Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp,Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.     

Light microscopical studies of the acute toxic effects of N-nitrosodimethylamine on the marine mussel, Mytilus edulis

Mussels, Mytilus edulis, exposed to N-nitrosodimethylamine (DMN), 100 mg DMN/liter sea water, exhibited the following tissue responses: Congestion of the larger vessels in the Leydig cell tissue and infiltration of the tissue with numerous hemocytes, well-demarcated lesions composed of hemocytes in the Leydig cell tissue, degeneration of the epithelial lining of the ducts of the digestive diverticula, and congestion of the branchial vessels with numerous hemocytes accompanied by distortion of the cilia and the ciliated columnar epithelium of the gills. Furthermore, deposition of collagen-like material in the walls of the vessels was observed. DMN also induced spawning prior to the normal spawning time.     

Molecular cloning and expression of IRAK-4, IL-17 and I-κB genes in Haliotis rufescens challenged with Vibrio anguillarum

The candidate genes interleukin-1 receptor associated kinase 4 (IRAK-4), Interleukin 17 (IL-17) and Inhibitor of NF-κB (I-κB) were cloned and evaluated in Californian abalone (Haliotis rufescens) hemocytes in response to Vibrio anguillarum. Molecular characterization evidenced that HrI-κB has a full cDNA sequence of 3027 bp with an encoding region of 401 amino acids (aa), HrIRAK-4 comprised 1969 bp that encoded for 516 aa, and Hr-IL17 had a full sequence of 806 bp encoding for 165 aa. qPCR analysis showed the higher constitutive expression level of Hr-IL17 in hemocytes; meanwhile Hr-IκB and Hr-IRAK4 gene expression levels were higher in gills and mantle. The assessment of gene expression in hemocytes after infection with V. anguillarum evidences the immune responses of Hr-IκB, Hr-IRAK4, and Hr-IL17 and their relationships through the NF-κB signaling pathway.     

Cellular defense reactions of the shore crab, Carcinus maenas: in vivo hemocytic and histopathological responses to injected bacteria

The cellular defense reactions of the shore crab, Carcinus maenas, were studied, following injections of the bacteria Bacillus cereus and Moraxella sp., by histological and ultrastructural examination of the gills, heart, and hepatopancreas. The majority of the bacteria were sequestered to the gills, but some were also later evident in the heart and hepatopancreas. The presence of the bacteria in the gills initiated the formation of numerous small cell clumps, composed of both refractile and phagocytic cells, which entrapped many microorganisms. The clumps reached a maximum size 6 hr after inoculation and although some were cleared from the gills others persisted for 7 days, becoming more compact and necrotic during this period. Clump formation appears to occur following recognition of the bacteria as foreign and results in the hemocytes becoming sticky and adherent. The response is very effective in rapidly immobilizing the bacteria, thus restraining the spread of infection. It is proposed that this phenomenon may be a significant component of crustacean cellular host defenses.     

The roles of carbohydrates in aggregation and adhesion of hemocytes from the California mussel (Mytilus calif ornianus)

Forty-seven carbohydrates failed to inhibit the cell aggregation and substrate adhesion behaviors of Mytilus californianus hemocytes. Treatment of hemocytes with sodium periodate and with YV-acetyl glucosaminidase interfered with these behaviors. Trypsin slightly reduced adhesion, and had no effect on aggregation. Hyaluronidase and neuraminidase failed to inhibit either behavior, and slightly enhanced them. An inhibitory effect of caffeine was not altered by hyaluronidase of N-acetyl-glucosaminidase, but neuraminidase partially reversed this inhibition. Inhibition by the calcium channel blocker verapamil was reversed by hyaluronidase. Cell-cell and cell-substrate adhesion behaviors probably involve complex carbohydrates in molluscan hemocytes.     

Histopathology of Aerococcus viridans var. homari infection (Gaffkemia) in the lobster, Homarus americanus, and a comparison with histological reactions to a gram-negative species, Pseudomonas perolens

Histological response of lobsters to injection of Aerococcus viridans var. homari, cause of gaffkemia, was followed over a 14-day period. Salient features in infected lobsters, Homarus americanus, were: aggregations of hemocytes occurring in hemal spaces throughout the tissues and increasing in number and size with time; the early phagocytosis of bacteria by the system of fixed phagocytes (FPs) present in hemal spaces of the hepatopancreas; and premature release of differentiating hemocytes from the hemopoietic tissue, so that by 14 days that tissue consisted mainly of large stem cells. Mass release of differentiating hemocytes presumably occurred to replace hemocytes lost from the circulation by their incorporation into aggregations or by lysis of individual cells ruptured through the pressure of phagocytized bacteria that were multiplying in them. Bacteria and their remains were present in FPs at 2 days but not visible in single or aggregated hemocytes until 6 days, when free bacteria were also present in the hemolymph. By 6 days, all bacteria, whether phagocytized or free, appeared normal and were surrounded by nonstaining halos that extended well beyond the stainable capsular material. As predicted earlier in physiological studies, gaffkemia is a nontoxic, noninvasive bacteremia. There was hemal stasis and consequent injury in the antennal gland due to free and aggregated hemocytes that occluded hemal spaces of that organ, but other tissues and organs appeared normal except for depletion of glycogen. Aggregations of hemocytes were present in lobsters 2 and 12 days after injection of a nonpathogenic, Gram-negative bacterium, Pseudomonas perolens. Unlike the case with gaffkemia, necrotic hemocytes were common in the aggregations, presumably in response to damage by endotoxin. A further difference was that aggregations were common in the heart of P. perolens-injected lobsters but rare in the heart of gaffkemic lobsters. Bacteria were not seen in hemolymph, hemocytes, or other cells of P. perolens-injected lobsters.     

Perkinsus marinus Extracellular Protease Modulates Survival of Vibrio vulnificus in Eastern Oyster (Crassostrea virginica) Hemocytes

The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues.     

Observations on the pathogenesis of the imperfect fungus,Fusarium solani,in the California brown shrimp,Penaeus californiensis

The pathogenesis of Fusarium solani infections was studied using a highly susceptible species, Penaeus californiensis. In two experiments, F. solani infections were established in artificially wounded and infected juvenile and adults of P. californiensis, while similar control groups were wounded but not artificially infected. In the first experiment, the progress of F. solani infections in 15-g cultured juveniles of P. californiensis was followed by gross inspection and by light and electron microscopical study of the wound area. Developing F. solani infections were produced with a success rate of 100% within 14 days postinfection, and deaths due to disease of greater than 50% of the artificially infected group had occurred by 24 days postinfection. Infections were histologically characterized by (1) expansion of lesions into the tissue surrounding the point of entry, (2) destruction of invaded tissues by host granuloma formation and possibly by fungal enzymes and toxins, and (3) hemocyte encapsulation of hyphae with eventual melanization of the encapsulating hemocytes. In the second study, wild adult P. californiensis were artificially infected with F. solani, and changes in several of their hemolymph constituents [alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hematopoietic tissue mitotic index] were determined as the disease developed. Significant differences occurred in these hemolymph parameters of shrimp with advanced F. solani infections compared to those of uninfected, unwounded control shrimp or those with early F. solani infections. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate.     

Tissue specificities of Thelohania duorara,Agmasoma penaei, and Pleistophora sp., microsporidian parasites of pink shrimp, Penaeus duorarum

The pathology of pink shrimp, Penaeus duorarum, infected with the microsporidians Thelohania duorara, Agmasoma penaei, and Pleistophora sp. was described. Infections of T. duorara were widespread in most tissues; spores were located throughout the hemocoel, at the periphery of all striated muscle bundles, and in muscle and connective tissue surrounding the digestive tract. A. penaei infections invaded only dorsal abdominal muscles, muscles adjacent to blood vessels, and ovaries. Infected muscles and ovaries were eventually completely destroyed. Masses of A. penaei spores were often engulfed by hemocytes. Pleistophora sp. infected the interior of all striated muscles. Infected muscles were never completely destroyed but were often atrophied.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Acidophilic granulocytes in the gills of gilthead seabream Sparus aurata: evidence for their responses to a natural infection by a copepod ectoparasite

Immunohistochemical and ultrastructural studies were conducted on the gills of gilthead seabream, Sparus aurata L., naturally infected with the copepod ectoparasite Ergasilus lizae (Krøyer, 1863) in order to assess pathology and the host immune cell response. Gills of 56 gilthead seabream were screened for ectoparasites; 36 specimens (64.3%) harbored E. lizae. Intensity of infection was 32.7±8.7 (mean ± SE). Pathological alterations to the gills of the host were more pronounced in close proximity to the copepod site of attachment. The parasite attached to the gills by means of its modified second antennae, occluded the arteries, provoked epithelial hyperplasia and hemorrhages and most often caused lamellar disruption. Numerous granular cells were encountered near the site of E. lizae attachment. In both infected and uninfected gills, the granular cells lay within the filaments and frequently occurred within the connective tissue inside and outside the blood vessels of the filaments. The type of granular cell was identified by immunohistochemical staining by using the monoclonal antibody G7 (mAb G7), which specifically recognizes acidophilic granulocytes (AGs) of S. aurata and with an anti-histamine antibody (as a marker for mast cells, MCs) on sections from 13 uninfected gills and 21 parasitized gills. The use of mAb G7 revealed that, in gills harboring copepods, the number of G7-positive cells (i.e., AGs; 32.9±3.9, mean number of cells per 45,000 μm² ± SE) was significantly higher than the density of the same cells in uninfected gills (15.3±3.8; ANOVA, P<0.05). Few histamine-positive granular cells (i.e., MCs) were found in the uninfected and parasitized gills. Here, we show, for the first time in S. aurata infected gills, that AGs rather than MCs are recruited and involved in the response to E. lizae infection in seabream.     

Hemocytic changes associated with the encapsulation and melanization of some insect parasites

The hemocytic changes that attend parasite encapsulation and melanization in larvae of Drosophila, Musca domestica, and Orthellia caesarion are very similar to those changes occurring in noninfected individuals at metamporhosis. Some of the changes include the precocious differentiation and migration of hemocytes. Comparative and quantitative hemocytological data suggest that some stimulus in infected larvae, acting directly on the hemocytes or on the mechanism(s) controlling their activity, causes the cells to leave areas of the body where they are normally found and to encapsulate parasites. Questions concerning the origin and mode of action of the initial stimulus are raised, and as a basis for future experimentation it is proposed that the cellular immune reactions of these insects against internal metazoan parasites result from a hormonal imbalance.