HIF-1 is involved in the regulation of expression of metallothionein and apoptosis incidence in different oxygen conditions in the white shrimp Litopenaeus vannamei.

The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1ß and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1ß decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.

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

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

First description of a widespread Mytilus trossulus-derived bivalve transmissible cancer lineage in M. trossulus itself.

Two lineages of bivalve transmissible neoplasia (BTN), BTN1 and BTN2, are known in blue mussels Mytilus. Both lineages derive from the Pacific mussel M. trossulus and are identified primarily by their unique genotypes of the nuclear gene EF1α. BTN1 is found in populations of M. trossulus from the Northeast Pacific, while BTN2 has been detected in populations of other Mytilus species worldwide but not in M. trossulus itself. Here we examined M. trossulus from the Sea of Japan (Northwest Pacific) for the presence of BTN. Using hemocytology and flow cytometry of the hemolymph, we confirmed the presence of disseminated neoplasia in our specimens. Cancerous mussels possessed the BTN2 EF1α genotype and two mitochondrial haplotypes with different recombinant control regions, similar to that of common BTN2 lineages. This is the first report of BTN2 in its original host species M. trossulus. A comparison of all available BTN and M. trossulus COI sequences suggests a common and recent origin of BTN2 diversity in populations of M. trossulus outside the Northeast Pacific, possibly in the Northwest Pacific.

The PirB toxin protein from Vibrio parahaemolyticus induces apoptosis in hemocytes of Penaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) is a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. The Pir toxin proteins secreted by a unique strain of Vibrio parahaemolyticus play an essential role in the pathogenesis of AHPND. At present, most studies on the effects of Pir toxin proteins in shrimp focus on digestive tissues or organs such as hepatopancreas, stomach, etc., with none on the immune organs. In the present study, two recombinant Pir toxin proteins (rPirA and rPirB) of V. parahaemolyticus were expressed with rPirB shown to enter shrimp hemocytes. Employing pull-down and LC-MS/MS analysis, GST-rPirB was found to interact with 13 proteins in hemocytes, including histone H3 and histone H4 and among which histone H4 had the highest protein score. Further analysis using GST pull-down and Far-Western blot analysis revealed that rPirB could interact with histone H4. In addition, using the purified nucleosome protein from Drosophila S2 cells, it was found that PirB protein could specifically bind to histones. When flow cytometry was applied, it was observed that the interaction between PirB and histones in shrimp hemocytes induces apoptosis, which results in the dephosphorylation of Serine 10 in histone H3. Collectively, the current study shows that in addition to its effect on the digestive tract of shrimp, the PirB toxin protein interacts with histones to affect the phosphorylation of histone H3-S10, thereby inducing apoptosis.

Enhancement of leukemia-like phenotypes in Drosophila mxc mutant larvae due to activation of the RAS-MAP kinase cascade possibly via down-regulation of DE-cadherin.

Loss of mxc gene function in mature hemocytes of Drosophila mxcmbn1 mutant results in malignant hyperplasia in larval hematopoietic tissues termed lymph glands (LGs) owing to over-proliferation of immature cells. This is a useful model for genetic analyses of leukemia progression. To identify other mutations that deteriorate the hyperplasia, we aimed to investigate whether hyper-activation of common signaling cascade enabled to enhance the phenotypes. Ectopic expression of the constitutively active forms of MAPK signaling factors in the mutant increased the hyperplasia and the number of circulating hemocytes, resulting in the production of LG fragments. The LG phenotype was related to the reduced DE-cadherin level in the mutants. Depletion of Drosophila MCRIP, involved in MAPK-induced silencing of cadherin gene expression, exhibited a similar enhancement of the mxcmbn1 phenotypes. Furthermore, expression of MMP1 proteinase that cleaves the extracellular matrix proteins increased in the mutant larvae harboring MAPK cascade activation. Depletion of Mmp1 and that of pnt (required for Mmp1 expression) suppressed the LG hyperplasia. Hence, we speculated that reduction in DE-cadherin level by either down-regulation of MCRIP or up-regulation of MMP1 was involved in the progression of the tumor phenotype. Our findings can contribute to understanding the mechanism underlying human leukemia progression.

Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

A novel WSSV responsive plasma protein from Litopenaeus vannamei contributes to hemocytes anti-apoptosis.

Litopenaeus vannamei, as an important marine aquaculture species, has attracted more and more attentions in past several years. More recently people got its genome fine mapping, which unveiled a gene treasure. In this study, we have identified a novel trypsin-like protein which came from previous WSSV-infected shrimp plasma iTRAQ data. This protein is a 39 kDa protein with 363 amino acids. It contains a conserved trypsin-domain and could be strongly induced with WSSV infection. Interestingly, knockdown of this protein made shrimps vulnerable to WSSV infection. Further exploration unveiled that this fragility was probably due to the fact that knockdown of this protein could cause shrimp hemocytes apoptosis, which indicated that this protein played key roles in preventing shrimp hemocytes from apoptosis. To further explore how LvTLAP protected shrimp hemocytes from apoptosis, GST pull down assay was applied to screen LvTLAP interacting protein in shrimp plasma. L. vannamei growth and transformation-dependent-like protein (LvGTD-like protein) was identified as a LvTLAP interacting protein, which played proapoptotic roles in cells. Thus, a possible explanation for LvTLAP anti-apoptosis activity was that this protein could block LvGTD-like protein proapoptotic activity to protect shrimp hemocytes from death. In general, our study has uncovered a novel WSSV responsive shrimp plasma protein, which played key roles in shrimp hemocytes anti-apoptosis and shrimp against WSSV infection.

Molecular characterization of troponin T in Scylla paramamosain and its role in Vibrio alginolyticus and white spot syndrome virus (WSSV) infection.

This study investigated the function of Troponin T (TnT) in the mud crab, Scylla paramamosain. The 1952 bp cDNA sequence of TnT was cloned from S. paramamosain using rapid amplification of cDNA ends (RACE) PCR. The quantitative real-time PCR analysis showed that TnT was highly expressed in the muscle and heart of S. paramamosain. Challenging with white spot syndrome virus (WSSV) or Vibrio alginolyticus (VA), two common pathogens that infect mud crabs, enhanced the expression of TnT in S. paramamosain. Knockdown of TnT using TnT-dsRNA led to up-regulating the expression of immune-related genes, such as c-type-lectin, toll-like-receptor, crustin antimicrobial peptide and prophenoloxidase. The cumulative mortality of WSSV- and VA-infected crabs was significantly increased following TnT knockdown. After WSSV or VA infection, TnT knockdown caused a significant reduction in phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC), indicating a regulatory role of TnT in the innate immune response of S. paramamosain to pathogens. Apoptosis of hemocytes was higher in crabs treated with TnT-dsRNA compared with control crabs treated with phosphate-buffered saline. Knockdown of TnT increased apoptosis of hemocytes following VA infection, but reduced hemocyte apoptosis following WSSV infection. In summary, TnT may enhance the immune response of S. paramamosain to WSSV infection by regulating apoptosis, THC, PO activity and SOD activity. And TnT may play a positive role in the immune response against VA infection by regulating apoptosis, THC, SOD activity and PO activity.

Litopenaeus vannamei sirtuin 6 homolog (LvSIRT6) is involved in immune response by modulating hemocytes ROS production and apoptosis.

The histone deacetylase, sirtuin 6 (SIRT6), plays an essential role in the regulation of oxidative stress, mitochondrial function and inflammation in mammals. However, the specific role of SIRT6 in invertebrate immunity has not been reported. Here, we characterized for the first time, a sirtuin 6 homolog in Litopenaeus vannamei (LvSIRT6), with full-length cDNA of 2919 bp and 1536 bp open reading frame (ORF) encoding a putative protein of 511 amino acids, which contains a typical SIR2 domain. Sequence and phylogenetic analysis revealed that LvSIRT6 shares a close evolutionary relationship with SIRT6 from invertebrates. Real-time quantitative PCR analysis of LvSIRT6 transcripts revealed that they were ubiquitously expressed in shrimp and induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting the involvement of LvSIRT6 in shrimp immune response. Moreover, knockdown of LvSIRT6 decreased mitochondrial membrane potential and increased total ROS level in hemocytes, especially upon V. parahaemolyticus challenge. Depletion of LvSIRT6 also increased hemocytes apoptosis in terms of decreased expression of pro-survival LvBcl-2, but increased expression of pro-apoptotic LvBax and LvCytochrome C, coupled with high LvCaspase3/7 activity. Shrimp were rendered more susceptible to V. parahaemolyticus infection upon LvSIRT6 knockdown. Taken together, our present data suggest that LvSIRT6 plays an important role in shrimp immune response by modulating hemocytes ROS production and apoptosis during pathogen challenge.

WSV152 induces apoptosis and promotes viral replication in Litopenaeus vannamei.

Previous studies have indicated that white spot syndrome virus (WSSV) infection induces apoptosis in many shrimp organs. However, the mechanism by which WSSV causes host apoptosis remains largely unknown. In this study, we demonstrated the function of wsv152, the first mitochondrial protein identified as encoded by WSSV. Glutathione S-transferase pulldown and co-immunoprecipitation analysis revealed that wsv152 interacts with the shrimp mitochondrial protein cytochrome c oxidase 5a (COX5a), a subunit of the COX complex. We also found that wsv152 expression significantly increased the rate of apoptosis, suggesting a role of wsv152 in WSSV-induced apoptosis in shrimp. Knockdown of wsv152 in vivo led to downregulation of several apoptosis-related shrimp genes, including cytochrome c, apoptosis-inducing factor and caspase-3. Suppression of wsv152 also resulted in significant reductions in the number of WSSV genome copies in tissues and in the mortality of WSSV-infected shrimp. Together, these results suggest that wsv152 targets host COX5a and is associated with the expression profiles of apoptosis-related shrimp genes. Wsv152 is likely also involved in WSSV-induced apoptosis, thereby facilitating virus infection and playing a complex role in WSSV pathogenesis.

The role of Astakine in Scylla paramamosain against Vibrio alginolyticus and white spot syndrome virus infection.

Astakine is a crucial factor in the proliferation and differentiation of hematopoietic stem cells and is directly involved in hematopoiesis in crustaceans. To assess the role of Astakine in the innate immune system of Scylla paramamosain, the immune responses in healthy and Astakine-inhibited S. paramamosain were investigated in the present study. The RNA transcripts of Astakine were widely distributed in all examined tissues, with significantly higher levels of expression in hemocytes of both healthy and challenged S. paramamosain with Vibrio alginolyticus and WSSV. When Astakine was knocked down by RNA interference technology, immune-related genes, including Janus kinase, prophenoloxidase, hemocyanin, ß-actin, myosin II essential light chain-like protein, signal transducer and activator of transcription, Relish, and C-type-lectin, were significantly down-regulated in hemocytes. The levels of phenoloxidaseactivity (PO), total hemocyte counts (THC) and hemocyte proliferation decreased significantly in hemocytes of Astakine-dsRNA treated S. paramamosain. After being challenged with V. alginolyticus and WSSV, the THC decreased significantly and the levels of hemocyte apoptosis increased significantly in Astakine-dsRNA treated S. paramamosain in comparison with those in infected groups without Astakine-dsRNA treatment. After being challenged with WSSV, the WSSV copies were significantly lower in Astakine-dsRNA treated groups than those in the WSSV infection group, which suggested that knockdown of Astakine was not conductive to WSSV replication and this might be associated with the decreasing THC. The results of survival analysis showed that the survival rate of V. alginolyticus or WSSV infected S. paramamosain decreased significantly following Astakine knockdown. These results suggested that RNA interference of Astakine might weaken the resistance of S. paramamosain to V. alginolyticus or WSSV infection. The weaken resistivity after knockdown Astakine might be related to the changes of important immune-related gene expression, THC, PO activity, proliferation and apoptosis of hemocytes.

The sensing pattern and antitoxic response of Crassostrea gigas against extracellular products of Vibrio splendidus.

Serious juvenile oyster disease induced by pathogenic Vibrio splendidus has resulted in tremendous economic loss, but the molecular mechanisms underlying this killing mechanism remain unclear. The resistance of adult oyster to V. splendidus or its virulence factors might provide a possible access to cognize the interaction between pathogen and host. In the present study, the extracellular products (ECP) from less virulent V. splendidus JZ6 were injected into adult Pacific oyster Crassostrea gigas, and the cellular and humoral immune response induced by ECP were investigated. The phagocytosis rate of hemocytes was significantly up-regulated (30.57%) at 6 h after ECP injection compared with that (21%) of control groups. And significantly high level of ROS production was also observed from 3 h to 12 h in ECP-injected oysters, concomitant with increased apoptosis rate of hemocytes (16.4% in ECP-injected group, p < 0.01) compared with control group (6.7%). By RT-PCR analysis, the expression level of antioxidant CgSOD in hemocytes significantly increased to 6.41-fold of that in control groups (p < 0.01) at 12 h post ECP injection. The expression levels of anti-toxic metalloprotease inhibitors CgTIMP629 and CgTIMP628 were also significantly up-regulated at the early (3-6 h) and late (6-24 h) stage of immune response, respectively. Moreover, after the ECP were incubated with serum proteins isolated from the ECP-injected oysters in vitro, the metalloprotease activity of ECP significantly declined by 21.39%, and less degraded serum proteins were detected by SDS-PAGE. When the primarily cultured hemocytes were stimulated with heat-inactivated ECP or fragments derived from ECP-degraded serum proteins, the expressions of CgTIMP629 (13.64 and 7.03-fold of that in saline group, respectively, p < 0.01) and CgTIMP628 (5.07 and 6.08-fold of that in saline group, respectively, p < 0.01) in hemocytes were all significantly induced. All the results indicated that the adult oysters could launch phagocytosis, antioxidant and anti-toxic response to resist the virulence of ECP, possibly by sensing heterologous ECP and ECP-induced endogenous alarm signals. These results provided a possible clue for the resistance mechanism of adult oysters towards the ECP of less virulent V. splendidus, which might be valuable for exploring strategies for the control of oyster disease.

Effects of dopamine on immune signaling pathway factors, phagocytosis and exocytosis in hemocytes of Litopenaeus vannamei.

Dopamine (DA) is an important neuroendocrine factor, which can act as neurotransmitter and neurohormone. In this study, we explored the immune defense mechanism in Litopenaeus vannamei with injection of dopamine at 10-7 and 10-6 mol shrimp-1, respectively. The genes expressions of dopamine receptor (DAR), G proteins (Gs, Gi, Gq), phagocytosis and exocytosis-related proteins, as well as intracellular signaling pathway factors, and immune defense parameters were measured. Results showed that mRNA expression levels of dopamine receptor D4 (D4), Gi, nuclear transcription factors and exocytosis-related proteins decreased significantly and reached the minimum at 3 h, while the genes expressions of Gs, Gq and phagocytosis-related proteins reached the highest and lowest levels at 3 h and 6 h, respectively. The second messenger synthetases increased significantly in treatment groups within 3 h. Simultaneously, the second messengers and protein kinases shared a similar trend, which were significantly elevated and reached the peak value at 3 h. Ultimately lead to the total hemocyte count (THC), proPO activity and phagocytic activity decreased significantly, reaching minimum values at 3 h, 3 h and 6 h, respectively. While PO activity showed obvious peak changes, which maximum value reached at 3 h. These results suggested that DA receptor could couple with G protein after DA injection and might regulate immunity through cAMP-PKA, DAG-PKC or CaM pathway.

Transcriptome analysis and histopathology of the mud crab (Scylla paramamosain) after air exposure.

The mud crab, Scylla paramamosain, is an economically-important crab in China. Air exposure is an important environmental stressor during mud crab culture and transportation. Adaptive mechanisms responding to air exposure in mud crabs are still poorly understood. In this study, mud crabs were exposed to air for 120 h. Air exposure decreased total hemocyte counts, led to cytological damage, and caused high mortality. Transcriptomic analysis was conducted at 0, 6 and 96 h after air exposure. A total of 3530 differentially expressed genes (DEGs) were identified. DEGs were mainly involved in the oxidative stress response, metabolism, cellular processes, signal transduction, and immune functions. Transcriptomic analysis also revealed that genes of glycolysis and of the tricarboxylic acid cycle were key factors in regulating the mud crab adaptation to air exposure.

Immune response to abamectin-induced oxidative stress in Chinese mitten crab, Eriocheir sinensis.

It is known that abamectin (ABM) inflicts oxidative damage on aquatic animals; however, knowledge about the immune response under pesticide-induced oxidative stress is incomplete. In the present study, several cellular and humoral immune parameters, including total haemocyte counts (THC), lysosomal membrane stability (LMS), activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LZM) were investigated to reveal the effects of ABM exposure on the immune defence mechanisms of the important freshwater crab, Erocheir sinensis. According to the results, a significant increase of THC was found in low concentration groups (0.03 and 0.06 mg/L), while dramatic decreases occurred in high concentration groups (0.12 and 0.24 mg/L) after 96 h of exposure. We also detected significant increases of reactive oxygen species (ROS) in haemocytes at 0.12 and 0.24 mg/L, and there was a dose- and time-dependent decrease of lysosomal membrane stability. These results suggest that the excessive generation of ROS induced by ABM may be leading the massive collapse of lysosomal membrane, which in turn may be causing the sharp drop of haemocyte counts in E. sinensis. The increase of hydrolytic enzymes ACP and AKP at low concentrations and the decrease at high concentrations also indicate an immune response associated with haemocytes status under stress. However, activities of LZM decreased significantly. After injection of Aeromonas hydrophil, mortalities increased under exposure to ABM and were positively related to ABM concentration. These results confirm that ABM exposure has the ability to impair immune defence and result in the host's susceptibility to pathogens.

Exogenous Ca2+ mitigates the toxic effects of TiO2 nanoparticles on phagocytosis, cell viability, and apoptosis in haemocytes of a marine bivalve mollusk, Tegillarca granosa.

Phagocytosis suppression induced by nanoparticles (NPs) exposure is increasingly reported in marine species. However, the mechanisms underlying this impact remain poorly understood. In order to improve our present understanding of the immunotoxicity of NPs, acute (96 h) TiO2 NP exposure and rescue trials via exogenous supply of Ca2+ were performed in the blood clam, Tegillarca granosa. The results show that the phagocytosis rate, cell viability, and intracellular Ca2+ concentration of haemocytes were significantly suppressed, whereas the intracellular ROS concentration of haemocytes significantly increased upon nTiO2 exposure. Exposure to nTiO2 also led to the significant downregulation of Caspase-3, Caspase-6, apoptosis regulator Bcl-2, Bcl-2-associated X, calmodulin kinase II, and calmodulin kinase kinase II. Furthermore, the toxic impacts of nTiO2 were partially mitigated by the addition of exogenous Ca2+, as indicated by the recovery tendency in almost all the measured parameters. The present study indicates that Ca2+ signaling could be one of the key pathways through which nTiO2 attacks phagocytosis.

Histologic Findings in Captive American Horseshoe Crabs (Limulus polyphemus).

Histopathology of 61 captive American horseshoe crabs (HSCs; Limulus polyphemus) is reviewed. HSC organs evaluated histologically included body wall (chitin, epidermis, dermis, and skeletal muscle), hepatopancreas, gut, gonads, book gills, eyes, heart, brain, and coxal gland. In descending order, lesions were most frequently identified in compound eye, body wall, book gills, hepatopancreas, chitinous gut, nonchitinous gut, heart, and brain; lesions were not observed in coxal gland or gonads. Hemocytes (also called amoebocytes) surrounded infectious agents and occluded ulcers. Large hemocyte aggregates had a central eosinophilic coagulum (ie, hemocyte coagulum). Cutaneous ulceration (34/60 cases), branchitis (29/48 cases), and ophthalmitis (17/20 cases) were common lesions and consistently associated with fungi, which were invasive into subjacent tissues, and/or bacteria, which were usually superficial. Fungal culture was performed in 3 cases and isolated Fusarium spp., although fungal morphology varied and multiple fungal species may have been present. Presumptive green algae were associated with ulceration in 1 case with minimal to no inflammation. Presumptive cyanobacteria were identified within a biofilm overlying the gills in 4 of 48 cases and were not invasive. Multifocal, random hepatopancreatitis was identified in 16 of 57 cases, 10 of which were associated with bacteria. Metacercarial cysts were identified in 25 of 61 cases and associated with minimal to no inflammation. Depleted eosinophilic globules in hepatopancreatic interstitial cells were interpreted as decreased nutritional status in 12 of 57 cases.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

SpBcl2 promotes WSSV infection by suppressing apoptotic activity of hemocytes in mud crab, Scylla paramamosain.

White spot syndrome virus (WSSV) is one of the most virulent and widespread pathogens that infect almost all marine crustaceans and therefore cause huge economic losses in aquaculture. The Bcl2 protein plays a key role in the mitochondrial apoptosis pathway, which is a crucial immune response in invertebrates. However, the role of Bcl2 in apoptosis and immunoregulation in mud crab, Scylla paramamosain, is poorly understood. Here, the Bcl2 homolog (SpBcl2) in S. paramamosain was cloned and its role in WSSV infection explored. The expression of SpBcl2 increased at both the transcriptional level and post-transcriptional level after WSSV infection, while the hemocytes apoptosis decreased significantly. Furthermore, there was increase in the level of cytochrome c coupled with an upregulation in the expression of SpBcl2. These results indicated that SpBcl2 suppressed apoptosis by preventing the release of cytochrome c from mitochondria, thereby promoting WSSV replication in mud crab. The findings here therefore provide novel insight into the immune response of mud crabs to WSSV infection.

Exposure to a nanosilver-enabled consumer product results in similar accumulation and toxicity of silver nanoparticles in the marine mussel Mytilus galloprovincialis.

The incorporation of silver nanoparticles (AgNPs) in commercial products is increasing rapidly. The consequent release of AgNPs into domestic and industrial wastewater raises environmental concerns due to their anti-microbial properties and toxicity to non-target aquatic organisms. The aim of the present study was to investigate the effects of nanArgen™ (Nanotek S.A.), a AgNP-enabled consumer product, in the marine bivalve Mytilus galloprovincialis. Two environmentally relevant concentrations of nanArgen™ (1 and 10 µg/L) were tested in vivo for 96 h, and Ag was quantified in mussel soft tissue and natural seawater (NSW). nanArgen™ suspensions were characterized via TEM, SEM, EDS, DLS, and UV-vis optical analysis. Several molecular and biochemical responses were investigated in exposed mussels: lysosomal membrane stability by Neutral Red Retention Time (NRRT) assay; micronucleus (MN) frequency in hemocytes; metallothionein (MT) protein content and gene expression (mt10 and mt20); catalase (CAT) and glutathione-S-transferase (GST) activities; malondialdehyde (MDA) accumulation in digestive glands; and efflux activity of ATP-binding cassette transport proteins (ABC) in gill biopsies. SEM, TEM and DLS analyses confirmed the presence of well-defined AgNPs in nanArgen™ which were roughly spherical with an average particle size of approx. 30 ± 10 nm. DLS analysis revealed the formation of AgNP aggregates in nanArgen™ suspension in NSW (Z-average of 547.80 ± 90.23 nm; PDI of 0.044). A significant concentration-dependent accumulation of Ag was found in mussels' whole soft tissue in agreement with a concentration-dependent decrease in NRRT and an increase of MN frequency in hemocytes and GST activities in digestive glands. A significant increase in MDA levels and MT via both molecular and biochemical tests, were also observed but only at the highest nanArgen™ concentration (10 µg/L). No changes were observed in CAT activities. ABC efflux activities in gill biopsies showed a significant decrease (p < 0.05) only at the lowest concentration (1 µg/L). On such basis, nanArgen™ is shown to be able to induce toxicity and Ag accumulation in marine mussels similarly to AgNPs and in short-term exposure conditions at environmentally relevant concentrations. AgNP-enabled products, instead of pristine AgNPs, should be the focus of future ecotoxicity studies in order to address any risks associated to their widespread use, disposal and uncontrolled release into the aquatic environment for non target species.