Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies (original) (raw)
Related papers
Fish & Shellfish Immunology, 1995
In order to ensure shrimp aquaculture production, immunology has become a priority in terms of disease control and prevention. Research must be devoted to the identification of anti-infectious effectors at the cellular and molecular levels. In this review, recent results obtained in the shrimp Penaeus japonicus are presented and discussed with reference to the Crustacea and other arthropods. Various cellular and plasma haemolymph components were characterised both antigenically and functionally. Haemocyte subpopulations, separated by isopycnic centrifugation on Percoll were discriminated using two monoclonal antibodies, 40E2 and 40El0. The granular cells were recognised by the antibody 40E2 specific for a plasmatic protein. By comparison, the hyaline and semi-granular cells were recognised by the antibody 40El0, which immunoprecipitated a plasmatic protein identified as an agglutinin. Moreover, an a2-macroglobulin-like protein present both in plasma and in haemocytes was antigenically characterized in P. japonicus as well as a plasma clotting factor. Functional studies have been undertaken by considering antimicrobial effectors and haemocyte-mediated oxidative defence mechanisms studied by chemiluminescence. Prospects on findings in shrimp immunology are discussed with reference to their application to zoosanitary prophylaxis and disease prevention. ~$)
Fish & Shellfish Immunology, 1995
In order to ensure shrimp aquaculture production, immunology has become a priority in terms of disease control and prevention. Research must be devoted to the identification of anti-infectious effectors at the cellular and molecular levels. In this review, recent results obtained in the shrimp Penaeus japonicus are presented and discussed with reference to the Crustacea and other arthropods. Various cellular and plasma haemolymph components were characterised both antigenically and functionally. Haemocyte subpopulations, separated by isopycnic centrifugation on Percoll were discriminated using two monoclonal antibodies, 40E2 and 40E10. The granular cells were recognised by the antibody 40E2 specific for a plasmatic protein. By comparison, the hyaline and semi-granular cells were recognised by the antibody 40E10, which immunoprecipitated a plasmatic protein identified as an agglutinin. Moreover, an α2-macroglobulin-like protein present both in plasma and in haemocytes was antigenically characterized in P. japonicus as well as a plasma clotting factor. Functional studies have been undertaken by considering antimicrobial effectors and haemocyte-mediated oxidative defence mechanisms studied by chemiluminescence. Prospects on findings in shrimp immunology are discussed with reference to their application to zoosanitary prophylaxis and disease prevention.
Monoclonal antibodies specific to haemocytes of black tiger prawn Penaeus monodon
Fish & Shellfish Immunology, 2005
Monoclonal antibodies specific to haemocytes of Penaeus monodon were generated from a mouse immunized with a mixture of SDS-treated and formalin-fixed haemocytes. Hybridoma clones were selected by immunohistochemistry against fixed haemocytes, heart, lymphoid organ, and haemopoietic tissue, and Western blot against haemocyte extract and haemolymph. Sixteen monoclonal antibodies specific to haemocytes were obtained and could be divided into six groups according to their binding capacities to various haemocyte proteins in Western blot analyses, 102, 43, approximately 20, 61, 175 and approximately 230 kDa, and their differences in recognition of haemocyte sub-populations. The first group of antibodies strongly recognized a small subset of semi-granulocytes (SG) and hyalinocytes (H) but occasionally stained lightly a very small population of granulocytes (G). The antibodies also bound to a group of cells in haemopoietic tissue as well as cells located at the inner layers of the tubules in the lymphoid organ but not in the spheroid. The second group of antibodies strongly bound to a large sub-population of G and SG with coarse granules but did not bind to most of the H. This group of antibodies also cross-reacted with cells in the outer layer of the tubules in the lymphoid organ. The third group of antibodies recognized all G and only a small portion of SG. The fourth, fifth and sixth groups bound to sub-populations of G, SG and H in similar proportions. None of the antibodies showed any cross-reactivity to other components in haemolymph. The common antigens recognized by the first and the second groups of antibodies in the haemopoietic tissue and the lymphoid organ may reflect relationships among these organs in the development of the sub-populations of G and SG. Haemopoietic tissue may be the site for haemocyte production and the lymphoid organ may be the site for further differentiation of at least two different lines of haemocytes.
Haemolytic activity in the brown shrimp (Penaeus californiensis holmes) haemolymph
Comparative Biochemistry and Physiology Part A: Physiology, 1993
Natural haemolytic activity in brown shrimp (Penaeus californiensis) haemolymph was detected using mouse erythrocytes as target cells. This activity is unrelated to agglutinating and phenoloxidase activity, but it is another probable component of the shrimp defence system. 2. The haemolytic reaction is time and dose dependent, and a serine-protease is involved. 3. The haemolytic factor is thermolabile and has an apparent molecular weight of 23.5 kDa.
Fish Pathology, 2003
Morphology and immunological roles of the three distinct types of hemocytes, hyaline, small granular and large granular cells in black tiger shrimp Penaeus monodon were stud ied. The study of functions of these hemocytes in the elimination of injected yeast (Saccharomy ces cerevisiae) or Vibrio harveyi as foreign bodies showed a rapid response against those particles. Together with fixed phagocytes, the blood cells removed the yeast through the process of phagocy tosis, nodule formation and encapsulation, which occurred at almost all parts of the body. The injection of the yeast caused a marked reduction in the blood cell counts in the hemolymph during the first 1 h. V. harveyi was efficiently removed within 3 h after injection. The granular cells (small granular and large granular hemocytes) were the major blood cells that are associated with phenoloxidase activity. The role of hemocytes and fixed phagocytes on defense mechanism in the shrimp were discussed.
Selected morpho-chemical features of hemocytes in farmed shrimp, Fenneropenaeus indicus in Iran
The aim of the present study was to determine various types of hemocytes, total and differential hemocyte count and to show some indices of phagocytosis, including percent phagocytosis and phagocytic index in Indian white shrimp, Fenneropenaeus indicus in Iranian shrimp farms. The hemolymph was extracted from the shrimps and mixed with anticoagulant. It then stained and Yeast was added as foreign particles to samples. Based on the cell size and presence of the granules and nucleocytoplasmic ratio, three major groups of hemocytes, including hyaline cells, small granular cells (SGC) and large granular cells (LGC) were identified. Hyaline cell (HC) was the smallest hemocyte. HC (hyaline cell) about was 10-15%, lower quantity in comparison to some species and those of LGC and SGC were 20-25% and 60-65%, respectively, suggesting that relative percentage of differential hemocyte count in Fenneropenaeus indicus is different from other decapods. Also, in this study, it was shown that SGH and LGH could engulf the yeast particles. In Contrast to some previous studies, no engulfment was observed by hyaline cells in vitro. In this study it was showed that there are some variations in total and differential hemocyte count compare to other species in the family of Penaeidae.
CYTOCHEMICAL FEATURES OF SHRIMP HEMOCYTES
1987
Morphological studies suggest that there are several types ofdecapod hemocytes; however, distinguishing criteria based on conventional staining techniques are often subtle or ambiguous. Cytochemical features ofridgeback prawn (Penaeidae: Sicyonia ingentis) hemocytes were studied using specific stains forlysosomes, cytoplasmic con tents, and granule enzymes. This approach facilitates the differentiation ofcell types in the ridgeback prawn and provides information on the functions of and relation ships among different cell types. Agranular hemocytes and a subgroup of small granule hemocytes contain exten sive cytoplasmic glycoprotein deposits which display smudgy, intense staining with Sudan black B. As previously shown, coagulogenâ€"the clotting material in deca podsâ€"stains with Sudan black B when extracted from lysed hemocytes. Other hemo cyte types display light staining limited to granule membranes.
Aquaculture, 2012
For studying shrimp immunity, in vitro haemocyte cultures are essential. Despite various reported attempts, well-described and reproducible culture techniques are lacking. The current work aimed to establish two in vitro haemocyte culture systems for Penaeus (Litopenaeus) vannamei. Haemocyte suspensions were either seeded in conventional Nunc® Nunclon™Δ Surface 24-well cell culture plates with glass cover slips (haemocytes in attachment) or in Nunc® Hydrocell Surface 24-well cell culture plates (haemocytes in suspension). The culture medium was based on L-15 (Leibovitz), and was further supplemented with L-glutathione and protease inhibitors in an attempt to improve haemocyte survival. Parameters such as number of living adherent and non-adherent single cells, number and average diameter of clusters and survival of cells inside clusters were evaluated. Additionally, live-cell imaging videos were recorded. It was found that haemocytes cultured for 1 h on glass coverslips in Nunc® Nunclon™Δ Surface plates could be separated in two cell fractions: adherent or non-adherent. Shrimp haemocytes cultured in Nunc® Hydrocell Surface plates remained in suspension and over time formed cell clusters which melanised. L-glutathione supplementation clearly improved haemocyte survival up to 48 h and delayed clustering and melanisation; addition of protease inhibitors did not. To validate the system, the phagocytic and antibacterial activities of adherent haemocytes towards Vibrio campbellii were evaluated. After 1 h of co-culture, 11.5 ± 0.14% of haemocytes showed phagocytosis with an average of 2.4 ± 0.1 bacteria internalised per haemocyte. Furthermore, haemocytes clearly demonstrated an antibacterial activity. It was concluded that these systems were reproducible and could keep haemocytes functionally active during the time required for the study of innate immune processes. Consequently, these techniques represent powerful tools for studying a variety of cell-mediated and humoral immune responses of shrimp in vitro.
Comparative Biochemistry and Physiology Part A: Physiology, 1992
The blue shrimp (Penaeus stylirostris) haemolymph is capable of agglutinating the red blood cells of several vertebrates to different titres. However, the haemagglutinin is considered non-specific because it is incapable of differentiating erythrocytes of human blood types A, B and 0. 2. Haemagglutinating activity and serum protein content were determined for male and female blue shrimp ranging in size from 8.5 to 16 cm. Haemagglutinating activity decreased significantly with animal size, while protein content was unaffected. 3. The above finding is probably related to maturation of the immune system and could explain the higher susceptibility of young shrimp to parasitic and viral diseases.
Fish & Shellfish Immunology, 2007
The clottable protein (CP) involved in Penaeus monodon haemolymph coagulation has previously been characterized and cloned. Polyclonal antibodies against purified CP were also prepared from rabbit serum. By Western blot analyses, we showed occurrence of CP in the shrimp central nervous system, gill, and lymphoid organ. Results of RT-PCR further indicated that the central nervous system, gill, and lymphoid organ transcribed more CP, heart and hepatopancreas transcribed less, while the haemocytes and the muscle did not. We further analyzed the CP distribution within shrimp lymphoid organ by immunohistochemical method, CP was found to localise in stromal cells of lymphoid organ rather than in the developing haemocytes. In addition, concentrations and regulation of the plasma CP under normal and artificially traumatic conditions were studied with rocket immunoelectrophoresis. The average plasma CP concentration in normal intermolt shrimps was elevated from 3 mg ml À1 to above 12 mg ml À1 after successive blood-withdrawing for a week. The production and secretion of CP apparently were increased more than 4 folds to compensate its loss. Our result also suggested that the shrimp sinus gland endocrine system is not directly required for the expression and up-regulation of CP.