Characterisation of monoclonal antibodies to haemocyte types of scallop (Chlamys farreri)

Four monoclonal antibodies (MAbs) (1E7, 1F12, 2H5, 2C6) against haemocytes of scallop (Chlamys farreri) were produced by immunising Balb/C mice. Analysed by the indirect immunofluorescence assay test (IIFAT), immunocytochemical assay, flow cytometry (FCM) and Western-blotting, they showed specificity for more than one haemocyte type (hyalinocyte and granulocyte) and various haemocyte components of scallop. Using IIFAT to detect monolayers separated from gradient density centrifugation, the four MAbs were positive with haemocytes at different interfaces. The percentage of positive cells (percent reactivity, PR) that MAb 1E7 reacted with at the 20-30%, 30-40% and 40-50% interfaces were 43.50%, 41.25% and 60.00% respectively, PR of MAb 1F12 were 31.00%, 63.50% and 41.00%, MAb 2C6 were 11.00%, 51.00%, 77.00%, and MAb 2H5 were 20.25%, 34.75%, 38.25%. For the immunocytochemical assay, MAb 1E7 1F12 and 2H5 was positive with the cytoplasm of both hyalinocyte and granulocyte, 2C6 was positive with the membrane and cytoplasm of hyalinocyte and granulocyte. Analysed by FCM, the PR of the four MAbs (1E7, 1F12, 2H5, 2C6) with haemocytes were 54.23%, 38.56%, 56.4%, and 79.7% respectively; moreover, the PR with different haemocyte types was variable. The results of Western-blotting showed that MAb 1E7 recognised an antigen of molecular weight 200 kDa, MAb 2C6 an antigen of 60 kDa, however, MAb 1F12 reacted with antigens of 70 kDa, 60 kDa and 45 kDa. There was no protein band that MAb 2H5 detected. In conclusion, 2C6 seems to be a very promising MAb to identify and differentiate granulocytes, and the four MAbs will be used in further studies on cellular defence mechanism research.     

用流式细胞仪测定扇贝血细胞吞噬活性

采用流式细胞仪技术对栉孔扇贝(Chlamysfarreri)和海湾扇贝(Argopectenirradians)血细胞吞噬活性进行了测定。通过对缓冲体系的筛选和比较,建立了扇贝血细胞吞噬活性的流式细胞仪检测方法,并应用该技术对两种扇贝血细胞的吞噬活性进行了比较研究。结果发现在TBS(0.05mol.L-1Tris-HCl,pH7.4;2%glucose;2%NaCl;20mmol.L-1EDTA)和PBS体系中,扇贝血细胞的吞噬活性几乎完全受到抑制,在CMPBS(0.1mol.L-1PBS,pH7.4;2%NaCl;2%glucose;1.0mmol.L-1CaCl2;0.5mmol.L-1MgCl2)体系中细胞吞噬活性略有升高,而在海水缓冲液中细胞吞噬活性最高,其中海湾扇贝和栉孔扇贝血细胞的吞噬率分别达到了26.73%和19.89%,且海湾扇贝血细胞的吞噬率显著高于栉孔扇贝(P<0.05)。研究结果为进一步探讨扇贝血细胞吞噬功能提供了方法依据。     

Oxidative burst in hard clam (Mercenaria mercenaria) haemocytes

Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorganisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.     

First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum

In the present study, we report on haemocyte distribution, determined by a Coulter Counter, in the clam Tapes philippinarum. In addition, cytoskeleton components of haemocytes were examined using specific probes for F-actin and alpha-tubulin. The mean number of circulating haemocytes was 5 (x10(6))cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2-3microm in diameter and 10-100fl in volume; and large cells, 6-10microm in diameter and 150-400fl in volume. Analysis of the haemocyte cytoskeleton revealed bundles of actin filaments oriented according to the cell major axis, and microtubules radiating from the microtubule-organizing centre in proximity of the nucleus. Interestingly, mitotic spindles were also found radiating from the microtubule-organizing centres, located at the spindle poles (centrosomes) of undifferentiated cells. On the basis of both our previous findings regarding circulating stem cells (Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol 10, 677-693) and new information from the present study, we suggest that haemoblasts are able to divide in the haemolymph of T. philippinarum. To our knowledge, this is the first report of mitotic spindles in circulating haemocytes from a bivalve species.     

四十里湾栉孔扇贝清滤率、摄食率和吸收效率的现场研究

运用生物沉积法在四十里湾不同海区对栉孔扇贝的生理生态学特征进行了研究。 1龄栉孔扇贝 (4 1 .1± 4.1 mm,软体干重 0 .48± 0 .1 0 g/ ind)清滤率变化范围为 0 .72～ 2 .5 4(平均 1 .2 7) L/ (ind· h)或 1 .65～ 5 .97(平均 2 .61 ) L/ (g· h)。清滤率受 TPM的变化影响不大 ,而摄食率却随 TPM的升高而升高。 2龄扇贝 (软体干重 1 .91± 0 .3 2 g/ ind)清滤率为2 .0 9～ 3 .99(平均 3 .1 0 ) L/ (ind· h)。栉孔扇贝吸收速率与 POM呈正相关关系 ,而与饵料质量 (POM/ TPM)无明显的相关性。1龄扇贝和 2龄扇贝吸收效率没有显著差别 ;扇贝对 POM的吸收效率与 TPM (或 POM)关系不大 ,却与饵料质量呈明显的正相关关系 ;扇贝对 POC、PON和 PP的吸收效率平均分别为 68.9%、64.0 %和 63 .6%。在沿岸养殖海域 ,栉孔扇贝通过大量的滤水摄食以及较高的吸收效率对生态系统的能量流动和物质循环产生影响。     

Flow cytometric comparison of haemocytes from three species of bivalve molluscs

Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.     

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: I. Haemocyte changes following in vitro challenge

In microbial infections, the interaction between microorganisms and phagocytic cells is a crucial determinant in the outcome of the disease process. We used flow cytometry to study the in vitro interactions between Vibrio tapetis, the bacterium responsible for Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum, and haemocytes from three bivalve species: the Manila clam (susceptible to BRD), the hard clam Mercenaria mercenaria and the eastern oyster Crassostrea virginica (both non-susceptible to BRD). Results demonstrated that V. tapetis cells and extracellular products elicit major changes in the haemocytes of R. philippinarum, including decreased viability and phagocytic activity, and altered size and internal structure. V. tapetis was able to kill haemocytes from M. mercenaria and C. virginica but to a far lesser extent than those of R. philippinarum. These results suggest that disease resistance is not solely dependent on a host activity against the pathogen, but is also a function and magnitude of the injury to the host cell by a given pathogen.     

Bacterial lipopolysaccharide modulates protein kinase C signalling in Lymnaea stagnalis haemocytes

Our knowledge of cell signalling pathways in the molluscan immune system and their response to immunological challenge is currently poor. The present study focused on the Protein Kinase C (PKC) pathway in the immune cells (haemocytes) of Lymnaea stagnalis and its response following exposure to bacterial lipopolysaccharide (LPS). Western blotting of haemocyte proteins with either anti-PKC (pan) or anti-phospho-PKC (Ser 660) antibodies revealed the presence of two PKC-like immuno-reactive proteins of approximately 76 and 85 kDa. Challenge of haemocytes with LPS transiently increased the phosphorylation of the 85 kDa isoform, with a 2.2-fold increase in phosphorylation levels at 5 min and a return to basal levels after 20 min. This LPS-mediated response was blocked following treatment of haemocytes with GF109203X. PKC activities measured in anti-phospho-PKC immunocomplexes following haemocyte treatment with LPS and GF109203X correlated well with the observed PKC phosphorylation levels. These data show for the first time that the activity of the PKC pathway in molluscan immune cells is modulated by LPS, as it is in mammals, and suggest that cell signalling in the innate immune response may have been conserved through evolution.     

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (> 300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with different relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens.     

Protein kinase A activity and protein phosphorylation in the haemocytes of immune-challenged Galleria mellonella larvae

Protein kinase A (PKA) activity was detected in the haemocytes of greater wax moth, Galleria mellonella larvae using a specific peptide substrate--kemptide. The enzyme was activated in vitro by 1 microM concentration of cAMP, 8-Br-cAMP, 8-Chl-cAMP and BzcMP, whereas in the case of cGMP 10 microM concentration was necessary. Immune challenge of G. mellonella larvae with bacteria led to changes in haemocyte PKA activity. Gram-positive M. luteus was a better inducer of PKA activity than Gram-negative E. coli. The kinetics of activity changes was dependent on the bacteria used and considerably differed from that observed in water-treated insects. Inhibition of PKA activity by cell-permeable, specific inhibitor, Rp-8-Br-cAMPS, induced changes in haemocyte morphology resembling those caused by live bacteria. Four potential PKA substrates of 155 kDa, 44 kDa, 40 kDa and 22 kDa were recognized in the haemocytes of naive larvae by phospho-motif antibodies for PKA phosphorylation consensus site. The modification level of 40 kDa protein changed after water treatment and immune challenge of G. mellonella larvae, whereas that of 155 kDa protein changed only after E. coli and LPS injections. Additionally, in the haemocytes of bacteria- and LPS-challenged insects a transient phosphorylation of 36 kDa protein was detected.     

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P相似文献   

Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L)

Data are presented that demonstrate the application of the neutral red retention assay (NRR) to monitor the effects of a bacterial inoculation on the haemocyte lysosomes of the European flat oyster Ostrea edulis, Pacific oyster Crassostrea gigas and scallop Pecten maximus. Bivalves were acclimated to three temperature regimes (5, 15 and 25 degrees C), at constant salinity for 7 days in the laboratory. Once baseline responses to acclimation temperature had been established, the effects of an in vivo inoculation on haemocyte lysosomal stability were assessed using the NRR assay. Lysosomal membrane stability was reduced in the presence of bacteria for all three species of bivalve, but destabilisation of C. gigas haemocyte lysosomes appeared to be most sensitive to the presence of the bacterium Listonella anguillarum. For all three bivalve species, the reduction in lysosomal stability appeared to be proportional to the growth of the bacterial inoculate. Using appropriate controls, the NRR assay was demonstrated to have great potential as a tool with which to make rapid initial assessments of the immune status of bivalve molluscs.     

Chromosomal rearrangement in pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops, using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.     

The antioxidants dimethylsulfoxide and dimethylthiourea affect the immediate adhesion responses of larval haemocytes from 3 lepidopteran insect species

Antioxidants, dimethylsulfoxide (DMSO) and dimethylthiourea (DMTU), at concentrations not affecting the viability of blood cells (haemocytes) from the larval stage of 3 lepidopteran insects - Galleria mellonella, Lymantria dispar, and Malacosoma disstria - differed in their influence on the innate binding of haemocytes to glass, bacteria to haemocytes, and on humoral responses to alien materials. In vitro DMSO had little effect, whereas DMTU substantially impaired the adhesion of the haemocyte types, the plasmatocytes and granular cells, to slides as well as the attachment of Bacillus subtilis to these haemocytes. Although both antioxidants increased lysozyme and phenoloxidase activities, there was no correlation of enzyme activity and haemocyte adhesion responses, possibly reflecting sequestered radicals. Nitric oxide and hydroxyl radicals offset the DMTU effect. In the absence of antioxidants, inactivate protein kinases A (PKA) and C (PKC) enhanced haemocyte aggregation. In general, DMSO, as opposed to DMTU, did not alter the effects of PKA and PKC activators and inhibitors on haemocyte aggregation or of PKC and PKA activities. High concentrations of DMSO and all levels of DMTU, although inhibiting PKA and PKC, inhibited haemocyte adhesion to slides. Comparable results occurred for DMTU-treated haemocytes incubated with B. subtilis. In vivo DMSO, unlike DMTU, did not impair plasmatocyte or granular cell responses to foreign materials, including bacterial removal from the haemolymph and nodulation.     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Monoclonal antibodies specific to haemocytes of black tiger prawn Penaeus monodon

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.     

Himasthla elongata: Effect of infection on expression of the LUSTR-like receptor mRNA in common periwinkle haemocytes

The first mollusc mRNA coding G-protein-coupled transmembrane receptor (GPСR), homologous to human receptors LUSTR 1 (GPR107) and LUSTR 2 (GPR108), was isolated from haemocytes of common periwinkle Littorina littorea. The analyses showed that the full-length cDNA is 1935 bp long and is predicted to encode a 614 amino acid protein (named Lit-LUSTR) with a calculated molecular mass of 69.6 kDa and theoretical isoelectric point 7.59. Pair-wise comparisons between Lit-LUSTR and LUSTR proteins from human or mouse have approximately 38% identity and 56% similarity. Lit-LUSTR clusters with LUSTR-A sub-family proteins and is a first characterization of proteins containing Lung7TM-R domain in Mollusca. Significant differences were found between the Lit-LUSTR mRNA levels in haemocytes of healthy periwinkles and those naturally infected with the echinostome trematode Himasthla elongata. Down regulated expression of the LUSTR-like receptor caused by infection illustrates modification of the haemocyte receptor system and may be attributed to the previously demonstrated greater numbers of “immature” haemocytes in the circulation of infected snails.