Hemocyte Lysate Research Articles

Immune responses of hemocytes in the blood clam Tegillarca granosa in response to in vivo Vibrio harveyi infection

Aquaculture of the blood clam Tegillarca granosa accounts for approximately 50% of Arcidae (ark shell) production in China. Vibrio infection severely threatens the sustainability of the clam aquaculture industry. Exposure to Vibrio induces an immune response in blood clams. However, the underlying mechanism remains poorly understood. In this study, immune responses of hemocytes in blood clams were detected after Vibrio infection; the immersion method was used in vivo to mimic the clam's natural infection process. After 24 h of exposure to Vibrio infection, the Vibrio load in hemolymph fluid in both the treatment Ⅰ (25,033.33 ± 19,563.11 CFU/mL) and treatment Ⅱ (122,163.33 ± 194,409.49 CFU/mL) groups were significantly higher, than that in the control group (13.67 ± 37.73 CFU/mL) (P < 0.05). Correspondingly, the production of intracellular reactive oxygen species was approximately 1.40 (treatment Ⅰ) and 2.12 (treatment Ⅱ) fold higher than that in the control group (P < 0.05), and the induced DNA damage showed a similar trend (P < 0.05). Vibrio infection also significantly increased lysozyme content, adenosine triphosphate content, and peroxidase isozyme activity, in both the serum and hemocyte lysates (P < 0.05). The expression of immune-associated genes (ABCA3, c-Myc, Caspase 3, and HSP70) was upregulated under infection conditions. The phagocytic activity was approximately 1.99 (treatment Ⅰ) and 2.57 (treatment Ⅱ) fold that in control clams (P < 0.05). In addition, the total hemocyte count and red granulocyte percentage both significantly decreased by approximately 75–90% after Vibrio infection. These results provided novel insights into the mechanism of hemocyte immunity in T. granosa against Vibrio infection, which may aid in the future prevention and control of Vibrio infection in vivo.

Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrioparahaemolyticus

Vibrio parahaemolyticus (Vpara) is the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS) in shrimp. Shrimp, like other invertebrates, lack an adaptive immune system and depend solely on innate immunity against invading pathogens. To better understand the defense mechanisms of shrimp to this problematic pathogen, we evaluated the changes in hematology, immunology and biochemical values of the hemolymph from shrimp challenged with V. parahaemolyticus up to 8 days post-challenge. Thirty-six shrimp (12 g) were distributed in 9 tanks (75 L), divided into three groups (non-challenged, challenged with 5 × 102 cfu/shrimp and challenged with 1 × 103 cfu/shrimp) in triplicate. Pacific white shrimp, Litopenaeus vannamei, were administered an inoculum of V. parahaemolyticus under the shell between the 5th and 6th abdominal segment to assess cellular and humoral immune responses. Total hemocyte count (THC) significantly decreased in shrimp challenged with Vpara at 6 h, 12 h and 24 h-post infection. Hemocyte lysate phenoloxidase (PO) activity in Vpara-challenged shrimp at 48 h post challenge was significantly increased compared to that of control shrimp. No significant differences were observed in total plasma protein between plasma from control and Vpara-challenged shrimp. However, shrimp challenged with 5 × 102, and 1 × 103 cfu/shrimp had significantly lower hemocyanin at 6 h and 48 h sampling point, respectively. At 24 h post-challenge, the ≥140 kDa and 70 kDa bands from SDS-PAGE of hemocyanin-concentrated hemolymph lysate samples showed a higher and lower intensity, respectively, in Vpara-challenged group than those of the control group. Plasma from Vpara-challenged shrimp at 6 h and 12 h-post infection significantly suppressed V. parahaemolyticus growth. However, significantly less bacterial growth suppression was observed in plasma of shrimp challenged with higher dose compared to control shrimp at the 192 h post-challenge point. Plasma chemistry parameters did not significantly differ among treatments. The changes observed in hemolymph parameters may be useful indicators of the health status of shrimp.

Heat Shock Protein 70 Is a Damage-Associated Molecular Pattern That by Binding to Lipopolysaccharide and β-1,3-Glucan-Binding Protein Activates the Prophenoloxidase System in Shrimp.

Recent studies have initiated a paradigm shift in understanding heat shock protein 70 (HSP70) functions in the shrimp immune system. However, the mechanism by which Litopenaeus vannamei (Lv)HSP70 modulates the innate immune response remains unclear. This study shows that LvHSP70 binds to the pattern recognition receptor LPS and β-1,3-glucan-binding protein (LvLGBP), and subsequently leads to the activation of the prophenoloxidase system. Injection of shrimp with rLvHSP70 significantly (p < 0.05) upregulated the gene and protein expression of the key pattern recognition receptor LvLGBP. A coimmunoprecipitation and ELISA-based binding assay strongly confirmed the binding of LvHSP70 to LvLGBP at polysaccharide recognition motifs (PLS motifs) with a K d of 4.44 μM and its competitive binding with LPS (IC50) is 8.036 μM. Conversely, LPS efficiently competed with LvHSP70 for binding to LvLGBP in a concentration-dependent manner with an IC50 of 7.662 μM, indicating that both are ligands of LvLGBP and likely bind at the same site. Binding of LvHSP70 to LvLGBP highly activated phenoloxidase activity in shrimp hemocyte lysate supernatants. Gene silencing of LvLGBP impaired the activation of phenoloxidase activity in shrimp by rLvHSP70, indicating that LvHSP70-LvLGBP interaction was essential for stimulating the immune cascade. Taken together, these results demonstrated that LvHSP70 is a ligand of LvLGBP similar to LPS and acts as a damage-associated molecular pattern to modulate the shrimp immune system via the prophenoloxidase system, eventually leading to the production of melanin and toxic reactive intermediates against invading pathogens.

Cellular and molecular immune response and production performance of Indian white shrimp Penaeus indicus (H. Milne-Edwards, 1837), reared in a biofloc-based system with different protein levels of feed

The present study focuses on the immunity and growth of Penaeus indicus fed with varying protein levels (25%, 30%, and 35%) in a biofloc based rearing system. A 120 days growth trial was carried out using juvenile Penaeus indicus (0.71 ± 0.01) with dietary protein level, 25% (LP), 30% (MP), and 35% (HP), and a control diet-fed with 35% acted as control group resulting in 4 treatments each with four replicates and were randomly assigned 16 tank units (7500 L each). A combination of different carbon sources (molasses, wheat flour, and rice bran in 2:1:1 ratio), yeast and a probiotic (Bacillus sp.) consortium were used for the development of biofloc. At the end of the trial, the growth parameters of shrimps viz., initial weight, feed conversion ratio (FCR), and daily growth coefficient (DGC) were computed. The results indicated that shrimp fed with medium (30%) protein (MP) diet recorded significantly (P < 0.05) improved growth performance compared to high protein fed group (35%) and low protein (25%) fed group (LP) in a biofloc system and control group (35%). The immunological parameters such as hemagglutination activity (HA) assay, serum protein, lysozyme, phenol oxidase (PO), and inhibition of superoxide dismutase (SOD) activity were observed in serum, plasma, and hemocyte lysate supernatant (HLS). The HA activity, PO activity in plasma was found to be higher in high protein fed animals, whereas medium protein resulted in enhanced PO activity in serum. Similarly, lysozyme and SOD were inhibited well in high protein fed animals compared to the low protein fed group. The vital immune genes's mRNA profiling showed a potential rise in the expressional pattern in MP and HP treatments compared to LP and control. BGBP (beta-1,3-glucan binding protein) and hemocyanin mRNA transcript levels were highly upregulated in the HP (5 fold) and moderately expressed in MP (2 fold) and LP (1–2 fold). The transcripts of peroxinectin, antimicrobial peptides like crustin showed significant upregulation in HP followed by in MP and LP and control. Likewise, other immune genes, such as SOD, prophenoloxidase (proPO), showed a similar trend in a marginal way, indicating immunomodulation in the biofloc groups. This study suggested that biofloc with high protein (35%) supplementation can substantially enhance the immune response of shrimps, although medium protein level (30%) is optimum for improving the survival, growth, and in turn economic return in Indian white shrimp.

Effect of microencapsulated phenolic compound extracts of Maclura tinctoria (L.) Steud on growth performance and humoral immunity markers of white leg shrimp (Litopenaeus vannamei, Boone, 1931) juveniles

Aim of study: The effect of microencapsulated phenolic compound extracts of Maclura tinctoria (MTBE) on growth performance and humoral immunity markers of the white leg shrimp Litopenaeus vannamei juveniles (0.5 ± 0.2 g initial weight) was studied.Area of study: M. tinctoria was collected from Hampolol, Campeche, and Arroyo del Agua, Culiacan, Sinaloa, Mexico.Material and methods: Three MTBE inclusions (0.5, 1, and 2.5 g MTBE/kg, Purina®) were compared with a control commercial feed (Purina®) during 30 days. Nine phenolic acids, nine flavonols, four dihydro-flavonoids, four flavones, and seven unidentified phenolic compounds were determined fin the MTBE using a Perkin Elmer® HPLC chromatograph and diode array-detection.Main results: The mean concentrations of total phenolic compounds, total flavonoid compounds, and condensed tannins were 198.05 ± 5.59 mg gallic acid equivalent (GAE) g-1 dw, 78.57 ± 1.80 quercetin equivalent g-1, and 28.32 ± 0.33 mg epicatechin equivalent g-1, respectively. The ferric reducing antioxidant power and the total antioxidant capacity, respectively, averaged 28.32 mg GAE mL-1 and 10.9 mg ascorbic acid equivalent mL-1. Survival, weight gain, and specific growth rate of L. vannamei were similar among the experimental diets. The dietary inclusion of MTBE at 0.5 g/kg of food showed significant higher (p &lt; 0.05) plasma hemocyte lysate protein (1.35 ± 0.055 µg mL-1), prophenoloxidase (0.47 ± 0.15, Abs. 492 nm), and superoxide anion (O2.-) activity (0.21 ± 0.07, Abs. 630 nm).Research highlights: The supplementation of MTBE at 0.5 g/kg of food could be considered as a potential alternative additive for L. vannamei diet in the juvenile production, since it improved the response of the humoral immunity markers at post larval life stages, when cultivated shrimp are more susceptible to be infected by pathogens.

Effects of a mixture of glyphosate, 17α-ethynylestradiol and amyl salicylate on cellular and biochemical parameters of the mussel Mytilus galloprovincialis

In this study the effects of a mixture of glyphosate (herbicide), 17a-ethinylestradiol (synthetic estrogen) and amyl salicylate (fragrance) to the mussel Mytilus galloprovincialis were evaluated. Mussels were exposed for 7 days to two realistic concentrations of the mixture (10 and 100 ng/L) and the effects on total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate lysozyme activity were measured. In addition, superoxide dismutase, catalase, acetylcholinesterase, glutathione-S-transferase and glutathione reductase activities were measured in gills and digestive gland. The survival-in-air test was also performed. Results demonstrated that the mixture affected both cellular and biochemical biomarkers, but not tolerance to aerial exposure of M. galloprovincialis. The negative effects recorded in this study suggested that more efforts should be done to assess the ecotoxicological risks posed by contaminant mixture to aquatic invertebrates.

Biochemical characterization of a hemolymph phenoloxidase and its endogenous inhibitor in the larvae of an invasive moth, Cydalima perspectalis Walker (Lepidoptera: Crambidae)

• A phenoloxidase was purified from the hemolymph of C. perspectalis with the molecular weight of ~ 78.7. • The optimal values of 7 and 30–35 °C were evaluated for pH and temperature dependency of the purified PO. • Syntehtic and endogenous inhibitors significantly affected PO activity both in vitro and in vivo. Phenoloxidase system is a crucial component of insect innate immunity which contribute to oxidize phenols to quinones and to generate reactive oxygen and nitrogen intermediates. In the current study, a phenoloxidase (PO) was extracted by hemocyte lysate preparation and purified through ammonium sulfate precipitation, Sepharyl G-100, and DEAE-Cellulose fast flow columns. At the end of the purification process, an enzyme was purified with a specific activity of 0.462 U/mg protein, recovery of 40.47%, purification fold of 14.43 and molecular weight of ~78.7 kDa. The optimal activity was recorded at pH 7 while the optimal temperature was recorded at 30–35 °C, 35 °C and 25–35 °C, using L-dihydroxyphenylalanine, hydroquinone, and pyrocatechol, respectively. The highest V max of PO was obtained using L-dopa while the lowest K m value was gained using hydroquinone. Among used synthetic inhibitors of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N, N,N0,N0-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA), EDTA and DTC inhibited more than 60% of the enzyme activity. Moreover, an endogenous phenoloxidase inhibitor (POI) was purified by twice processing of Sepharyl G-100 chromatography with the molecular weight of ~52 kDa. The IC 50 of POI was found 31.3 mg against the purified PO of C. perspectalis and led to a higher value of K m . Finally, larval injection by DTC and POI demonstrated significant inhibition of PO over the time of exposure. A comprehensive understanding of insect’s POs may better clarify the ways of their survival within infected areas and to potentially target them by specific and selective compounds.

Identification of a mannose‐binding lectin‐like protein recognized by the anti‐CD25 antibody in haemocytes fromCherax quadricarinatus

The crustacean haemolymph contains three main cell populations; however, it is not clear which mechanisms participate in the regulation of cells related to innate immunity. This work aimed to identify potential interleukin-like receptors that could regulate cellular responses in Cherax quadricarinatus. By histochemical analysis with murine anti-CD25 staining (targeting the α-chain of the IL-2 receptor), we identified that this antibody recognizes cytoplasmic granules in semigranular and granular haemocytes. In haemocytes stimulated with phorbol myristate acetate (PMA), increased fluorescence was observed in these cytoplasmic granules, whereas staining with a human IL-2 antibody after stimulation with 1–10 ng/ml PMA revealed no overexpression of the receptor or oxidative burst in haemocytes. Two-dimensional Western blot analysis of haemocyte lysates showed that anti-CD25 identified a 27.4-kDa protein with an isoelectric point (pI) of 7.7 and a 46-kDa protein with a pI of 6.9. De novo sequencing of these proteins identified that they had 32% homology with a mannose-binding lectin (MBL) from Pacifastacus leniusculus. Our results indicate that a mannose-binding lectin-like protein could exert a protective effect that prevents damage from other activated immune responses.

Comparative study on phenoloxidase activity of biofloc-reared pacific white shrimp Penaeus vannamei and Indian white shrimp Penaeus indicus on graded protein diet

Shrimp aquaculture demands technological advancement that boosts production and environmental sustainability. This study evaluated and compared the growth performance and innate immunity of two species of shrimp (Pacific White shrimp Penaeus vannamei and India white shrimp Penaeus indicus) reared under biofloc technology with different dietary protein levels (Low-25%, Medium-30% and High-35%). As prophenoloxidase (proPO) activity is an important parameter in immune response of shrimp, we focused on the dynamics of prophenoloxidase induced by three different activators (Sodium dodecyle sulfate, (SDS, Trypsin & Laminarin). At the end of the experiment, the effect of activators on proPO activity in serum, plasma and hemocyte lysate (HLS) was evaluated in both the species under conventional and biofloc systems with graded proteins. This experiment lasted for a month as nursery rearing followed by 60 days of grow-out culture. There was no significant difference (p < 0.05) in growth performances of both the species reared under biofloc, whereas control group exhibited lower growth. Significantly higher average body weight (ABW), survival and specific growth rate (SGR) were observed in medium protein groups reared under biofloc than the other biofloc groups and control. The medium protein groups also showed significantly lower (p < 0.01) TAN, NO2–N, and NO3–N levels compared to other treatments and the control exhibited considerably higher TAN, NO2–N and NO3–N levels. The proPO activity in serum of both the shrimps were found to be significantly higher (p < 0.05) than the control in which highest proPO activity was observed in SDS activated groups irrespective of the graded protein levels. A similar trend was observed in the plasma of P. indicus in which SDS activated group fed with high protein level showed highest proPO activity, whereas in plasma of P. vannamei trypsin activated groups fed with low protein level showed higher proPO activity than SDS and laminarin activated groups. The HLS of P. vannamei showed highest proPO functioning in trypsin activated group fed with high protein level, whereas in P. indicus, the highest proPO activity was observed in SDS activated group fed with high protein diet. Though in many cases, the groups fed with high protein diet showed highest proPO activity, the low protein and the medium protein groups also exhibited upregulation in the immune response parameter reared under biofloc irrespective of the species. SOD activity of the hemocytes were influenced by different dietary protein levels and better inhibition was observed in HP when compared to control and other treated groups. Thus the native non-SPF Indian white shrimp P. indicus has comparable potential as the SPF Pacific white shrimp P. vannamei in terms of innate immunity, immune response, and growth when reared under biofloc conditions.

Ecotoxicological hazard of a mixture of glyphosate and aminomethylphosphonic acid to the mussel Mytilus galloprovincialis (Lamarck 1819)

Assessment of the effects of chemical mixtures is a very important objective of the ecotoxicological risk assessment. This study was aimed at evaluating for the first time the effects of a mixture of glyphosate and its main breakdown product aminomethylphosphonic acid (AMPA) on various biomarkers in the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days to either 100 µg/L of glyphosate, 100 µg/L of AMPA or a mixture of both (100 + 100 µg/L). Various haemocyte parameters, such as total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate acid phosphatase activities were measured. In addition, the effects of exposure on the activity of antioxidant enzymes, acetylcholinesterase and glutathione-S-transferase were evaluated in gills and digestive gland from mussels. On the whole, this study demonstrated that the variables considered in the experimental plan, namely treatment, exposure time and their interaction, affect significantly biomarker responses in M. galloprovincialis. The effects of the mixture were comparable to those of the individual compounds, whereas their synergistic effects were occasionally observed, under the experimental conditions tested at least.

Salmo Salar glucocorticoid receptors analysis of alternative splicing variants under stress conditions

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F1-ATP synthase β-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.

Cell surface transglutaminase required for nodavirus entry into freshwater prawn hemocytes

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F1-ATP synthase β-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.

Glyphosate affects haemocyte parameters in the clam Ruditapes philippinarum

This study was aimed at evaluating the effects of glyphosate on haemocyte parameters of the clam Ruditapes philippinarum. Clams were exposed for 7 days to differing glyphosate concentrations (10, 100 and 1000 μg/L) and various haemocyte parameters were measured, such as total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities. Glyphosate reduced significantly THC values, while increased both diameter and volume of haemocytes. Exposure to the highest herbicide concentration increased significantly haemocyte proliferation. No significant effects on haemolymph lactate dehydrogenase and haemocyte lysate lysozyme activities were observed, whereas haemocyte lysate acid phosphatase activity resulted significantly increased in clams exposed at 100 and 1000 μg/L. On the whole, this study demonstrated that glyphosate influenced significantly haemocyte parameters in R. philippinarum.

The impact of holding stressors on the immune function and haemolymph biochemistry of Southern Rock Lobsters (Jasus edwardsii)

Lobsters are fished world-wide due to their status as a high value, luxury seafood. A large proportion of the product is sold via live export, with lobsters subject to a range of stressors during holding post-capture. Improving the current understanding of the immune response to these stressors assists in improving efficiency and reducing loss in the chain between capture and consumption. In this study, the immune status of four treatment groups of Southern Rock Lobster (Jasus edwardsii) were studied: controls recently landed from a fishing boat, lobsters displaying advanced shell necrosis, lobsters in an unexplained moribund state and lobsters held in a processing facility for 10 weeks in standard conditions (i.e. high density, fasted). A total of 15 immune parameters and 19 haemolymph biochemical parameters were assayed. Phenoloxidase activity was only sporadically observed in haemocyte lysate and was consistently observed at a low level in the plasma with no difference between treatments for either. Haemocyte lysate prophenoloxidase activity was detected in most individuals, with no differences found between treatments. Prophenoloxidase in the plasma showed the highest level of activity, with the shell necrosis treatment demonstrating an elevated activity level relative to the other three treatments. Cell viability was not affected in any treatment. Lobsters with shell necrosis had a reduced capacity for phagocytosis, a significantly higher total haemocyte count, fewer hyalinocytes and more granulocytes and semigranulocytes. Fasted lobsters showed an opposite shift, with significantly more hyalinocytes compared to the other treatments and very few granulocytes and semigranulocytes. The balance of a range electrolytes, minerals metabolites and enzymes were affected in shell necrosis and fasted treatments, raising them as potential markers for immunocompromised lobsters. Multivariate analysis of all assayed parameters showed that all individuals in the necrosis treatment showed a similar, distinct immune response and that the fasted treatment, along with one control and one moribund individual, showed a separate intermediate response. The remainder of the control and moribund lobsters demonstrated a distinct “non-response” in comparison. These results offer a characterisation of the physiological response to common challenges during post-capture holding of rock lobsters, demonstrating the differential response to pathogenic bacterial infection, long term fasting, non-specific moribundity and the stress of capture and transport.

Effects of aminomethylphosphonic acid, the main breakdown product of glyphosate, on cellular and biochemical parameters of the mussel Mytilus galloprovincialis

The effects of the breakdown products of herbicides on aquatic species are largely unknown. In a recent study, we evaluated the effects of glyphosate on the mussel Mytilus galloprovincialis. This study was designed to evaluate for the first time the impact of aminomethylphosphonic acid (AMPA) - the main breakdown product of glyphosate - on cellular and biochemical parameters of the mussel Mytilus galloprovincialis. Bivalves were exposed for 7, 14 and 21 days to 1, 10 and 100 μg/L of AMPA and various biomarkers were measured in haemolymph (total haemocyte counts, haemocyte diameter and volume, haemolymph pH, haemocyte proliferation, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities), as well as in gills and digestive gland (superoxide dismutase, catalase, glutathione S-transferase and acetylcholinesterase activities). AMPA concentrations in seawater samples from the experimental tanks were also measured in order to correlate the biomarker responses of mussels with their exposure to the actual concentrations of AMPA. The MANOVA analysis demonstrated that the experimental variables considered (exposure dose, exposure duration, and their interaction) affected significantly biomarker responses. Nevertheless, the two-way ANOVA analysis revealed significant effects of AMPA on most of the biomarkers measured. The overall results of this study demonstrated that AMPA can affect cellular and biochemical parameters in mussels, similarly to glyphosate.

A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.

Immune status of the spiny lobster Jasus edwardsii with tail fan necrosis.

Tail fan necrosis (TFN), a disorder commonly found in some populations of commercially fished and cultured lobsters, is thought to be initiated by injuries caused by handling and containment. The unsightly appearance of affected lobster tails significantly lowers their commercial value. Knowledge about TFN is limited. In this study we describe the morphological features of TFN and apply 6 common methods for evaluating the immune status of wild-caught Australasian red spiny lobsters Jasus edwardsii with and without TFN. The disease was more frequent in uropods than in telsons of the tail fan, and more extensive on the ventral versus the dorsal surfaces of the tail fan. Missing appendages (i.e. antenna, pereiopod or pleopod) were significantly more common and greater in number for individual lobsters affected with TFN versus those without, possibly as a result of handling in the fishery or as an indirect effect of the disease. Two immune parameters, total haemocyte count and phenoloxidase activity in the haemocyte lysate supernatant (HLS), were significantly compromised in lobsters with TFN. No differences were found in the other immune parameters, i.e. haemocyte viability, haemolymph bacterial count and the protein content of haemolymph plasma and HLS. The results are consistent with injury sustained during prior capture and handling that initiates TFN in these natural caught lobsters. These results raise some potential concerns about the fitness of lobsters in natural populations that are affected by TFN, and some potential solutions are proposed.

Ecotoxicological risk assessment for the herbicide glyphosate to non-target aquatic species: A case study with the mussel Mytilus galloprovincialis.

Glyphosate (GLY) is one of the most used herbicide worldwide. Considering that information concerning the impact of GLY on bivalves is scarce, in this study we evaluated for the first time the effects of environmentally realistic concentrations of GLY (10, 100 and 1000μg/L) to the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days and several biomarkers were measured in haemocytes/haemolymph (total haemocyte counts, haemocyte diameter and volume, haemolymph pH, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities), as well as in gills and digestive gland (antioxidant enzyme and acetylcholinesterase activities). The concentrations of GLY and its main metabolite aminomethylphosphonic acid in the experimental tanks were also measured. The MANOVA analysis demonstrated that the experimental variables considered (exposure concentration, exposure duration, and their interaction) affected significantly biomarker responses. In addition, the two-way ANOVA analysis indicated that GLY was able to affect most of the cellular parameters measured, whereas antioxidant enzyme activities resulted to be influenced moderately. Interestingly, exposure to GLY reduced significantly acetylcholinesterase activity in gills. Although preliminary, the results of this study demonstrated that GLY can affect both cellular and biochemical parameters in mussels, highlighting a potential risk for aquatic invertebrates.

Immune response of anti-lectin Pjlec antibody in freshwater crab Paratelphusa jacquemontii

Sialic acid specific lectin Pjlec isolated from serum of the freshwater crab Paratelphusa jacquemontii served as an antigen for the production of immunoglobulin (Ig) in Balb/c mice sera. Enzyme-linked immunosorbent assay (ELISA) of mice anti-sera with Pjlec lectin affirmed the induction and production of antibody. Anti-Pjlec antibody was isolated from the antisera of mice by Protein A Sepharose affinity chromatography and checked for purity by immunoblot with lectin. Mass spectrometry (MS/MS) of papain digethe peptide sequence of antigen binding fragment (Fab) and fragment crystallizable (Fc). Coatingsted anti-Pjlec revealed of anti-Pjlec to the target cell, rabbit erythrocyte failed to enhance in vitro phagocytosis in the crab. However, inoculation of anti-Pjlec in the hemolymph of the crab elicited in vitro phagocytosis. Proteins in hemocyte lysate supernatant (HLS) were separated by electrophoresis failed to immunoblot with Pjlec or anti-Pjlec. Peptide sequences of trypsin digested lectin protein appeared homologous to deuterostome chordate. The protostome crab that lack the ability to synthesize sialic acid however bind to sialic acid a deuterostome sugar to suggest the complexity in innate immune system of invertebrates. The application of lectin and its antibody require further study on application of pathological conditions associated with alterations in sialylated cell surface.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).