Hemocyte cell types of the Cortes Geoduck, Panopea globosa (Dall 1898), from the Gulf of California, Mexico.

The geoduck Panopea globosa is an endemic and economic valuable species from the Mexican Northwest coast whose biology has been little studied. No information exists about their hemocytes to date, which is highly important to assess the welfare of wild and cultured organisms. In this study, hemocytes of adult P. globosa were characterized at the morphological, ultrastructural and functional level. The mean number of hemocytes in the hemolymph of P. globosa was 6 × 105 ± 2 × 105 cells mL-1. The cells were identified as granulocytes (Gr) and hyalinocytes (H). The former accounted for 28% of adhered cells in the hemolymph, measured 6-18 µm, showed numerous basophilic granules in the cytoplasm, with round and eccentric nuclei, and a nucleus:cytoplasm ratio of 0.44 ± 0.01. Hyalinocytes were the most abundant cells in the hemolymph of P. globosa (72% adhered cells) and were subdivided, according to their size, in small (Hs) 4-12 µm and large (HL) 6-18 µm. Hyalinocytes were eosinophilic round or ovoid cells with a central or eccentric nucleus, few or no granules in the cytoplasm and similar nucleus:cytoplasm ratio (Hs: 0.63 and HL: 061). Lysosomes and lipids were observed in Gr, while carbohydrates were the most abundant energy substrate in H. Both hemocytic cell types, mainly Gr, were capable to ingest particles and yield superoxide (P > 0.05). The present study shows for the first time the cell types, abundance and immune activities of hemocytes present in the hemolymph of P. globosa. This information provides a useful baseline to carry out further research on the cellular immune response of the clam to potential pathogens or changes in environmental factors.

Immune and biochemical responses in hemolymph and gills of the Patagonian freshwater mussel Diplodon chilensis, against two microbiological challenges: Saccharomyces cerevisiae and Escherichia coli.

Immune cell characterization, immunological response and the associated gill oxidative balance were studied in the Patagonian freshwater mussel, Diplodon chilensis, using two microbiological immunostimulant models: Saccharomyces cerevisiae and Escherichia coli. Mussels were collected out of the breeding season in Paimún Lake and acclimated in the laboratory. Two exposure experiments were performed during two consecutive weeks: (1) mussels challenged with 500 yeast cells mL-1; and (2) mussels challenged with 1000 bacteria cells mL-1. Microorganisms were added in the water every two days, alternating with 6000 lyophilized cells of the green algae Scenedesmus vacuolatus mL-1. A control group, fed with S. vacuolatus, was set for each treatment. Morphological cell characterization was carried out in adherent hemocytes of D. chilensis hemolymph under control conditions. The most important cell type observed were the hyalinocytes (representing ca. 98% of the circulating cells), agranular cells with non-central polymorphic nucleus surrounded by cytoplasm; granulocytes (cells with cytoplasmic granules and non-central rounded nucleus) represented ca. 2%. Another two cell types were occasionally detected, binucleated hyalinocytes and hemoblast-like cells but were not considered for the analyses. Both adherent hyalinocytes and granulocytes exhibit phagocytic activity towards Congo red stained yeast, which was two-fold higher in granulocytes than in hyalinocytes, regardless of the applied challenge. Total hemocyte counts were diminished in mussels challenged with S. cerevisiae or E. coli. Hydrolytic and defense cellular enzyme activities were analyzed only for hyalinocytes. Both, S. cerevisiae and E. coli increased acid phosphatase activity. E. coli challenge diminished hemocyte lysosomal membrane stability and increased humoral phenoloxidase activity, while S. cerevisiae challenge did not affect any of these variables. Mussels challenged with E. coli showed increased gill antioxidant response without oxidative damage, while those challenged with S. cerevisiae showed no change in these variables.

A potential microRNA regulation of immune-related genes in invertebrate haemocytes.

Bivalve mollusks have been employed as sentinel organisms in environmental health programs due to their sedentary lifestyle, filter-feeding behavior and their ability to accumulate pathogens or toxin molecules inside tissues. Endocrine disrupting chemicals (EDCs) can be up taken and bioaccumulated, and due to sensibility of mollusks to these EDCs, being able to cause immune alterations. Recently, microRNAs (miRNAs) were shown to be involved in modulation and buffering developmental processes against the effects of environmental alterations and pathogenic microorganisms. Moreover, it is suggested that this miRNAs are incorporated into the estrogen-controlled immune network, regulating mechanism of immune gene expression at the posttranscriptional level, modulating immune responses as phagocytosis, redox reaction and apoptosis in bivalve haemocytes. Thus, miRNAs can be used as biomarkers that specifically elucidate immunotoxic effects caused by exogenous biotic or abiotic factors, and can act as useful tools in integrated monitoring environmental health programs. In this review, we aim to describe the investigations that have been carried out on miRNAs in bivalve mollusks, especially those associated with immune responses against infectious agents and xenobiotic exposure.

Modulation of immune and antioxidant responses by azinphos-methyl in the freshwater mussel Diplodon chilensis challenged with Escherichia coli.

The aim of the present study was to characterize the immune response-total hemocyte number, cell type proportion, hemocyte viability, lysosomal membrane stability, phagocytic activity, cellular acid and alkaline phosphatase activity, and humoral bacteriolytic and phenoloxidase activity--in Diplodon chilensis exposed to 0.2 mg/L of azinphos-methyl (AZM), using Escherichia coli as immunological and pro-oxidant challenges. In addition, glutathione-S-transferase and lipid peroxidation thiobarbituric acid reactive substances were analyzed in gill tissue. Mussels from an unpolluted site were treated for 3 d as follows: 1) experimental control; 2) solvent effects control (acetone 0.01%); 3) bacterial challenge effects control (E. coli, 5 cells/mL × 104 cells/mL); 4) pesticide effects control (AZM in acetone); 5) control for combined effects of solvent and bacterial challenge; and 6) exposed to AZM, then challenged with E. coli. The results showed increased granulocyte proportion and phagocytic activity. Partial reversion of deleterious effects of E. coli on lysosomal membranes was observed in mussels exposed to AZM and then challenged with E. coli. Total hemocyte number and humoral bacteriolytic activity were increased only by E. coli challenge. Acid phosphatase activity was increased by both E. coli and AZM, whereas the stimulating effect of E. coli on alkaline phosphatase activity was negatively modulated by AZM. Azinphos-methyl inhibited phenoloxidase activity regardless of the E. coli challenge. Gill glutathione-S-transferase activity was increased by E. coli treatment either alone or pretreated with acetone or AZM and by AZM alone. Thiobarbituric acid reactive substance levels were reduced by AZM alone or combined with the E. coli challenge and by acetone followed by the E. coli challenge. Both acetone and AZM seem to be important modulators of immune and antioxidant responses in D. chilensis. Environ Toxicol Chem 2017;36:1785-1794. © 2016 SETAC.

Immunological function and antibacterial activity of two ferritin proteins from the freshwater pearl mussel Hyriopsis schlegelii.

Ferritin is a conserved iron-binding protein involved in host defense and cellular iron metabolism in most organisms. We investigated the expression profiles of two ferritin genes (designated HsFer-1 and HsFer-2) in the hemocytes, gonad, and hepatopancreas of Hyriopsis schlegelii, when challenged with bacteria and metal ions. HsFer gene transcription increased 1.8-7.7- and 1.9-6.1-fold in these tissues after stimulation with Staphylococcus aureus and Vibrio anguillarum, respectively. In addition, following exposure to Fe3+, expression of HsFer-1 and HsFer-2 was elevated by 1.5-6.1- and 3.6-10.1-fold, respectively. Levels of HsFer-1 and -2 mRNA also increased significantly after treatment with Cu2+ and Pb2+ at certain concentrations. Moreover, recombinant HsFer-1 and -2 were able to inhibit the growth of two strains of bacteria, and the former efficiently chelated Fe3+. From these results, we conclude that HsFer-1 and -2 may be involved in iron metabolism and immune defense by inhibiting the growth of bacteria.

Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D. chilensis, continuous and long term feeding with E. gracilis cells favors immune response and reduces the damage caused by sewage pollution exposure on hemocytes. Nevertheless, diet and transplantation procedures may produce negative effects on the oxidative balance of gills and digestive gland and should be taken into account for bioremediation strategies.

Immunological Profile of the Yellow Clam Mesodesma mactroides (Mesodesmatidae) from the Southern Coast of Rio Grande do Sul, Brazil.

The yellow clam Mesodesma mactroides (Mesodesmatidae) is a sandy beach bivalve that is distributed from Rio de Janeiro, Brazil, to the south of Buenos Aires province, Argentina. The yellow clam population has been declining in recent decades. To increase our understanding of this species, we evaluated the immunological status of yellow clams collected during different seasons from various areas in the state of Rio Grande do Sul, Brazil. We characterized the hemocytes, determined the differential hemocyte counts (DHCs), calculated the apoptotic index, and evaluated the incidence of parasites in yellow clams through histological analysis. We identified two types of hemocyte (hyaline and granular) that showed significant variation in DHCs among sampling areas during the summer and winter. The apoptotic index only exhibited significant variation during the summer. Histopathological analysis results did not significantly differ among sampling areas. This work demonstrated that environmental variation (e.g., temperature and salinity) associated with anthropogenic actions may be affecting the immune system of yellow clams. However, more studies are needed to determine the full influence of these factors on the yellow clam's immune system and thus contribute to future management and aquaculture of the species. Received May 10, 2015; accepted October 28, 2015.

Identification and expression of antioxidant and immune defense genes in the surf clam Mesodesma donacium challenged with Vibrio anguillarum.

The immune system in marine invertebrates is mediated through cellular and humoral components, which act together to address the action of potential pathogenic microorganisms. In bivalve mollusks biomolecules implicated in oxidative stress and recognition of pathogens have been involved in the innate immune response. To better understand the molecular basis of the immune response of surf clam Mesodesma donacium, qPCR approaches were used to identify genes related to its immune response against Vibrio anguillarum infection. Genes related to oxidative stress response and recognition of pathogens like superoxide dismutase (MdSOD), catalase (MdCAT), ferritin (MdFER) and filamin (MdFLMN) were identified from 454-pyrosequencing cDNA library of M. donacium and were evaluated in mantle, adductor muscle and gills. The results for transcripts expression indicated that MdSOD, MdFLMN and MdFER were primarily expressed in the muscle, while MdCAT was more expressed in gills. Challenge experiments with the pathogen V. anguillarum had showed that levels of transcript expression for MdSOD, MdCAT, MdFER, and MdFLMN were positively regulated by pathogen, following a time-dependent expression pattern with significant statistical differences between control and challenge group responses (p<0.05). These results suggest that superoxide dismutase, catalase, ferritin and filamin, could be contributing to the innate immune response of M. donacium against the pathogen V. anguillarum.

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Lipopolysaccharide and ß-1,3-glucan binding protein in the hard clam (Meretrix meretrix): molecular characterization and expression analysis.

Pattern recognition molecules play an important role in innate immunity by recognizing conserved molecular patterns that are present on the surface of invading microorganisms. In this study, a lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) gene was cloned from the hard clam Meretrix meretrix (designated as Mm-LGBP) by the expressed sequence tags and rapid amplification of cDNA ends method. The cDNA was 1827 bp in length, consisting of a 71-bp 5'-terminal untranslated region, a 62-bp 3'UTR, and a 1734-bp open reading frame encoding a 577-amino acid polypeptide with an estimated molecular mass of 60.7 kDa and a theoretical isoelectric point of 5.56. Characteristic potential polysaccharide binding, cell adhesion, and glucanase motifs were identified in the Mm-LGBP, indicating that Mm-LGBP should be a new member of the LGBP family. Quantitative real-time polymerase chain reaction was developed to detect the mRNA expression level of Mm-LGBP in 6 different tissues. Higher-level mRNA expression of Mm-LGBP was detected in the gill and digestive gland tissues. The upregulation of Mm-LGBP mRNA after Vibrio anguillarum challenge showed that Mm-LGBP play a pivotal role in antibacterial immunity.

Health status and bioremediation capacity of wild freshwater mussels (Diplodon chilensis) exposed to sewage water pollution in a glacial Patagonian lake.

Deleterious effects on health and fitness are expected in mussels chronically exposed to sewage water pollution. Diplodon chilensis inhabiting SMA, an area affected by untreated and treated sewage water, shows increased hemocyte number and phagocytic activity, while bacteriolytic and phenoloxidase activities in plasma and reactive oxygen species production in hemocytes are lower compared to mussels from an unpolluted area (Yuco). There are not differences in cell viability, lysosomal membrane stability, lipid peroxidation and total oxygen scavenging capacity between SMA and Yuco mussels' hemocytes. Energetic reserves and digestive gland mass do not show differences between groups; although the condition factor is higher in SMA than in Yuco mussels. Gills of SMA mussels show an increase in mass and micronuclei frequency compared to those of Yuco. Mussels from both sites reduce bacterial loads in polluted water and sediments, improving their quality with similar feeding performance. These findings suggest that mussels exposed to sewage pollution modulate physiological responses by long-term exposure; although, gills are sensitive to these conditions and suffer chronic damage. Bioremediation potential found in D. chilensis widens the field of work for remediation of sewage bacterial pollution in water and sediments by filtering bivalves.

Molecular characterization of two kazal-type serine proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum.

This study reports two kazal-type serine protease inhibitors (KPI) identified in a cDNA library from the surf clam Mesodesma donacium, and characterized through Rapid Amplification of cDNA Ends (RACE). The KPIs, denoted as MdSPI-1 and MdSPI-2, presented full sequences of 1139 bp and 781 bp respectively. MdSPI-1 had a 5'untranslated region (UTR) of 175 bp, a 3'UTR of 283 bp and an open reading frame (ORF) of 681 pb that encodes for 227 amino acids. MdSPI-2 showed a 5'UTR of 70 bp, a 3'UTR of 279 bp and an ORF of 432 bp that encodes for 144 amino acids. Both sequences presented two kazal-type tandem domains. Phylogenetic analysis of MdSPI-1 and MdSPI-2 shows a main clade composed by other bivalve species and closely related crustaceans. Real time PCR analysis showed that MdSPI-1 is mainly up-regulated in mantle, foot, gills and muscle tissues, while MdSPI-2 is expressed principally in foot tissue. Moreover, to evaluate the immune response of MdSPI-1 and MdSPI-2, infections with Vibrio anguillarum were performed. Herein, MdSPI-1 and MdSPI-2 transcription expression were significantly up-regulated at 2 and 8 h post-challenge. Our results suggest that MdSPI-1 and MdSPI-2 are important humoral factors of innate immunity in M. donacium.

Comparative study of various immune parameters in three bivalve species during a natural bloom of Dinophysis acuminata in Santa Catarina Island, Brazil.

This study aimed to verify if Dinophysis acuminata natural blooms affected the immune system of three bivalves: the oyster, Crassostrea gigas, the mussel, Perna perna, and the clam, Anomalocardia brasiliana. Animals were obtained from a renowned mariculture farm in the southern bay of Santa Catarina Island during, and 30 days after (controls), an algal bloom. Various immunological parameters were assessed in the hemolymph of the animals: total and differential hemocyte counts, percentage of apoptotic hemocytes, protein concentration, hemagglutinating titer and phenoloxidase activity. The results showed that the mussel was the most affected species, with several altered immune parameters, whereas the immunological profile of clams and oysters was partially and completely unaffected, respectively.