White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.     

Trichobilharzia ocellata in Lymnaea stagnalis: a flow cytometric approach to study its effects on hemocytes.

Flow cytometric analysis was performed on hemocytes in suspension derived from individual Lymnaea stagnalis. The distribution of cell sizes within the hemocyte population was comparable in all 40 specimens studied. The size distribution of circulating hemocytes is unimodal and continuous, with no discrete subpopulations, and is not affected by age or by infection with Trichobilharzia ocellata. Flow cytometry proved to be a very useful technique in analysis of hemocyte populations in snails and anti-hemocyte monoclonal antibodies can be employed in these studies. The use of individual instead of pooled hemolymph samples in studying hemocyte populations of molluscs is stressed.     

A flow cytometric approach to the study of crustacean cellular immunity

Responses of hemocytes from the crayfish Procambarus zonangulus to stimulation by fungal cell walls (zymosan A) were measured by flow cytometry. Changes in hemocyte physical characteristics were assessed flow cytometrically using forward- and side-scatter light parameters, and viability was measured by two-color fluorescent staining with calcein-AM and ethidium homodimer 1. The main effects of zymosan A on crayfish hemocytes were reduction in cell size and viability compared to control mixtures (hemocytes in buffer only). Adding diethyldithiocarbamic acid, an inhibitor of phenoloxidase, to hemocyte and zymosan mixtures delayed the time course of cell size reduction and cell death compared to zymosan-positive controls. The inclusion of trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade, along with prophenoloxidase activation, played a key role in generating signal molecules which mediate these cellular responses. In addition to traditional methods such as microscopy and protein chemistry, flow cytometry can provide a simple, reproducible, and sensitive method for evaluating invertebrate hemocyte responses to immunological stimuli.     

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.     

Characterisation of Mytilus edulis hemocyte subpopulations by single cell time-lapse motility imaging

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 μm min^?1. Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte–hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed.     

中国鲎和圆尾鲎血淋巴细胞分类和特征的比较研究

为了更好地了解中国鲎(Tachpleus tridentatus)和圆尾鲎(Carcinoscorpius rotundicauda)血淋巴细胞的种类组成和特征差异,综合运用光学显微镜、扫描电镜和粒度仪,较为系统地对两种鲎的血淋巴细胞进行了分类和特征研究,从而为两种鲎的血淋巴细胞和分子生物学研究提供基础资料。根据血淋巴细胞大小、核质比、细胞着色特点、细胞中颗粒存在与否、颗粒的密集程度等,中国鲎和圆尾鲎的血淋巴细胞均可分为大颗粒细胞、小颗粒细胞和透明细胞三种主要类型,且两种鲎的血淋巴细胞均以颗粒细胞为主,透明细胞在血淋巴细胞中所占比例最小,但具有高核质比。两种鲎的同类血淋巴细胞在染色和形态上无显著性差异,但在同一种鲎中,血淋巴细胞密度存在显著的雌雄差异。       

Bacterial clearance rate and a new differential hemocyte staining method to assess immunostimulant activity in shrimp

New methods were developed to assess immunostimulant efficacy in the black tiger shrimp Penaeus monodon. Test shrimp were fed with 2 or 4 % yeast extract (YE)-coated feed while controls were fed non-coated feed. After 4 wk of feeding, individual shrimp were assessed for total hemocyte counts (THC), the number of granular hemocytes (GH) and rate of bacterial clearance. For hemocyte counts, formalin-fixed hemolymph was stained with 1.2 % Rose Bengal in 50 % ethanol for 20 min at room temperature. Some of this mixture was used for THC with a hemocytometer while some was smeared on a microscope slide and left to dry before counterstaining with hematoxylin for GH counts. By this technique, high quality smears were obtained for accurate differential counts. Bacterial clearance assays were used to assess the sum effect of humoral and cellular defense mechanisms. Vibrio harveyi was injected intramuscularly at 1 x 10(8) cells per shrimp and hemolymph was collected in anticoagulant at 0, 15, 30 and 60 min post-injection for quadruplicate drop counts (20 microl) on TCBS agar. Total hemocyte counts for shrimp fed with 4 % YE were significantly higher (p < 0.05) than those for shrimp fed with non-coated feed. The percentage of granular cells and the rates of bacterial clearance for the YE-fed shrimp were higher than those for shrimp fed the control diet. These 2 methods provide a simple and rapid comparison of shrimp groups for differences in anti-bacterial defense capacity.     

New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes

Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.     

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda,Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.Extensive hemocyte aggregates (''islets'') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.     

Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi)

We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pH_i), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pH_e) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pH_i) within hyalinocytes among pH treatments and the mean pH_i (7.26) was lower than the mean pH_e. In contrast, there were significant differences among treatments in pH_i of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pH_i compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction.     

MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.     

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.     

Honey Bee Hemocyte Profiling by Flow Cytometry

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.     

Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includens

Insect hemocytes have historically been identified on the basis of morphology, ultrastructure and hypothesized function. Among insects in the order Lepidoptera, five hemocyte classes are usually recognized: granular cells, plasmatocytes, spherule cells, oenocytoids and prohemocytes. We have generated a panel of monoclonal antibodies (mAbs) against hemocytes of the moth Pseudoplusia includens. In this study, hemocyte identification using 16 different mAbs was compared to identification methods using morphological characters. Three main categories of mAb binding activity were identified: (1) mAbs that specifically labeled only one morphological class of hemocytes, (2) mAbs that labeled granular cells and spherule cells, and (3) mAbs that labeled plasmatocytes and oenocytoids. With one exception, none of the antibodies bound to other tissues in P. includens. However, certain mAbs that specifically labeled granular cells and/or spherule cells in separated hemocyte populations also labeled plasmatocytes co-cultured with granular cells or cultured in granular cell conditioned medium. Overall, our results suggest that granular cells are antigenically related to spherule cells, and that plasmatocytes are antigenically related to oenocytoids. The use of mAbs as hemocyte markers are discussed.     

Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan

Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = 0 became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms.     

Activation of lobster hemocytes for phagocytosis

Activation of lobster (Homarus americanus) hemocytes for phagocytosis of sheep erythrocytes (SRBC) was demonstrated in vitro by incubation with lipopolysaccharide and by prolonged adherence to glass coverslips. Morphological changes, which preceded phagocytic activation, were detected by phase microscopy and Nomarski interference microscopy. These included spreading, the formation of filopodia and pseudopodia, granular darkening and dispersion, and vacuolation. Hemolymph serum opsonin greatly enhanced the recognition and phagocytosis of SRBC by activated hemocytes. Increases of 15 to 20 times background levels were observed both in the proportion of hemocytes which were actively phagocytic, and the percent of rosette-forming hemocytes. This suggested that the enhanced phagocytosis was the result of both the recruitment of a quiescent precursor population during activation, and an increase in the availability of opsonin binding sites on hemocyte membranes.     

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min.An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.     

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzymatic assay following electrophoretic analysis on polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE indicated that hemocyte lysate presented orthodiphenoloxidase (catecholase) activity. The enzymes from the three species differed in molecular size, activating substances and trypsin sensitivity.     

Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus)

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.     

转cry1Ab基因水稻对二化螟幼虫血细胞的影响

研究了转cry1Ab基因水稻“克螟稻1号”对二化螟Chilo suppressalis幼虫细胞免疫系统的影响。结果表明,转cry1Ab基因水稻对二化螟幼虫的血细胞影响明显,取食转cry1Ab基因水稻后,二化螟幼虫各类血细胞都明显低于取食非转基因水稻“秀水11”的对照组（原血细胞和囊血细胞在取食初期例外）,随取食时间延长,各类血细胞数量及血细胞总数均呈递减的趋势。从各类血细胞所占血细胞总数的百分比来看,原血细胞在取食36 h后锐减,而浆血细胞和粒血细胞则比例增加,其余珠血细胞、囊血细胞的变化不明显。另外,血细胞还出现空泡化、肿胀等病态变化,致使血细胞快速破裂。由此推测转cry1Ab基因水稻自身表达的毒蛋白能严重干扰靶标昆虫二化螟幼虫的细胞免疫系统。