However, its heterogeneous responses under Cu exposure have not been studied. Single-cell transcriptome profiling (scRNA-seq) provides high-resolution visual insights into tissue dynamics and environmental responses. Here, we used scRNA-seq to study the responses of different cell populations of hemocytes under Cu exposure in an estuarine oyster Crassostrea hongkongensis. The 1900 population-specific Cu-responsive genes were identified in 12 clusters of hemocytes, which provided a more sensitive technique for examining Cu exposure. The granulocyte, semigranulocyte, and hyalinocyte had specific responses, while the granulocyte was the most important responsive cell type and displayed heterogeneity responses of its two subtypes. In one subtype, Cu was transported with metal transporters and chelated with Cu chaperons in the cytoplasm. Excess Cu disturbed oxidative phosphorylation and induced reactive oxygen species production. However, in the other subtype, endocytosis was mainly responsible for Cu internalization, which was sequestered in membrane-bound granules. Collectively, our results provided the first mRNA expression profile of hemocytes in oysters and revealed the heterogeneity responses under Cu exposure.

Trace elements (Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) in Mytilus galloprovincialis and Tapes decussatus from Faro and Ganzirri Lakes (Sicily, Italy): Flow cytometry applied for hemocytes analysis.

BACKGROUND: Trace elements present in sessile molluscs, are important because they are used in human consumption and it has significantly increased in recent years. While their filtering of the water can lead to their build-up of organic and inorganic materials that can be sampled and analyzed, this can also lead to bioaccumulation of harmful substances, such as essential and non-essential elements, that can harm the human health if in taken in high concentrations or for a long period of time. METHODS: In the present study, the trace metal content (Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) of two sessile crustaceans, 20 Mytilus galloprovincialis (mussel) and 20 Tapes decussatus (clam) in Faro and Ganzirri Lakes (Messina, Sicily, Italy) were analyzed. Haemolymph samples were taken on both molluscs in order to analyze the haemocyte population by flow cytometric analysis. Unpaired t-tests were used to determine significant differences for the essential and non-essential metallic elements concentrations in the lake waters and in the tissues of M. galloprovincialis and T. decussatus and for hemocyte populations R1 (halinocytes) and R2 (granulocytes). RESULTS: The results suggested that that in Faro Lake, the tissue Al, Cr and Pb levels in M. galloprovincialis were higher than those for T. decussatus, in contrast to Mn, Fe, Ni, Cu, Zn and Cd, which were higher in T. decussatus. Unpaired t-tests showed that there were significantly higher proportions of halinocytes in M. galloprovincialis versus T. decussatus for both Faro Lake (41.8 % vs. 24.3 %; P < 0.001) and Ganzirri Lake (43.0 % vs. 22.4 %; P < 0.001). In contrast, while there were significantly higher proportions of granulocytes in Faro Lake (21.2 % vs. 9.1 %; P < 0.001), this difference was not seen for the granulocytes of M. galloprovincialis versus T. decussatus in Ganzirri Lake (9.6 % vs. 13.0 %). CONCLUSION: This study shows that M. galloprovincialis and T. decussatus can indeed bioaccumulate some of these metal, such that activation of the immune responses is specific to certain cell types. Future research must focus on the balance of trace elements in the consumption of these shellfish, and analyzes with more sophisticated tools can be used to diagnose the increased concentration of trace elements and the quantification of trace metals from shellfish to clams.

Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process.

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

iTRAQ-based quantitative proteomic profiling of the immune response of the South African abalone, Haliotis midae.

The South African abalone Haliotis midae is a commercially important species farmed at high densities in land-based aquaculture systems. Disease outbreaks have had a severe financial impact on the abalone industry yet the molecular mechanisms underlying the immune response of H. midae remain obscure. In this study, a comparative shotgun proteomics approach using iTRAQ coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A total of 118 non-redundant, unique haemocyte proteins were identified and quantified, with 16 proteins significantly regulated. Hierarchical clustering and pathway analysis uncovered a coordinated response dominated by calcium and cAMP signalling via activation of MAPK cascades. Early up-regulated biological processes involve phagocytosis, nitric oxide production and ATP-synthesis, whilst down-regulated responses were predominantly involved in the regulation of apoptosis. The late up-regulated response involved protein kinase activity and detoxification processes. Expression of selected proteins was validated by Western blot. A putative allograft inflammatory factor-1 protein was further selected to establish its functional molecular role in haemocytes. Confocal imaging revealed that allograft inflammatory factor-1 regulates phagocytosis via a functional interaction with filamentous actin. This is the first time a high-throughput proteomics approach has been used to investigate the immune response of H. midae.

Toward the Molecular Deciphering of Pomacea canaliculata Immunity: First Proteomic Analysis of Circulating Hemocytes.

Pomacea canaliculata is a freshwater snail with interesting biological features that include invasiveness, human parasite hosting, and adult regeneration. Its immune system may represent the target for strategies aimed at controlling the spread of the snail population and its hosting of the human parasite Angiostrongylus cantonensis. Moreover, immune functions likely have a role in the snail's ability to wound heal and regenerate. Despite its importance in multiple processes, very little is known about the molecular basis of P. canaliculata immunity. Aiming to contribute to filling this gap, the ultrastructure of circulating hemocytes in healthy snails is studied and the first proteomic analysis of these cells is performed, evidencing 83 unique proteins, 96% of which have identifiable homologs in other species. Fifteen proteins are retrieved as potentially involved in immune-related signaling pathways, such as hemocyanin, C1q-like protein, and HSP90 together with cytoskeleton and cytoskeleton-related proteins involved in cell motility and membrane dynamics. This first proteome study on non-stimulated hemocytes provides a valid reference for future investigations on the molecular changes under stressful circumstances, like pathogen exposure, wounding, or environmental changes.

Are semi-terrestrial crabs threatened by human noise? Assessment of behavioural and biochemical responses of Neohelice granulata (Brachyura, Varunidae) in tank.

This study examined the effects of human lab-generated noise (sweep tone) on the behaviour and biochemistry of a semi-terrestrial crab (Neohelice granulata). The experiment was carried out in tanks equipped with video- and audio-recording systems on a total of seventy-eight specimens. In total, 42 experimental trials with sweep-tone exposure and control conditions were performed using crabs in single and group layouts. After a habituation period of 30 min, the locomotor and acoustic (sound signals emitted by the crabs) behaviours were monitored for 30 min. During this time, the animals in sweep-tone conditions were exposed to ascending sweeps in a bandwidth range of 2.5-25 kHz. Exposure to sweep-tone noise produced significant changes in the number of signals emitted, locomotor behaviours and plasma parameters, such as haemolymph total haemocyte count and glucose, lactate and total protein concentrations, revealing that human noise could represent a disturbance for this crustacean species.

Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.

Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.

Bombyx mori hemocyte extract has anti-inflammatory effects on human phorbol myristate acetate-differentiated THP­1 cells via TLR4-mediated suppression of the NF-κB signaling pathway.

Hemolymph is the circulating fluid of insects and is a key component of their immune system. However, little is known concerning hemocyte identification, development, differentiation and related cellular immune responses. The present study aimed to determine whether a hemocyte extract prepared from Bombyx mori larvae had anti­inflammatory effects; THP­1 (a human monocytic leukemia cell line) cells that had been differentiated into macrophage­like cells by treatment with phorbol myristate acetate (PMA) were used. THP­1 cells were cultured with different concentrations of a B. mori hemocyte extract prior to exposure to lipopolysaccharide (LPS) to induce an inflammatory response. The effects of the B. mori hemocyte extract on anti­inflammatory pathways were determined using reverse transcription­quantitative polymerase chain reaction and western blotting to assess the expression of pro­inflammatory molecules. The B. mori hemocyte extract inhibited the LPS­induced mRNA expression of Toll­like receptor 4 in addition to LPS­induced interleukin (IL)­1ß, IL­6, IL­8 and tumor necrosis factor­α. Treatment of PMA­differentiated THP­1 cells with B. mori hemocyte extract also inhibited inducible nitric oxide synthase and cyclooxygenase­2 transcription and translation. Nuclear factor­κB activation and phosphorylation also decreased. Further in­depth functional studies are required to understand the mechanism underlying the anti­inflammatory effects of silkworm hemocyte extract.

Rapid Assessment of Genotoxicity by Flow Cytometric Detection of Cell Cycle Alterations.

Flow cytometry is a convenient method for the determination of genotoxic effects of environmental pollution and can reveal genotoxic compounds in unknown environmental mixtures. It is especially suitable for the analyses of large numbers of samples during monitoring programs. The speed of detection is one of the advantages of this technique which permits the acquisition of 104-105 cells per sample in 5 min. This method can rapidly detect cell cycle alterations resulting from DNA damage. The outcome of such an analysis is a diagram of DNA content across the cell cycle which indicates cell proliferation, G2 arrests, G1 delays, apoptosis, and ploidy.Here, we present the flow cytometric procedure for rapid assessment of genotoxicity via detection of cell cycle alterations. The described protocol simplifies the analysis of genotoxic effects in marine environments and is suitable for monitoring purposes. It uses marine mussel cells in the analysis and can be adapted to investigations on a broad range of marine invertebrates.

Role of purified ß-1, 3 glucan binding protein (ß-GBP) from Paratelphusa hydrodromus and their anti-inflammatory, antioxidant and antibiofilm properties.

ß- 1, 3-glucan binding protein (ß-GBP), a pattern recognition protein (PRP), plays a critical role in triggering the innate immune response by detecting ß-glucan found on the surface of microbes. In the present study, ß-GBP was purified from the haemolymph of rice field crab Paratelphusa hydrodromus by affinity column chromatography. The monomeric protein Ph-ß-GBP appeared as a single band with a molecular weight of approximately 95 kDa in SDS-PAGE analysis and its purity was determined to be 89% by HPLC. MALDI-TOF/TOF analysis revealed that, the purified 95 kDa protein display 36% similarity with ß-GBP of crayfish Astacus lepidodactylus. Purified Ph-ß-GBP exhibited increased agglutination, phagocytic activity and encapsulation in a dose-dependent manner, indicating the involvement of Ph-ß-GBP in cellular immune response against pathogens in crustaceans. Moreover, addition of Ph-ß-GBP increased the prophenoloxidase (proPO) and serine protease activity, possibly contributing to the clearance of pathogens. The antioxidant activity of Ph-ß-GBP was determined by DPPH radical scavenging activity demonstrates maximum scavenging activity of 78.4%. In addition, RBC membrane stabilization and inhibition of protein (albumin) denaturation proved anti-inflammatory property of Ph-ß-GBP. Furthermore, light microscopic and confocal laser scanning microscopic analysis revealed that the reactive compound (laminarin and Ph-ß-GBP) reduced the biofilm thickness of Gram-positive (Enterococcus faecalis) and Gram-negative (Vibrio parahaemolyticus) bacteria at the concentration of 25 µg/ml. Taken together, our results demonstrate that, the ß-GBP triggers proPO activating system in rice field crab P. hydrodromus and plays a vital role in innate defense mechanism against invading pathogens.

Validation of Comet assay in Oregon-R and Wild type strains of Drosophila melanogaster exposed to a natural radioactive environment in Brazilian semiarid region.

Natural radiation of geological origin is a common phenomenon in Brazil, a country where radioactive agents such as uranium may be often found. As an unstable atom, uranium undergoes radioactive decay with the generation of a series of decay by-products, including radon, which may be highly genotoxic and trigger several pathological processes, among which cancer. Because it is a gas, radon may move freely between cracks and gaps in the ground, seeping upwards into the buildings and in the environment. In this study, two Drosophila melanogaster Meigen (Diptera, Drosophilidae) strains called Oregon-R and Wild (collected in a non-radioactive environment) were exposed to atmospheric radiation in the Lajes Pintadas city, in the semiarid zone of northeastern Brazil. After six days of environmental exposure, the organisms presented genetic damage significantly higher than that of the negative control group. The genotoxic effects observed reinforce the findings of other studies carried out in the same region, which warn about the environmental risks related to natural radioactivity occurrence. The results also validate the use of the Comet assay in hemocytes of D. melanogaster as a sensitive test to detect genotoxicity caused by natural radiation, and the use of a recently collected D. melanogaster strain in the environmental of radon.

Exploration of the Innate Immune System of Styela clava: Zn2+ Binding Enhances the Antimicrobial Activity of the Tunicate Peptide Clavanin A.

Tunicates have been used as primitive models for understanding cell-mediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn2+ ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, ß-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn2+ added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn2+ import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn2+ has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill.

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea.

Structural Studies of a Lipid-Binding Peptide from Tunicate Hemocytes with Anti-Biofilm Activity.

Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.

Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi).

We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pHi), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pHe) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pHi) within hyalinocytes among pH treatments and the mean pHi (7.26) was lower than the mean pHe. In contrast, there were significant differences among treatments in pHi of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pHi compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction.

Identification of a thioredoxin peroxidase gene involved in resistance to nucleopolyhedrovirus infection in Helicoverpa armigera with RNA interference.

Thioredoxin peroxidases (Tpxs) play a crucial role in protection against oxidative damage in several insect species. However, studies on the characteristics and functions of Tpxs in Helicoverpa armigera are lacking. In this study, a novel 2-Cys Tpx gene from H. armigera (HaTpx) was identified. Sequence analysis revealed that HaTpx is highly conserved and shares two catalysis regions (VCP) with other insect species. HaTpx mRNA was found to be expressed in an age-dependent manner and was ubiquitous in all tissues examined. Hormone treatment showed that the expression of HaTpx is clearly induced by 20-hydroxyecdysone but repressed by Juvenile hormone. Additionally, extreme temperature, ultraviolet light, mechanical injury, Escherichia coli, Metarhizium anisopliae, nucleopolyhedrovirus (NPV) infection, and H2O2 treatment markedly induced HaTpx gene expression. Reactive oxygen species (ROS) levels in hemocytes and MDA concentrations in the hemolymph after NPV infection were evaluated, and the results indicated that NPV infection causes excessive ROS generation. After knockdown of HaTpx by RNA interference, the expression of three antioxidant genes (Cu/ZnSOD, Trx, and TrxR) was increased, whereas two antioxidant genes (CAT and GPX) showed decreased expression. Moreover, the susceptibility of H. armigera to NPV infection increased after HaTpx knockdown. These results indicated that HaTpx contributes to the susceptibility of H. armigera to NPV, and the results also provide a theoretical basis for a novel strategy for developing new chemicals and microbial pesticides that target HaTpx gene for controlling H. armigera.

Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species.

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is hijacked by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. Screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). Phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. Glycan array analysis, however, revealed that, unlike CvGal1 which preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Furthermore, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and ß-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for Perkinsus chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of Perkinsus species by the oyster galectins is consistent with their relative prevalence in oyster and clam species and supports their role in facilitating parasite entry and infectivity in a host-preferential manner.

Purification and Characterization of a Cysteine-Rich 14-kDa Antibacterial Peptide from the Granular Hemocytes of Mangrove Crab Episesarma tetragonum and Its Antibiofilm Activity.

Antimicrobial peptide (AMP) crustin is a type of immune molecule present in the immune system of crustaceans and response against microbial invasion. In the present study, we have identified and characterized the cationic, amphipathic structure consisting of AMP crustin from a mangrove crab Episesarma tetragonum using CM Sepharose-based cation exchange column chromatography. E. tetragonum crustin showed a single band of 14 kDa on SDS-PAGE and the homogeneity showed retention time of 8.4 min in RP-HPLC. Functional studies of E. tetragonum crustin exhibits the antibacterial activity (2-4 µg/ml) and biofilm inhibition (20 µg/ml) against the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis. Hydrophobicity and extrapolysaccharide production of Gram-positive bacteria were inhibited through the bactericidal inhibitory concentration. In situ visualization analysis of biofilm inhibition was observed through light and confocal laser scanning microscopy. Surface morphology and the bacterial biofilm inhibition were viewed by scanning electron and atomic force microscopy. This study emphasizes the potential activity of E. tetragonum crustin, an interesting candidate for the development of novel broad-spectrum antimicrobial agent against bacterial pathogens. Graphical Abstract Antimicrobial peptide synthesis and host-pathogen interaction lead to production of immune molecules directed to destruction of pathogens.

Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes.

The residual contaminators such as ammonia and nitrite are widely considered as relevant sources of aquatic environmental pollutants, posing a great threat to shrimp survival. To study the toxicological effects of ammonia and nitrite exposure on the innate immune response in invertebrates, we investigated the oxidative stress and apoptosis in haemocytes of freshwater prawn (Macrobrachium rosenbergii) under isolated and combined exposure to ammonia and nitrite in order to provide useful information about adult prawn immune responses. M. rosenbergii (13.44 ± 2.75 g) were exposed to 0, 5, and 25 mg/L total ammonia-N (TAN) and 0, 5, and 20 mg/L nitrite-N for 24 h. All ammonia concentrations were combined with all nitrite concentrations, making a total of nine treatments studied. Following the exposure treatment, antioxidant enzyme activity, reactive oxygen species (ROS) generation, nitric oxide (NO) generation, and apoptotic cell ratio of haemocytes were measured using flow cytometry. Results indicated that ROS generation was sensitive to the combined effect of ammonia and nitrite, which subsequently affected the Cu-Zn SOD activity. In addition, CAT showed the highest activity at 5 mg/L TAN while GPx decreased at 5 mg/L TAN and returned towards baseline at 25 mg/L. NO generation synchronized with the apoptotic cell ratio in haemocytes, indicating that NO production was closely associated with programmed cell death. Both NO production and apoptotic ratios significantly decreased following 25 mg/L TAN, which may be due to the antagonistic regulation of NO and GPx. We hypothesized that the toxicological effect of nitrite exhibited less change in physiological changes compared to that of ammonia, because of the high tolerance to nitrite exposure in mature M. rosenbergii and/or the competitive effects of chloride ions. Taken together, these results showed that ammonia and nitrite caused a series of combined oxidative stress and apoptosis in M. rosenbergi, but further studies are of great need to explain the mechanisms.

Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori.

Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system.