Proteins In Hemocytes Research Articles

Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria

Estuarine organisms are exposed to multiple stressors including large fluctuations in partial pressure of carbon dioxide (PCO2) and concentrations of trace metals such as cadmium (Cd) that can affect their survival and fitness. Ocean acidification due to the increasing atmospheric PCO2 leads to a decrease in pH and shifts in the carbonate chemistry of seawater which can change bioavailability and toxicity of metals. We studied the interactive effects of PCO2 and Cd exposure on metal levels, metabolism and immune-related functions in hemocytes of two ecologically and economically important bivalve species, Mercenaria mercenaria (hard shell clam) and Crassostrea virginica (Eastern oyster). Clams and oysters were exposed to combinations of three PCO2 levels (∼400, 800 and 2000 μatm PCO2, corresponding to the present day conditions and the projections for the years 2100 and 2250, respectively) and two Cd concentrations (0 and 50 μg l−1) in seawater. Following four weeks of exposure to Cd, hemolymph of both species contained similar Cd levels (50–70 μg l−1), whereas hemocytes accumulated intracellular Cd burdens up to 15–42 mg l−1, regardless of the exposure PCO2. Clam hemocytes had considerably lower Cd burdens than those of oysters (0.7–1 ng 10−6 cells vs. 4–6 ng 10−6 cells, respectively). Cd exposure suppressed hemocyte metabolism and increased the rates of mitochondrial proton leak in normocapnia indicating partial mitochondrial uncoupling. This Cd-induced mitochondrial uncoupling was alleviated in hypercapnia. Cd exposure suppressed immune-related functions in hemocytes of clams and oysters, and these effects were exacerbated at elevated PCO2. Thus, elevated PCO2 combined with Cd exposure resulted in decrease in phagocytic activity and adhesion capacity as well as lower expression of mRNA for lectin and heat shock protein (HSP70) in clam and oyster hemocytes. In oysters, combined exposure to elevated PCO2 and Cd also led to reduced activity of lysozyme in hemocytes and hemolymph. Overall, our study shows that moderately elevated PCO2 (∼800–2000 μatm PCO2) potentiates the negative effects of Cd on immunity and thus may sensitize clams and oysters to pathogens and diseases during seasonal hypercapnia and/or ocean acidification in polluted estuaries.

Proteomic Analysis of Differentially Expressed Proteins in Fenneropenaeus chinensis Hemocytes upon White Spot Syndrome Virus Infection

To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (Fenneropenaeus chinensis) at 24 h post infection (hpi). Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO) 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase) and one down-regulated protein (prophenoloxidase) were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection.

Arginine kinase of Litopenaeus vannamei involved in white spot syndrome virus infection

Virus–host interaction is important for virus infection. White spot syndrome virus VP14 contains transmembrane and signal peptides domain, which is considered to be important for virus infection. Until now, the function of this protein remains undefined. In this study, we explored the interaction of VP14 with host cell. A new shrimp protein (arginine kinase of Litopenaeus vannamei, LvAK) is selected and its localization in shrimp cells is also confirmed.Cellular localization of LvAK protein in shrimp hemocytes showed that LvAK was primarily located at the periphery of hemocytes and was scarcely detectable in the nucleus. Tissue distribution indicated that arginine kinase gene was spread commonly in the tissues and was highly present in shrimp muscle tissue. The expression of LvAK mRNA in muscle was significantly up-regulated after WSSV stimulation. Indirect immunofluorescence assay showed that LvAK interacted with VP14 in WSSV-infected shrimp. Injection of LvAK protein enhanced the mortality of shrimp infected with white spot syndrome virus (WSSV). These results showed that LvAK is involved in WSSV infection. Future research on this topic will help to reveal the molecular mechanism of WSSV infection.

Expression kinetics of β-integrin in Chinese shrimp (Fenneropenaeus chinensis) hemocytes following infection with white spot syndrome virus

Our previous study has demonstrated that an integrin β subunit of Chinese shrimp (Fenneropenaeus chinensis) (FcβInt) involved in WSSV infection. In order to further elucidate the potential role of the FcβInt in the WSSV infection, expression response of FcβInt to WSSV infection in shrimp hemocytes was investigated after intra-muscular injection with the virus. Following time-course hemocytes sampling, the expression variation of FcβInt in hemocytes was examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA) using the monoclonal antibody (Mab) 2C5 against FcβInt, which was successfully produced with recombinant partial FcβInt and exhibited binding to a 120 kDa hemocyte protein. Meanwhile, the dynamic state of FcβInt mRNA level and WSSV copies in hemocytes were determined by quantitative real-time PCR. The result of FCIFA showed that FcβInt was mainly expressed on the semi-granular and granular cells, which was down-regulated at 6 h post infection (p.i.), and significantly increased to the peak level at 12 h p.i., then decreased to the control level by 24 h. However, FcβInt on the hyaline cells was lowly expressed and didn't show active response to the viral infection. The variation of FcβInt concentrations in total hemocytes determined by ELISA was roughly in accordance with the changing tendency of FcβInt expressed on the semi-granular and granular cells. FcβInt mRNA level in total hemocytes was significantly up-regulated to the peak level at 12 h p.i. Moreover, the number of WSSV copies in hemocytes began to exhibit a significant increase at 24 h p.i. The present study demonstrated that WSSV infection would induce a differential regulation of FcβInt expression in different type hemocytes, which provided valuable evidences for the close correlation between FcβInt and WSSV infection.

Gene expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48h exposure, respectively. HSP70 expression level significantly increased at 8 and 72h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48h, while CAT mRNA expression level increased at 24 and 48h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12h exposure. Expression level of TRx reached its highest level after 48h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.

Comparative proteomic identification of the hemocyte response to cold stress in white shrimp, Litopenaeus vannamei

To understand molecular responses of crustacean hemocytes to cold stress, flow cytometer analysis and two-dimensional electrophoresis proteomic approach were used to investigate altered proteins in hemocytes of Litopenaeus vannamei during cold stress treatment. Through flow cytometer analysis, 13°C for 24h post-cold stress was selected as the suitable temperature and condition for cold stress treatment. MALDI-TOF/TOF MS analysis revealed that 6 forms of 6 proteins were significantly up-regulated, including three enzymes (cystathionase, glyceraldehyde 3-phosphate dehydrogenase and glyoxalase 1) and one immune-related protein (oncoprotein nm23), whereas 24 forms of 3 proteins were significantly down-regulated in the treated shrimp (hemocyanin, hemocyte transglutaminase and transketolase). There were 20 spots identified as hemocyanin meaning that it may play important roles in environmental regulation in shrimp. Real-time fluorescence quantitative PCR confirmed that the levels of transcription of the hemocyanin, partial mRNA for hemocyanin, cystathionase, glyoxalase 1 and oncoprotein nm23 genes were found to relate well with that of their translation products after cold stress treated, while only the levels of hemocyte transglutaminase transcripts were not corresponded with that of their translation products. Further investigation of these data may lead to better understanding of the molecular responses of crustacean hemocytes to cold stress.

Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates.

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval Schistosoma mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability of hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations.

Production and characterization of monoclonal antibodies to the hemocytes of mud crab, Scylla serrata

Monoclonal antibodies (MAbs) to hemocytes of mud crab, Scylla serrata, were produced by immunizing mice with formalin-fixed hemocytes. Of the six MAbs produced, two (MAb 1D11 and MAb 1A2) reacted specifically with hemocyte proteins in western blot. MAb 1A2 showed strong immunofluorescent reaction with granular hemocytes and a weak reaction with semigranular cells. However, hyaline cells did not show any reaction. The MAbs also showed strong cross-reactivity with the hemocytes of different crab species but not with other crustaceans. The MAbs produced can be used as a marker for granular hemocytes of crabs.

Characterization of two isoforms of antiliopolysacchride factors (Sp-ALFs) from the mud crab Scylla paramamosain

In the previous study of the mud crab (Scylla paramamosain) hemocyte proteins, which interacted with a bacterium, Vibrio parahaemolyticus, a protein known as antilipopolysaccharide factor (Sp-ALF) was isolated in addition to a serine proteinase homolog (Sp-SPH) protein. In the present study, we further reported the characterization of two isoforms of the mud crab ALF – Sp-ALFs genes (designated as Sp-ALF1 and Sp-ALF2, respectively) based on our previous result. The Sp-ALF1 and Sp-ALF2 cDNA contained 1070 bp and 731 bp, respectively, with 123 deduced amino acid residues. Alignment of deduced amino acid sequences showed that Sp-ALFs possessed high identity with other known ALFs from crustaceans and exhibited an overall similarity of 57.7% to those of ALFs compared. Phylogenetic tree analysis revealed a clear group of each species and also suggested that ALFs from Scylla genus and those from Portunus genus were closely related. Tissue distribution analysis in adult crab implied that both Sp-ALF1 and Sp-ALF2 were mainly expressed in hemocytes. The mRNA transcripts were also found in embryo (I, II, III and V), zoea-I and juvenile crab, but were rarely observed in the megalopa stage. To further identify the biological activity of Sp-ALFs, recombinant proteins (rSp-ALFs: designated as rSp-ALF1 and rSp-ALF2, respectively) were obtained by expression in Pichia pastris, and the synthetic peptide fragments (sSp-ALFs: designated as sSp-ALF1 and sSp-ALF2, respectively) including the putative LPS binding loop were also prepared for antimicrobial test. The results indicated that both rSp-ALFs and sSp-ALFs were highly effective against most of the Gram-positive bacteria and Gram-negative bacteria tested. In contrast to cecropin P1, a membrane integrity assay revealed that Sp-ALFs did not affect the Escherichia coli by disruption of membrane integrity. Additionally, the recombinant Sp-ALFs proteins exhibited strong antiviral activity against an important aquaculture pathogen, white spot syndrome virus, in crustaceans. Taken together, these data suggested that Sp-ALFs might play a key role in immune defense against microbial infection in the mud crab S. paramamosain.

Salinity a significant environmental factor forVibrio harveyivirulence inFenneropenaeus indicus

The effect of ambient salinity on the haemolymph variables of Fenneropenaeus indicus and its susceptibility to Vibrio harveyi infection under salinity stress has been studied. Adult shrimps were acclimated to 5‰ (hypo osmotic), 25‰ (iso osmotic) and 35‰ (hyper osmotic) salinity levels and the animals were injected with a mid logarithmic culture of V. harveyi at sub lethal level and haemolymph parameters were analysed. Haemolymph proteins, intracellular superoxide anion production, phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were found to be at elevated level both at 5‰ and 35‰ post challenge. The haematological responses showed a progressive increase (P < 0.05) up to post challenge day 5 (PCD 5) followed by a considerable decline at all salinities with the lowest being at 35‰. The alterations in the variables were higher in shrimps held at 5‰. However, the V. harveyi infection was severe in animals held at 35‰. The reduction in the parameters could be correlated with the decrease in survival rate of shrimps at 35‰ with a concurrent increase in V. harveyi at this salinity. Multiple regression analysis revealed that ACP (P < 0.001), haemocyte protein HCP (P < 0.001) and PO (P < 0.05) could explain 91% variability in the shrimp survival. These parameters may be used as effective shrimp health indicators. It is evident from the study that ambient salinity alters the haemolymph variables, modulates the virulence in V. harveyi and makes the shrimps more vulnerable to infection at higher salinity. The virulence of V. harveyi is increased at 35‰ salinity as being evidenced from the high mortality at this salinity. The study emphasizes the importance of salinity as an important environmental factor both in terms of host susceptibility and virulence of the pathogen.

Accumulation and toxicity of aluminium-contaminated food in the freshwater crayfish, Pacifastacus leniusculus

The accumulation and toxicity of aluminium in freshwater organisms have primarily been examined following aqueous exposure. This study investigated the uptake, excretion and toxicity of aluminium when presented as aluminium-contaminated food. Adult Pacifastacus leniusculus were fed control (3 μg aluminium/g) or aluminium-spiked pellets (420 μg aluminium/g) over 28 days. Half the crayfish in each group were then killed and the remainder fed control pellets for a further 10 days (clearance period). Concentrations of aluminium plus the essential metals calcium, copper, potassium and sodium were measured in the gill, hepatopancreas, flexor muscle, antennal gland (kidney) and haemolymph. Histopathological analysis of tissue damage and sub-cellular distribution of aluminium were examined in the hepatopancreas. Haemocyte number and protein concentration in the haemolymph were analysed as indicators of toxicity. The hepatopancreas of aluminium-fed crayfish contained significantly more aluminium than controls on days 28 and 38, and this amount was positively correlated with the amount ingested. More than 50% of the aluminium in the hepatopancreas of aluminium-fed crayfish was located in sub-cellular fractions thought to be involved in metal detoxification. Aluminium concentrations were also high in the antennal glands of aluminium-fed crayfish suggesting that some of the aluminium lost from the hepatopancreas is excreted. Aluminium exposure via contaminated food caused inflammation in the hepatopancreas but did not affect the number of circulating haemocytes, haemolymph ion concentrations or protein levels. In conclusion, crayfish accumulate, store and excrete aluminium from contaminated food with only localised toxicity.

Haemocyte protein expression profiling of scallop Chlamys farreri response to acute viral necrosis virus (AVNV) infection

Acute viral necrosis virus (AVNV) was newly reported as one causative agent responsible for mass mortality of adult Chinese scallop Chlamys farreri, which is widely cultured on northern China coast. Unfortunately, the interaction between virus and host is largely unknown. According to these, this study was undertaken to deeply explore the immune response of haemocyte against AVNV. Two-dimensional gel electrophoresis (2-DE) was introduced to produce protein expression profiles from samples taken at 24 h post-infection (hpi) from the haemocytes of C. farreri that were either specific pathogen free or else infected with AVNV. Forty-eight protein spots, which consistently showed either a marked change (≥1.5-fold difference) in accumulated levels or else were highly expressed in haemocytes, were selected for further investigation. In-gel trypsin digestion was conducted followed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). Matching search was subsequently performed throughout bioinformatics databases. A total of 42 proteins were identified, all of which were classified into eight categories according to their Gene Ontology annotations of biological processes and molecular functions, i.e. cytoskeleton proteins, proteins involved in metabolism, proteins related to calcium homeostasis, chaperone, proteins involved in immunity, proteins involved in transcriptional regulation, proteins related to signal transduction, and ungrouped proteins. The possible biological significance of some observed proteins in the host response to AVNV was discussed. These studies could be served as the first global analysis of differentially expressed proteins in haemocytes from AVNV-infected C. farreri, and in addition to increasing our understanding of the pathogenesis of this virus-associated scallop disease, the results presented here should be useful both for potential biomarkers identification and anti-virus approaches development as well.

Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

BackgroundViral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time.ResultsThe two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR.ConclusionsThis work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14-3-3 protein epsilon. The information is useful for understanding the immune system of shrimp against pathogenic bacteria.

Lipopolysaccharide-induced phosphorylation and partial amino acid sequence of a hemocyte protein in the silkworm, Bombyx mori

Lipopolysaccharide-induced phosphorylation and partial amino acid sequence of a hemocyte protein in the silkworm, Bombyx mori

Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera, Apidae, Meliponini): Cell types and their role in phagocytosis

Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. In the current investigation the cellular in the hemolymph third instar larvae of M. scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.5 μm fluorescence beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris.

C-Terminal Hemocyanin from Hemocytes of Penaeus vannamei Interacts with ERK1/2 and Undergoes Serine Phosphorylation

To understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up-regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (pI), whereas the N-terminal fragments had less acidic pI. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co-immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin.

Immune function in the Arctic Scallop, Chlamys islandica, following dispersed oil exposure

With the current expansion of offshore oil activities in Arctic regions, there is an urgent need to establish the potential effects of oil-related compounds on Arctic organisms. As susceptibility to growth, disease and survival is determined partly by the condition of an organism's immune system, measurement of endpoints linked to the latter system provide important early warning signals of the sub-lethal effects of exposure to contaminants. This study assessed the impact of dispersed oil exposure on immune endpoints in the Arctic Scallop Chlamys islandica, using a combination of cellular and humoral biological responses. Laboratory exposures of C. islandica to sub-lethal dispersed oil concentrations (0.06 and 0.25 mg l −1) were conducted over 15 days, followed by a 7-day recovery period in clean, filtered seawater. Cellular endpoints were significantly altered following dispersed oil exposure: haemocyte counts ( P < 0.01) and protein levels ( P < 0.01) were significantly elevated, whilst cell membrane stability ( P < 0.001) and phagocytosis ( P < 0.01) demonstrated a significant reduction. Whilst these results indicate alteration in the immune endpoints measured, this appears to be reversible upon removal of the contaminant stress. However, the impact of long-term continuous exposure and high-level acute exposure to oil is still unknown, and may have consequences for disease resistance and hence survival.

Mortality and blood loss by blue swimmer crabs (Portunus pelagicus) after simulated capture and discarding from gillnets

Abstract Uhlmann, S. S., Broadhurst, M. K., Paterson, B. D., Mayer, D. G., Butcher, P., and Brand, C. P. 2009. Mortality and blood loss by blue swimmer crabs (Portunus pelagicus) after simulated capture and discarding from gillnets. – ICES Journal of Marine Science, 66: 455–461. Two laboratory experiments were carried out to quantify the mortality and physiological responses of juvenile blue swimmer crabs (Portunus pelagicus) after simulated gillnet entanglement, air exposure, disentanglement, and discarding. In both experiments, all but control blue swimmer crabs were entangled in 1-m2 gillnet panels for 1 h, exposed to air for 2 min, subjected to various treatments of disentanglement ranging between the forceful removal of none, one, two, and four appendages, then “discarded” into individual experimental tanks and monitored for 10 d. In Experiment 1, mortalities were associated with the number of appendages removed and the occurrence of unsealed wounds. In Experiment 2, live blue swimmer crabs were sampled for blood at 2 min and 6, 24, and 72 h post-discarding to test for the effects of disentanglement and appendage removal on total haemocyte counts, clotting times, protein levels (by refractive index), and blood ion concentrations. Compared with blue swimmer crabs that had sealed or no wounds, those with unsealed wounds had lower total haemocyte counts, protein, and calcium concentrations and increased clotting times and magnesium and sodium levels. Induced autotomy, as opposed to the arbitrary, forceful removal of appendages has the potential to minimize the mortality and stress of discarded, juvenile blue swimmer crabs.

Visualization of shell matrix proteins in hemocytes and tissues of the Eastern oyster, Crassostrea virginica

The tissues of the oyster were examined for the presence of shell matrix proteins (SMPs) using a combination of Western, proteomic, and epi-fluorescent microscopy techniques. SMP, including 48 and 55 kDa phosphoproteins, was detected in the epithelial cells of mantle, gill, heart, and adductor muscle and linings of arteries and veins. The 48 kDa SMP circulates continuously within the hemolymph, and is present in the immune system hemocytes. It appears to be secreted from hemocytes on induction of shell repair. We suggest that the 48 and 55 kDa proteins are multifunctional and bridge the process of soft tissue repair and shell formation by mediating cellular activities during immune response as well as interacting with the mineral phase during deposition.

Proteomic analysis of differentially expressed proteins in Penaeus vannamei hemocytes upon Taura syndrome virus infection

To understand molecular responses of crustacean hemocytes to virus infection, we applied 2-DE proteomics approach to investigate altered proteins in hemocytes of Penaeus vannamei during Taura syndrome virus (TSV) infection. At 24 h postinfection, quantitative intensity analysis and nano-LC-ESI-MS/MS revealed 11 forms of 8 proteins that were significantly up-regulated, whereas 9 forms of 5 proteins were significantly down-regulated in the infected shrimps. These altered proteins play important roles in host defense (hemocyanin, catalase, carboxylesterase, transglutaminase, and glutathione transferase), signal transduction (14-3-3 zeta), carbohydrate metabolism (acetylglucosamine pyrophosphorylase), cellular structure and integrity (beta-tubulin, beta-actin, tropomyosin, and myosin), and ER-stress response (protein disulfide isomerase). Semiquantitative RT-PCR and Western blot analysis confirmed the upregulation of 14-3-3 at both mRNA and protein levels. Interestingly, several altered protein spots were identified as fragments of hemocyanin. Mass spectrometric analysis showed that the hemocyanin spots at acidic and basic regions represented the C- and N-terminal hemocyanin fragments, respectively. As three-quarters of C-terminal fragments were up-regulated, whereas two-thirds of N-terminal hemocyanin fragments were down-regulated, we therefore hypothesize that C- and N-terminal hemocyanin fragments may have differential roles in hemocytes. Further investigation of these data may lead to better understanding of the molecular responses of crustacean hemocytes to TSV infection.