Hemocyte parameters of the Pacific oyster Crassostrea gigas a year after the Hebei Spirit oil spill off the west coast of Korea

In marine bivalves, hemocytes support various physiological functions, including immune defense, nutrient transport, shell repair, and homeostatic maintenance. Although the effects of marine contaminants on the immunological functions of bivalves have been extensively investigated, the impacts of oil spills are not well understood. Therefore, we investigated hemocyte parameters in the Pacific oyster Crassostrea gigas 13 months after the Hebei Spirit oil spill (December 2007) off the west coast of Korea. The parameters studied included hemocyte concentration and mortality, relative proportion of hemocyte populations, and immunological functions such as phagocytosis and oxidative activity using flow cytometry. These immune-related parameters in oysters damaged by the oil spill were also compared to control oysters that were collected from an area unaffected by the spill. The flow cytometry study indicated that granulocyte population, phagocytic capacity, and reactive oxygen species production in oysters exposed to crude oil 13 months prior were depressed compared to the unexposed control oysters. Our data suggest that immunocompetence in oysters affected by the oil spill had not fully recovered 1 year after the accident, although more detailed studies on the physiology and disease resistance should be performed.     

Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays

Summary. Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin β1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall–plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall–plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall–plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays. Correspondence: J. S. Liang, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China.     

Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations

Oysters, Crassostrea virginica, from two populations, one from a coastal pond experiencing repeated dinoflagellate blooms (native), and the other from another site where blooms have not been observed (non-native), were analyzed for cellular immune system profiles before and during natural and simulated (by adding cultured algae to natural plankton) blooms of the dinoflagellate Prorocentrum minimum. Significant differences in hemocytes between the two oyster populations, before and after the blooms, were found with ANOVA, principal components analysis (PCA) and ANOVA applied to PCA components. Stress associated with blooms of P. minimum included an increase in hemocyte number, especially granulocytes and small granulocytes, and an increase in phagocytosis associated with a decrease in aggregation and mortality of the hemocytes, as compared with oysters in pre-bloom analyses. Non-native oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance. The effect of P. minimum on respiratory burst was different according to the origin of the oysters, with the dinoflagellate causing a 35% increase in the respiratory burst of the native oysters but having no effect on that of the non-native oysters. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum.     

A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus

Invertebrates display effective innate immune responses for defense against microbial infection. However, the protozoan parasite Perkinsus marinus causes Dermo disease in the eastern oyster Crassostrea virginica and is responsible for catastrophic damage to shellfisheries and the estuarine environment in North America. The infection mechanisms remain unclear, but it is likely that, while filter feeding, the healthy oysters ingest P. marinus trophozoites released to the water column by the infected neighboring individuals. Inside oyster hemocytes, trophozoites resist oxidative killing, proliferate, and spread throughout the host. However, the mechanism(s) for parasite entry into the hemocyte are unknown. In this study, we show that oyster hemocytes recognize P. marinus via a novel galectin (C. virginica galectin (CvGal)) of unique structure. The biological roles of galectins have only been partly elucidated, mostly encompassing embryogenesis and indirect roles in innate and adaptive immunity mediated by the binding to endogenous ligands. CvGal recognized a variety of potential microbial pathogens and unicellular algae, and preferentially, Perkinsus spp. trophozoites. Attachment and spreading of hemocytes to foreign surfaces induced localization of CvGal to the cell periphery, its secretion and binding to the plasma membrane. Exposure of hemocytes to Perkinsus spp. trophozoites enhanced this process further, and their phagocytosis could be partially inhibited by pretreatment of the hemocytes with anti-CvGal Abs. The evidence presented indicates that CvGal facilitates recognition of selected microbes and algae, thereby promoting phagocytosis of both potential infectious challenges and phytoplankton components, and that P. marinus subverts the host's immune/feeding recognition mechanism to passively gain entry into the hemocytes.     

Direct effects of physical stress can be counteracted by indirect benefits: oyster growth on a tidal elevation gradient

The paradigmatic gradient for intertidal marine organisms of increasing physical stress from low to high elevation has long served as the basis for using direct effects of duration of water coverage to predict many biological patterns. Accordingly, changes in potential feeding time may predict the direction and magnitude of differences between elevations in individual growth rates of sessile marine invertebrates. Oysters (triploid Crassostrea ariakensis) experimentally introduced at intertidal (MLW+0.05 m) and subtidal (MLW–0.25 m) elevations in racks provided a test of the ability to use duration of water coverage to predict changes in growth. During early-to-mid winter, a depression of 38–47% in shell growth of intertidal oysters matched the 36% reduction in available feeding time relative to subtidal oysters. In late winter as solar heating of exposed oysters increased, growth differences of 52–55% departed only slightly from the predicted 39%. In spring, however, duration of water coverage failed to predict even the correct direction of growth change with elevation as intertidal oysters grew 34% faster despite 39% less feeding time. Intense seasonal development of shell fouling by other suspension feeders like ascidians, mussels, and barnacles on subtidal (94% incidence) but not on aerially exposed intertidal (21–38% incidence) oysters may explain why duration of water cover failed to predict spring growth differences. Less intense fouling develops on intertidal oysters due to the physiological stress of aerial exposure on settlers, especially during higher temperatures and longer solar exposures of spring. Fouling by suspension feeders is known to reduce growth of the host through localized competition for food and added energetic costs. Thus, in springtime, indirect effects of aerial exposure providing a partial refuge from biological enemies overwhelmed direct effects of reduced duration of water coverage to reverse the expected pattern of slower intertidal growth of a marine invertebrate.     

Evidence for a direct link between stress and immunity in the mollusc Haliotis tuberculata

Stress is thought to cause increased disease outbreaks and mortality in a number of invertebrates but currently very little information is available on mechanisms linking physiological states of stress and reduced disease resistance in these organisms. In the present study, we examined the possibility that stress alters immune functions, the principal line of defense against pathogens, in a molluscan model, the abalone Haliotis turbeculata. Immune parameters were investigated in abalones subjected to a 15 min mechanical disturbance which, as indicated by noradrenaline and dopamine hemolymphatic levels, resulted in a transient state of physiological stress. During the application of the stressor, immune parameters such as the number of circulating hemocytes, the migratory activity, the phagocytic capacity and the respiratory burst responses of hemocytes, decreased significantly. All parameters returned to initial values within 15-30 min after the end of the disturbance and a transient period of immunostimulation occurred between 100 and 480 min after the stress for all immune parameters except intracellular superoxide anion production. These results indicate that in the abalone H. tuberculata, as in vertebrates, a link exists between stress and the immune system. This may begin to answer why stress and disease outbreaks are linked in shellfish.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

Assessing the microbial oxidative stress mechanism of ozone treatment through the responses of Escherichia coli mutants

Aims:  To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli. Methods and Results:  Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 μg ml^?1 for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure. Conclusions:  The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress–related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone. Significance and Impact of the Study:  This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation.     

小肠结肠炎耶尔森菌对多粘菌素B的压力应答

【背景】小肠结肠炎耶尔森菌(Yersinia enterocolitica)是重要的人畜共患食源性病原菌。由于其生存环境与传染性生活方式,小肠结肠炎耶尔森菌暴露在各种生存压力中,而胞膜压力应答能力对维持其环境耐受性和毒力发挥着重要作用。【目的】探究小肠结肠炎耶尔森菌在胞膜压力应答中的调节机制。【方法】通过使用多粘菌素B破坏小肠结肠炎耶尔森菌细胞膜的稳定性,并从生长能力、运动能力、生物被膜形成能力以及相关基因表达的变化探讨Rcs (Regulator of Capsule Synthesis)系统对多粘菌素B产生的胞膜压力的应答。【结果】多粘菌素B引起的细胞胞膜压力抑制了小肠结肠炎耶尔森菌的运动和生物被膜形成能力;而阻断Rcs信号途径后,小肠结肠炎耶尔森菌的运动和生物被膜形成能力有所恢复。对flhC、hmsS、hmsT等关键下游表型基因的表达水平的分析结果表明Rcs双组分系统对由多粘菌素B诱导的胞膜压力作出响应,通过感知胞膜胁迫向胞内传递信号,积极地调控细菌增强对抗菌肽的抗性。【结论】明确了Rcs双组分系统在响应多粘菌素B压力胁迫中的特异性调控作用,加深了对小肠结肠炎耶尔森菌环境应答机制...     

Tissue responses exhibited by Biomphalaria alexandrina snails from different Egyptian localities following Schistosoma mansoni exposure

Snails’ susceptibilities to infection with Schistosoma mansoni were determined through observation of infection rates, total cercarial production and tissue responses of the first generation (F1) of Biomphalaria alexandrina snails, originally collected from different Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) and responses were compared between groups. The emergence of cercariae for a 3-month period and the calculation of survival and infection rates, in control (Schistosome Biological Supply Center; SBSC) and infected snails were evaluated. SBSC and Giza snails showed greater susceptibilities to infection and lower mortality rates. In addition, at 6 and 72 h post-exposure to miracidia all the snail groups showed no difference in the anatomical locations of sporocysts. The larvae were found in the head-foot, the mantle collar and the tentacles of the snails. Sporocysts showed normal development with low tissue reactions in SBSC and Giza snail groups infected with S. mansoni miracidia (SBSC). However, in Fayoum, Kafr El-Sheikh, Ismailia and Damietta snail groups, variable tissue responses were observed in which numerous hemocytes made direct contact with S. mansoni larvae forming capsules. The results suggested that, different responses of B. alexandrina snail’s hemocytes towards S. mansoni are related to the degree of susceptibility of these snails. So this is important in planning the strategy of schistosomiasis control.     

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.This work was supported in part by funding from the USDA-SBIR program, 4Cs Breeding Technologies, and the Louisiana Sea Grant College Program.     

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.     

Cytotoxic effects of parasitism and application of venom from the endoparasitoid Pimpla turionellae on hemocytes of the host Galleria mellonella

In parasitoid species devoid of polydnaviruses and virus‐like particles, venom appears to play a major role in suppression of host immunity. Venom from the pupal endoparasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae) has previously been shown to contain a mixture of biologically active components, which display potent paralytic, cytotoxic, and cytolytic effects toward lepidopteran and dipteran hosts. The current study was undertaken to investigate if parasitism and/or envenomation by P. turionellae affects the frequency of apoptotic and necrotic hemocytes, hemocyte viability and mitotic indices in Galleria mellonella L. (Lepidoptera: Pyralidae) pupae and larvae. Our study indicates that parasitism and experimental envenomation of G. mellonella by P. turionellae resulted in markedly different effects on the ratio of apoptotic hemocytes circulating in hemolymph depending on the host developmental stages. The ratio of early and late apoptotic hemocytes increased in G. mellonella pupae and larvae upon parasitization and at high doses of venom when compared to untreated, null and Phosphate Buffered Saline (PBS) injected controls. In contrast, an increase in necrotic hemocytes was only observed in parasitized pupae at 24 h and no difference was observed in larvae. The lowest hemocyte viability values were observed with pupae as 69.87%, 69.80%, and 72.47% at 4, 8, and 24 h post‐parasitism. The ratio of mitotic hemocytes also decreased in pupae and larvae upon parasitization and at high doses of venom. Staining of hemocytes with annexin V‐FITC revealed green fluorescent ‘halos’ along the plasma membranes of venom treated cells within 15 min following exposure to venom. By 1 h post‐venom – treatment, the majority of hemocytes displayed binding of this probe, indicative of early stage apoptosis. These same hemocytes also displayed a loss of plasma membrane integrity at the same time points as evidenced by accumulation of propidium iodide in nuclei.     

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.     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>, a model system for functional validation of abiotic stress responsive genes

Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress specific. Although many of these have been cloned, their functional significance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for different abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further confirmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be efficiently validated for diverse abiotic stresses using Chlamydomonas system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Hema and M. Senthil-Kumar contributed equally.     

Direct Action of Insulin on Plasma Membrane ATPase Activity in Human Lymphocytes

LITTLE is known about the effects of insulin on lymphocytes. Helmreich and Eisen¹ concluded that it has insignificant effects, but others^2–5 have made a case for a role in inflammatory and immunological responses. We^6,7 have demonstrated that noradrenaline enhances the uptake of both glucose and potassium by lymphocytes, as does insulin in several tissues. We have associated this action of noradrenaline with a direct effect on membrane adenosine triphosphatase (ATPase) activity⁷. The observation⁸ that insulin bound to ‘Sepharose’ polymers enhances glucose transport while in contact only with the plasma membrane indicated that insulin might have a direct action similar to that of noradrenaline on membrane ATPase. The observations reported here show that insulin stimulates ATPase activity and glucose uptake in the lymphocyte and suggest a relationship between membrane ATPase activity and glucose transport.     

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.     

Minchinia nelsoni (Haplosporida) Disease Syndrome in the American Oyster Crassostrea virginica

SYNOPSIS. Minchinia nelsoni disease was studied in 3 populations of oysters from Marumsco Bar, Pocomoke Sound, Maryland. The native population was sampled regularly beginning in 1961 shortly after the disease invaded this area. Two populations of healthy year-old oysters were introduced (one in 1965 and one in 1966) into Pocomoke Sound and studied concurrently with the native population to determine the course of development of the disease and epizootic differences in populations. Gross and microscopic examination of oysters showed progressive stages of infection. Initial infection occurred in epithelia of the filtering organs (gill, palp, water tubes) and spread into connective tissue where hyaline hemocytes infiltrated. Intermediate infection was characterized by local infection and infiltration of connective tissue in and adjacent to epithelia of gill, palp, esophagus, stomach, gut, diverticula, and gonadal alveoli. Advanced infection was recognized by the general invasion and infiltration of connective tissue and the circulatory system by hyaline hemocytes. Terminal infections showed histologically massive pyknosis of nuclei and necrosis of tissues before outward signs of death were apparent. Remission of the disease was recognized by diminution of infection intensity and infiltration; localization of parasites near external epithelia; increased pigment cell formation; and diapedesis and deposition of necrotic parasites and tissue against the shell, followed by external conchiolinous encapsulation. Death from M. nelsoni disease may be attributed in part to seasonal environmental or physiologic stresses, which infected oysters, weakened and in poor condition due to the pathologic manifestations of the disease, are unable to tolerate. The gross pathologic signs of disease were: pale digestive gland, poor condition, mantle recession, weakness, and conchiolinous shell depositions. The microscopic signs were: diapedesis, relative decrease in numbers of phagocytes; relative increase in numbers of hyaline hemocytes; phagocytosis, fibrosis, cellular infiltration, abscess, ulceration, excessive pigment cell formation, mechanical disruption, pyknosis, and necrosis.