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Mechanism of lysosomal enzyme release from Mercenaria mercenaria granulocytes: A scanning electron microscope study
Abstract
A scanning electron microscope study of Mercenaria mercenaria granulocytes at 1 and 2 hr postchallenge with a 0.02-ml Millipore-filtered, sterile sea water suspension of heat-killed Bacillus megaterium at a concentration of 4 × 106 bacteria/ml revealed that (1) granulocytes challenged with bacteria revealed more and larger lysosomes protruding from their surfaces than those of the control groups; (2) release of intact lysosomes from granulocytes into serum occurred concurrently with phagocytosis; (3) enzymes released from discharged lysosomes acted on bacterial cell walls causing their partial degradation, thereby enhancing endocytosis; and (4) degranulation is a normal process but is greatly enhanced when stimulated by phagocytosis of bacteria. This study also demonstrated that lysosomes budded off from the plasma membrane of granulocytes into serum, although the actual biochemical mechanism(s) that causes the detachment of lysosomes from the plasma membrane and the subsequent lysis of the double-membraned lysosomes to release the hydrolases remains unresolved.
... Lysosome is a main cell structure against several species of grampositive and -negative bacteria by releasing various hydrolytic enzymes Mohandas et al., 1985). It can synthesize lysozyme enzyme in bivalve hemocytes and then secreted into the hemolymph during phagocytosis (Cheng et al., 1975), and could be used as an indicator of the health status and the vitality of the defense system in bivalves (Chu and La Peyre, 1989). ...
... It can synthesize lysozyme enzyme in bivalve hemocytes and then secreted into the hemolymph during phagocytosis (Cheng et al., 1975), and could be used as an indicator of the health status and the vitality of the defense system in bivalves (Chu and La Peyre, 1989). Among the hemolymph constituents, lysosome participates in the inactivation of invading microbes and is generally affected by environmental stress (Liu et al., 2004;Mohandas et al., 1985). In this study, extension of the lysosomal compartment Table 3 Summary of one-way ANOVA results on effects of salinity (S) on hemocyte mortality (HM), phagocytosis (Pha), esterase (Est), reactive oxygen species (ROS), lysosome (Lyso) and total hemocyte counts (THC) at two dissolved oxygen (DO) levels, respectively. ...
Marine hypoxia owing to nutrient enrichment of water bodies by anthropogenic activities has become a global problem for several decades. Another marine environmental factor, salinity, plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where hypoxia frequently occurs. The present study evaluated the interactive effects of hypoxia and salinity on immune functions of hemocytes in the green-lipped mussel Perna viridis, which widely distributes in estuarine and coastal waters of the Indo-Pacific region. Mussels were subjected to six combinations of oxygen levels (hypoxia: 1.5 mg O2 l− 1, normoxia: 6.0 mg O2 l− 1) and salinities (15‰, 22‰ and 30‰) for 168 h. Mussel hemocytes were sampled after 24 h, 48 h, 96 h and 168 h exposure, for analysis of immune functions including hemocyte mortality, phagocytosis, esterase, reactive oxygen species (ROS) production, lysosomal content and total hemocyte counts using flow cytometric methods. Thereafter, all treatments were recovered to normoxic condition (6.0 mg O2 l− 1) and salinity 30‰ for 24 h to study the recovery of immune functions from the combined hypoxic and hyposaline stresses. Interactive effects of dissolved oxygen (DO) and salinity on all immune parameters were observed during the experiment. Generally, hemocyte mortality was higher at low salinity than that at high salinity levels; higher hemocyte mortality was also noted when mussels were exposed to hypoxia compared with normoxia. Phagocytosis at low salinities and hypoxia was decreased compared to normal DO and salinity treatment, respectively. The activity of esterase and ROS production at low salinity and hypoxia were reduced, respectively. Lysosomal content and total hemocyte counts were lower at low salinity and hypoxia compared to other treatments, respectively. After 24 h recovery, hypoxia and salinity effects were still significant among previous treatments, indicating immune functions of P. viridis could not recover from combined stresses in a short time.
... Acid dependency may also be a safety mechanism, ensuring that Perforin-2 remains in a latent state so as not to damage the vesicular and cellular membranes of the phagocyte. Acid dependency likely evolved very early as lower metazoans such as sponges, corals, and mollusks have been shown to express Mpeg1 and have macrophage-like cells that phagocytose and eliminate foreign invaders through the use of lysosomal enzymes, reactive oxygen species, and cellular acidification (98)(99)(100)(101)(102)(103). However, the exact mechanism of acid driven pore formation remains to be elucidated. ...
The membrane attack complex (MAC) of the complement system and Perforin-1 are well characterized innate immune effectors. MAC is composed of C9 and other complement proteins that target the envelope of gram-negative bacteria. Perforin-1 is deployed when killer lymphocytes degranulate to destroy virally infected or cancerous cells. These molecules polymerize with MAC-perforin/cholesterol-dependent cytolysin (MACPF/CDC) domains of each monomer deploying amphipathic β-strands to form pores through target lipid bilayers. In this review we discuss one of the most recently discovered members of this family; Perforin-2, the product of the Mpeg1 gene. Since their initial description more than 100 years ago, innumerable studies have made macrophages and other phagocytes some of the best understood cells of the immune system. Yet remarkably it was only recently revealed that Perforin-2 underpins a pivotal function of phagocytes; the destruction of phagocytosed microbes. Several studies have established that phagocytosed bacteria persist and in some cases flourish within phagocytes that lack Perforin-2. When challenged with either gram-negative or gram-positive pathogens Mpeg1 knockout mice succumb to infectious doses that the majority of wild-type mice survive. As expected by their immunocompromised phenotype, bacterial pathogens replicate and disseminate to deeper tissues of Mpeg1 knockout mice. Thus, this evolutionarily ancient gene endows phagocytes with potent bactericidal capability across taxa spanning sponges to humans. The recently elucidated structures of mammalian Perforin-2 reveal it to be a homopolymer that depends upon low pH, such as within phagosomes, to transition to its membrane-spanning pore conformation. Clinical manifestations of Mpeg1 missense mutations further highlight the pivotal role of Perforin-2 within phagocytes. Controversies and gaps within the field of Perforin-2 research are also discussed as well as animal models that may be used to resolve the outstanding issues. Our review concludes with a discussion of bacterial counter measures against Perforin-2.
... In phagocytosis a distinct sequence of events can be identified, with particle recognition followed by adherence, uptake, destruction and disposal of the invading body (Feng, 1988). Intracellular degradation occurs by a range of lysosomal enzymes, although extracelluar digestion by elevated lysozyme has been observed in haemolymph samples of Mercenaria mercenaria Foley and Cheng, 1977;Mohandas et al, 1985). Phagocytosis requires the movement of cells -frequently termed amoebocytes for the manner of their locomotion -the spreading and rate of locomotion of which can be quantified as a haemocytic index. ...
p>The aim of this study was to provide a comprehensive set of quantitative and qualitative baseline responses at physiological, metabolical and immunological levels, in the Pacific oyster Crassostrea gigas (Thunberg), the European flat oyster Ostrea eduli (L.), and the Manila clam Tapes philippinarum (Adams and Reeve). The energetics of these species were compared across a matrix of temperature and salinity conditions. Field trials examined the effect of exposure of three O. edulis populations to infection by the protozoan parasite Bonamia ostreae , and enzyme electrophoresis investigated the genetic basis for any differences. Changes in immunocompetence were monitored from field samples and with controlled Vibrio anguillarum bacterial challenges. Haemolymph and haemocytic responses were recorded.
Filtration rate had the most significant effect on scope for growth (SFG) indices measured in all species. C. gigas showed a much wider range of filtration rates than O. edulis and consequently had much higher SFG. Optimum environmental conditions for C.gigas occurred at 20-25<sup>o</sup>C and 19-25�, compared with 20<sup>o</sup>C and 33� for O. edulis , and 15-20<sup>o</sup>C at 33� in T. philippinarum . Separate winter and summer physiological behaviour was detected in C. gigas and O. edulis , with the change occurring at 15<sup>o</sup>C and 10-12<sup>o</sup>C respectively. Body condition indices were inversely proportional to SFG and were probably related to the reproductive cycle. Temperature was shown to have the most significant influence on energetic factors, with salinity having little effect.
Field trials investigating Bonamia effects in three O. edulis populations found a significant, inverse size relationship with most of the physiological measurements.</p
... There is evidence of a kind of 'compound exocytosis' (Pickett & Edwardson, 2006) leading to granulocyte degranulation in Pomacea canaliculata . Granulocyte degranulation in bivalves (Ciacci et al., 2009;Cheng et al., 1975;Foley & Cheng, 1977;Mohandas, Cheng & Cheng, 1985;Rebelo et al., 2013) has been related to the release of lysozyme and other hydrolytic enzymes that may kill bacteria and fungi, and Ottaviani (1991) has reported lysozyme from haemocytes of a gastropod. Therefore, it is likely that granulocytes occurring in the intercellular spaces of the gill epithelium are there serving a defensive role in Pomacea canaliculata. ...
Ampullariids are freshwater gastropods bearing a gill and a lung, thus showing different degrees of amphibiousness. In particular, Pomacea canaliculata (Caenogastropoda, Ampullariidae) is an obligate air-breather that relies mainly or solely on the lung for dwelling in poorly oxygenated water, for avoiding predators, while burying in the mud during aestivation, and for oviposition above water level. In this paper, we studied the morphological peculiarities of the gill in this species. We found (1) the gill and lung vasculature and innervation are
intimately related, allowing alternation between water and air respiration; (2) the gill epithelium has features typical of a transporting rather than a respiratory epithelium; and (3) the gill has resident granulocytes within intraepithelial spaces that may serve a role for immune defence. Thus, the role in oxygen uptake may be less significant than the roles in ionic/osmotic regulation and immunity. Also, our results provide a morphological background to understand the dependence on aerial respiration of Pomacea canaliculata. Finally, we consider these findings from a functional perspective in the light of the evolution of amphibiousness in the Ampullariidae, and discuss that master regulators may explain the phenotypic convergence of gill
structures amongst this molluscan species and those in other phyla.
... Lytic enzymes used to degrade phagocytized foreign particles inside the phagolysosome can be released to the extracellular space (Mohandas et al., 1985), constituting part of the humoral component of the immune response. Humoral defenses comprise constitutive or inducible biosynthesis of proteins with bacteriolytic and opsonic functions (Loker et al., 2004;Montaño et al., 2011). ...
Immune cell characterization, immunological response and the associated gill oxidative balance were studied in the Patagonian freshwater mussel, Diplodon chilensis, using two microbiological immunostimulant models: Saccharomyces cerevisiae and Escherichia coli. Mussels were collected out of the breeding season in Paimún Lake and acclimated in the laboratory. Two exposure experiments were performed during two consecutive weeks: (1) mussels challenged with 500 yeast cells mL-1; and (2) mussels challenged with 1000 bacteria cells mL-1. Microorganisms were added in the water every two days, alternating with 6000 lyophilized cells of the green algae Scenedesmus vacuolatus mL-1. A control group, fed with S. vacuolatus, was set for each treatment. Morphological cell characterization was carried out in adherent hemocytes of D. chilensis hemolymph under control conditions. The most important cell type observed were the hyalinocytes (representing ca. 98% of the circulating cells), agranular cells with non-central polymorphic nucleus surrounded by cytoplasm; granulocytes (cells with cytoplasmic granules and non-central rounded nucleus) represented ca. 2%. Another two cell types were occasionally detected, binucleated hyalinocytes and hemoblast-like cells but were not considered for the analyses. Both adherent hyalinocytes and granulocytes exhibit phagocytic activity towards Congo red stained yeast, which was two-fold higher in granulocytes than in hyalinocytes, regardless of the applied challenge. Total hemocyte counts were diminished in mussels challenged with S. cerevisiae or E. coli. Hydrolytic and defense cellular enzyme activities were analyzed only for hyalinocytes. Both, S. cerevisiae and E. coli increased acid phosphatase activity. E. coli challenge diminished hemocyte lysosomal membrane stability and increased humoral phenoloxidase activity, while S. cerevisiae challenge did not affect any of these variables. Mussels challenged with E. coli showed increased gill antioxidant response without oxidative damage, while those challenged with S. cerevisiae showed no change in these variables.
... Susceptibility to parasite infection was determined by the genetic background of snail and parasites . Professional phagocytic cells termed hemocytes, dwelling in the tissues or circulating with the blood fluid of gastropods and bivalves, phagocytose or encapsulate pathogens, eliminating these with cellmediated cytotoxicity involving lysosomal enzymes and production of reactive oxygen species (Adema et al., 1991;Granath and Yoshino, 1983;La Peyre et al., 1995;McKerrow et al., 1985;Mohandas et al., 1985;van der Knaap and Loker, 1990). Depending on the species, molluscs may have either a single type or several functionally different categories of hemocytes, and these cells may originate from connective tissue or specialized organs, termed the amoebocyte producing organ (APO) in gastropods (Jeong et al., 1983), or from the white body organ in cephalopods (Claes, 1996;Cowden, 1972). ...
... Observation of histological material shows that mature granulocytes appear to aggregate near foreign agents and degranulate in the same way as molluscan haemocytes. This process was studied in quahog (Mercenaria mercenaria) by Mohandas et al. (1985) who showed that bacteria stimulate haemocytes to extrude intact lysosomes into the haemolymph, a process referred to as degranulation. The resulting release of lysosomal hydrolases is assumed responsible for associated host and non-host tissue damage (Feng 1988, Hose & Martin 1989, Watanabe 1999) and may be one mechanism by which bactericidal activity is seen to rise in lobster haemolymph after inoculation of formalin-killed bacteria (see Sinderman (1971) for review). ...
There is little published information on the disease status of the prawns on the north-west
shelf, yet these prawns (Fenneropenaeus merguiensis, Metapenaeus endeavouri, Penaeus
esculentus and Melicertus latisulcatus) form the basis of a commercial fishery worth in excess
of A$42 million in 2001-2002. There are also stocks of P. monodon on the shelf which form
an important source of broodstock for the developing aquaculture industry in Western
Australia, and potentially also for the Northern Territory and Queensland.
Unfortunately, prawns are infected with a variety of viral diseases, many of which have been
translocated to new areas with movements of the host prawn – mainly for aquaculture but in
some cases through frozen product destined for human consumption.
There is a zoogeographic barrier at the Torres Strait so there is no reason to expect that the
prawns in Queensland and New South Wales will have the same diseases as those in Western
Australia. This is particularly so since the northwest shelf has had little, if any, exposure to
other areas through translocations. This presents a unique opportunity to study the viruses
and other diseases that may have co-evolved in the area with the prawns. This isolation is
already under threat, with, for example, the movement for aquaculture purposes of Gill
Associated Virus (GAV) infected post-larvae from Queensland into the Northern Territory.
Thus, there are two disease risks for which this project provides background data. The first is
the importation into Western Australia of prawns from other states and from the Northern
Territory. To assess adequately the disease risk posed by the imports, we need to understand
the local disease status and this has been achieved. The second risk is that diseases endemic in
Western Australia may pose a risk to aquaculture establishments in other States. This report
provides a basis on which those states can assess the risk to their own industries.
During the five years of the project over 2500 prawns have been examined for disease, mainly
by histology, but also by molecular techniques for White Spot Syndrome, Yellow Head Virus,
and Gill Associated Virus. Most of the prawns were sourced from the wild fishery, but both
P. monodon and P. esculentus are now being spawned and on-grown in Western Australia
under pilot scale or commercial conditions. The disease investigations associated with these
nursery or grow-out operations have also been used in compiling this report. The two
problems so far encountered in these prawns under aquaculture conditions are Monodon
baculovirus-like virus (MBV-like virus) (in P. esculentus only) and bacterial problems (both
species).
Overall, Western Australian prawns are exposed to MBV-like virus and Hepatopancreatic
parvo-virus (HPV). Based on limited electron microscopy, an eosinophilic virus-like inclusion
in epithelial cells particularly in the midgut, and similar to HPV but with different staining
characteristics, may be a fixation artefact. There are a number of syndromes that may be
associated with un-recognised viruses but could equally be due to autolysis and fixation
artefacts. Further work is clearly required on these. There is also a rich fauna of metazoan
parasites in most of the wild prawns. These are of little concern for aquaculture but probably
deserve some taxonomic attention. The lack of MBV-like virus and HPV in P. monodon and
the absence of GAV in any species are of particular note.
... Las enzimas lisosomales dentro de los hemocitos, matan y degradan las partículas fagocitadas y en algunos casos, son liberadas de la célula al plasma u otros tejidos donde pueden modificar la conformación molecular de la superficie de las partículas y con ello favorecer su reconocimiento y fagocitosis. Por este motivo, la actividad enzimática en la hemolinfa ha sido estudiada como uno de los indicadores de la capacidad inmune en muchas especies de bivalvos, incluyendo C. virginica, M. mercenaria, Mya arenaria, Ostrea edulis, C. gigas, M. galloprovincialis, R. decussatus, Corbicula japonica y Pinctada imbricata (Cheng 1975, Rodrick 1975, Mohandas et al. 1985, Dikkeboom et al. 1988, Pipe 1990a, Kumazawa et al. 1991, Oubella et al. 1994, Cajaraville y Pal 1995, Xue y Renault 2000, Luna-González et al. 2004, Nusetti et al. 2004). Enzimas lisosomales como lisozima, fosfatasa ácida, esterasa y bglucoronidasa han sido usadas para definir la función de los hemocitos, en diferentes especies de bivalvos (Feng y Canzonier 1970, Chu y La Peyre 1989, Beckmann et al. 1992, Torreilles et al. 1997, Luna-González et al. 2004, Nusetti et al. 2004. ...
... Thus pathogen-associated molecular pattern recognition is exerted by humoral and hemocyte membrane receptors, triggering mechanisms of foreign particles elimination, such as phagocytosis, cellular aggregation and encapsulation, and reactive oxygen species production (ROS) [11,12]. In addition, enzymatic components such as acid phosphatase, alkaline phosphatase, and lysozymes participate in intracellular and/or extracellular degradation processes [13], whereas phenoloxidase plays a central role in encapsulation/melanization [14]. Both stimulation and suppression of immunological response variables by pesticides have been reported for the freshwater prawn Macrobrachium rosenbergii exposed in vivo to the organophosphorus pesticide trichlorfon (0 mg/L, 0.2 mg/ L, and 0.4 mg/L during 3 h, 6 h, 12 h, and 24 h) [15] and for the blue mussel Mytilus edulis exposed to the organophosphorus pesticide azamethiphos (0.1 mg/L for 24 h) [16]. ...
The aim of this work was to characterize the immune response (total hemocyte number, cell type proportion, hemocyte viability, lysosomal membrane stability, phagocytic activity, cellular acid and alkaline phosphatase activity, and humoral bacteriolytic and phenoloxidase activity) in Diplodon chilensis exposed to 0.2 mg/L of azinphos-methyl (AZM), using Escherichia coli as immunological and prooxidant challenge. Additionally, glutathione-S-transferase (GST) and lipid peroxidation (TBARS) were analyzed in gill tissue. Mussels from an unpolluted site were treated for three days as follows: 1) Experimental control
... Lysosomal enzymes within haemocytes participate in the killing and degradation of phagocytosed materials (Cheng, 1975Cheng, , 1981). In some cases, lysosomal enzymes are released from haemocytes into the plasma or extracellular compartments of other tissues, to degrade foreign materials (Mohandas et al., 1985). The occurrence of lysosomal enzymes has been demonstrated in haemocytes of some bivalve molluscs, Crassostrea virginica and Mercenaria mercenaria (Cheng & Rodrick, 1975a), Mya arenaria (Rodrick, 1975), Ostrea edulis and Crassostrea gigas (Chagot, 1989), Mytilus edulis (Pipe, 1990), Corbicula japonica (Kumazawa et al., 1991) and Mytilus galloprovincialis (Cajaraville *Author to whom all correspondence should be addressed. ...
... Lysosomal enzymes within haemocytes participate in the killing and degradation of phagocytosed materials (Cheng, 1975Cheng, , 1981). In some cases, lysosomal enzymes are released from haemocytes into the plasma or extracellular compartments of other tissues, to degrade foreign materials (Mohandas et al., 1985). The occurrence of lysosomal enzymes has been demonstrated in haemocytes of some bivalve molluscs, Crassostrea virginica and Mercenaria mercenaria (Cheng & Rodrick, 1975a), Mya arenaria (Rodrick, 1975), Ostrea edulis and Crassostrea gigas (Chagot, 1989), Mytilus edulis (Pipe, 1990), Corbicula japonica (Kumazawa et al., 1991) and Mytilus galloprovincialis (Cajaraville *Author to whom all correspondence should be addressed. ...
... Lysosomal enzymes within haemocytes participate in the killing and degradation of phagocytosed materials (Cheng, 1975Cheng, , 1981). In some cases, lysosomal enzymes are released from haemocytes into the plasma or extracellular compartments of other tissues, to degrade foreign materials (Mohandas et al., 1985). The occurrence of lysosomal enzymes has been demonstrated in haemocytes of some bivalve molluscs, Crassostrea virginica and Mercenaria mercenaria (Cheng & Rodrick, 1975a), Mya arenaria (Rodrick, 1975), Ostrea edulis and Crassostrea gigas (Chagot, 1989), Mytilus edulis (Pipe, 1990), Corbicula japonica (Kumazawa et al., 1991) and Mytilus galloprovincialis (Cajaraville *Author to whom all correspondence should be addressed. ...
... Some non phagocytizing granulocytes showed gigantic granules of high electron density which were probably the result of R granules' fusion. It is likely that these merging granules were participating in a kind of 'compound exocytosis' [42] resulting in granulocyte degranulation, as it has been shown in bivalves (e.g., [43,[44][45][46][47]) and has been related to the release of lysozyme and other hydrolytic enzymes that may kill bacteria, either in the circulation or in tissue. Release of lyzozyme immunoreactive molecules was increased in serum of an heterobranch gastropod after bacterial challenge [48]. ...
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.
... Antimicrobial factors (antimicrobial peptides, lysosomal enzymes, lectins) are synthesized in the hemocytes (Mohandas et al. 1985, Tunkijjanukij & Olafsen 1998, Mitta et al. 1999, Mitta et al. 2000, where they destroy bacteria, and when released into plasma contribute to a systemic microbial response (Cheng et al. 1975, Cheng 1986, Mitta et al. 1999. Lysozyme is found in oyster hemocytes and serum (Cheng & Rodrick 1974) as well as in mantle mucus (McDade & Tripp 1967a). ...
Fluid and its associatedmucus from the pallial (mantle) cavity of eastern oysters Crassostrea virginica (Gmelin) from Black RockHarbor, Bridgeport, Connecticut, inhibited growth of both Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Serratia marcescens, and Vibrio parahaemolyticus) bacteria in antimicrobial assays. In the presence of oyster fluid, E. coli resulted in significant reduction in growth after 26 h. Soluble lysozyme activity in pallial cavity fluid of oysters collected in the fall was 3 times greater than that measured in combined winter–spring–summer samples (P = 0.0008). During the course of the study, copper concentrations in pallial cavity fluid ranged from 0.60–2.49 ppm and zinc concentrations ranged from 9.7–61.0 ppm. Copper concentrations remained relatively constant throughout the study; the highest zinc concentrations were recorded in the fall. Fall antimicrobial assays showed heightened antimicrobial activity compared with the spring, which may be the
result of increased lysozyme activity and higher zinc concentrations present in the pallial cavity fluid at that time of year. Results of this study suggest that pallial cavity fluid and its associated mucus likely serve an important role in defense-related functions as the first line of defense against infections from environmental pathogens in Crassostrea virginica.
... 포도당, 총단백질 및 중성지방은 생체내에 영양대사의 중요한 물질로 알려져 있는데 특히 총단 백질에 있어서 큰 차이를 보인다는 것은 혈액채취 시기에 다소 차이가 있었다는 것을 고려하더라도 여 윔증 개체와 병원성 균주에 감염된 개체가 생리학적 영양대사에 차이를 가지는 것으로 생각된다. Lysozyme 활성은 살균 및 항 염증성 특성을 가지고 있어 항 기생충 작용, 항바이러스 작용 및 오염물질의 작용 등에 관여함으로써 생체의 방어기작에 다양한 역할을 하는 것으로 알려져 있다(Mohandas et al., 1985). 본 연구에서 두 균주를 접종한 후 혈청과 점액 에서 lysozyme 활성을 분석한 결과, 대조구에 비해 JV1, JE1 및 JV1+JE1 실험구에서 면역활성이 떨어지 는 양상을 나타냈으나 시간이 경과함에 따라 점차 유의적으로 증가하였다(Fig. ...
Two bacteria strains were isolated from emaciated olive flounder (Paralichthys olivaceus) in aquafarm and were identificated as Vibrio harveyi (JV1) and Edwardsiella tarda (JE1), respectively. In the challenge experiments, we found 100% cumulative mortalities in all of olive flounder injected with JV1, JE1 or JV1+JE1 within eleven days after the injection. Two bacteria strains were reisolated from dead fish and were analyzed using the PCR method. In the physilogical analysis, the hematocrit, AST, ALT and cholesterol levels in experimental groups were increased significantly compared to those in control group, but the glucose, total protein and triglyceride levels were significantly decreased. Additionally, the lysozyme activity in the blood serum was decreased. The histopathological observations of the intestine showed that all groups had detachment and destruction of epithelial tissues except for the control group.
... Phagocytosis is one of the most welldocumented cell responses to non-self-material [1,2]. Upon phagocytosis, foreign particles are degraded by lysosomal hydrolytic enzymes [3e6], which can be also released into extracellular spaces to neutralise non-self-material [7]. In addition, haemocytes can produce reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and intermediate compounds, that act as bactericidal agents [4,8]. ...
The smooth venus clam Callista chione is a commercially exploited bivalve species that lives on the sandy bottom of the Italian coast of the Northern Adriatic Sea. Currently, no information is available in the literature about the haemocytes of this bivalve species. In this study, we performed a morpho-functional characterisation of the haemocytes of C. chione. In freshly collected haemocytes, the total haemocyte count (THC) (measured by a Coulter Counter) varied markedly among individuals, and the mean number of haemocytes was 1.2 (x10(6)) cells mL haemolymph(-1). The mean values for the haemocyte diameter and volume were 4.2 μm and 77.8 fL, respectively. In some cases, higher THC values were related to a smaller haemocyte size, but no correlation was detected between the THC and haemocyte diameter or between THC and cell volume. Conversely, a positive correlation was observed between cell diameter and volume. Two haemocyte types were distinguished by light microscopy: granulocytes (76%), with evident cytoplasmic granules, and hyalinocytes (24%), with a few or no granules. After adhesion to slides and fixation, the cell diameter was approximately 10 μm for granulocytes and 7 μm for hyalinocytes. The granules of the granulocytes were stained in vivo with Neutral Red, indicating that they were lysosomes. The granulocytes and hyalinocytes were further distinguished as basophils and acidophils. Both the granulocytes and the hyalinocytes were able to phagocytise yeast cells. Of 2643 cells that were counted, 2007 (76%) showed phagocytic activity. The granulocytes and hyalinocytes were both positive for some hydrolytic enzymes, whereas they were not positive for peroxidase or phenoloxidase. The two types of haemocytes also produced superoxide anion. Overall, this preliminary study indicates that both the granulocytes and hyalinocytes of C. chione are immune effector cells.
Copyright © 2014. Published by Elsevier Ltd.
... Epididymal secretory protein E1, also known as Niemann-Pick type C-2, facilitates cholesterol transport from lysosomes [85]. The upregulation of epididymal secretory protein e1 uniquely in susceptible oysters at days 5 and 15 may represent lysomal turnover as an aspect of a continuous response to bacteria [86]. Cadherin is involved in cell adhesion [87], and legumain is a cysteine protease associated with response to bacteria [88], antigen processing [89], and ECM remodeling [90] in mammals. ...
The eastern oyster Crassostrea virginica is an ecologically and economically important estuarine organism. Roseovarius Oyster Disease (ROD), caused by the bacterium Roseovarius crassostreae, went unreported before 1988 and presently affects oysters from the Long Island Sound north to Maine. The identification of potential genes and pathways of the effective host defense response in the eastern oyster to R. crassostreae is important not only to provide a basis for enhanced breeding techniques, but also contributes to the understanding of innate immunity in a broader, evolutionary sense. The goal of this study is to uncover genes and general processes potentially involved in disease resistance to ROD in the eastern oyster and identify diversified gene families involved in immunity. ROD-resistant and ROD-susceptible families of oysters were exposed to R. crassostreae and cDNA from samples collected at 1, 5, 15, and 30 days after challenge was sequenced using Illumina GAIIx. Patterns of differential gene expression were analyzed at the gene and gene family levels. We used sequence similarity clustering to define gene families and further explored the size, composition, and phylogeny of several groups of diversified proteins. The eastern oyster host defense response to R. crassostreae involved serine proteases, serine protease inhibitors, c-type lectins, C1q domain-containing proteins, fibrinogen domain-containing proteins (FREPs), interferon-induced protein 44 family proteins, scavenger receptors with class B SRCR domains, and GTPase of the immunity associated protein family (GIMAP) proteins. Further, similarity clustering of proteins and translated transcripts from diverse invertebrates suggested that GIMAP proteins are expanded in molluscs and IFI44 proteins are expanded in bivalves. We have also identified several diversified gene families of unknown identity that appear to be involved in oyster immunity against R. crassostreae. This research provides candidate genetic markers to aid in the breeding of disease-resistant strains of oysters.
... Epididymal secretory protein E1, also known as Niemann-Pick type C-2, facilitates cholesterol transport from lysosomes [85]. The upregulation of epididymal secretory protein e1 uniquely in susceptible oysters at days 5 and 15 may represent lysomal turnover as an aspect of a continuous response to bacteria [86]. Cadherin is involved in cell adhesion [87], and legumain is a cysteine protease associated with response to bacteria [88], antigen processing [89], and ECM remodeling [90] in mammals. ...
The American oyster Crassostrea virginica, an ecologically and economically important estuarine organism, can suffer high mortalities in areas in the Northeast United States due to Roseovarius Oyster Disease (ROD), caused by the gram-negative bacterial pathogen Roseovarius crassostreae. The goals of this research were to provide insights into: 1) the responses of American oysters to R. crassostreae, and 2) potential mechanisms of resistance or susceptibility to ROD. The responses of oysters to bacterial challenge were characterized by exposing oysters from ROD-resistant and susceptible families to R. crassostreae, followed by high-throughput sequencing of cDNA samples from various timepoints after disease challenge. Sequence data was assembled into a reference transcriptome and analyzed through differential gene expression and functional enrichment to uncover genes and processes potentially involved in responses to ROD in the American oyster. While susceptible oysters experienced constant levels of mortality when challenged with R. crassostreae, resistant oysters showed levels of mortality similar to non-challenged oysters. Oysters exposed to R. crassostreae showed differential expression of transcripts involved in immune recognition, signaling, protease inhibition, detoxification, and apoptosis. Transcripts involved in metabolism were enriched in susceptible oysters, suggesting that bacterial infection places a large metabolic demand on these oysters. Transcripts differentially expressed in resistant oysters in response to infection included the immune modulators IL-17 and arginase, as well as several genes involved in extracellular matrix remodeling. The identification of potential genes and processes responsible for defense against R. crassostreae in the American oyster provides insights into potential mechanisms of disease resistance.
... For example, killing mechanisms associated with haemocytes involve reactive oxygen molecules, such as the phenoloxidase cascade, that are now known to be important for phagocytosis, melanisation and encapsulation. Inducible serum antimicrobial factors including lysozyme (a bacteriolytic protein) are released by degranulation when phagocytosis occurs (Cheng et al., 1975;Mohandas, Cheng and Cheng, 1985;Xu-Tao Hong, Li-Xin Xiang and Jian-Zhong Shao, 2006). However, the distinction between immunomediators, hormones and neurotransmitters, has become blurred by the finding that haemocytes can synthesis neuroendocrine peptide hormones and also have receptors for these peptides (Ottaviani andFranceschi, 1998a, 1998b). ...
The study of molluscan diseases has a long history. The first publication on the redial
stages of a trematode appeared in the 18th century; early papers on molluscan phagocytosis
appeared in the last half of the 19th century and yet much work published before about
1975 does not appear in electronic abstract databases and is effectively “lost”. By contrast,
a recent search of a leading abstract database for the terms “mollusc” and “disease” shows
that the number of publications has exploded in the last eight years and the exponential
trend looks set to continue. Much of the increase has been driven by the introduction of
molecular technologies, the rediscovery that the immunology of invertebrates generally
is a rich hunting ground for new biochemical defence systems and thus potential medical
breakthroughs and the desire to publish multiple papers from the same project. As this
publication trend continues, it will become increasingly difficult to be knowledgeable on all
aspects of molluscan diseases and considerable specialisation is inevitable.
It is not only our knowledge about known mollusc diseases that has grown, since new
diseases continue to be reported as: aquaculture becomes more intensive; the Asia/Pacific
regional skills base develops; and international reporting becomes more accurate. Transfer
of disease between jurisdictions is also becoming more rapid as products are sent live around
the world both as broodstock and for human consumption. Thus, the work of the Network of
Aquaculture Centres in the Asia and the Pacific and the Food and Agriculture Organization
of the United Nations in awareness raising and skills development will continue to make
an impact. It is inevitable that, as the initial work on mollusc diseases developed around
shellfish growing areas in Europe and America, the next generation of molluscan disease
experts will be based in the Asia and the Pacific region.
... b - Mohandas et al.1985 Bacillus megaterium Cheng, 2000 API ZYM Lopez et al., 1997c 30∞C 10∞C 10∞C 10∞C Xing et al.2002 2007 Cheng, 2000 Proctoeces maculatus Feng, 1988 Wootton et al.2006 ...
Many bivalve species are cultured widely around the world due to their commercial importance. As a result of the increases in areas and production associated with intensive culture, mass mortalities of bivalves caused by a wide variety of agents have become a serious problem. Various infectious diseases represented by protozoan and bacterial diseases have been threatening shellfish culture industries, and hence, many investigations have been made on the defense mechanisms, as well as diseases, in bivalves. The molluscan defense mechanism is comprised of the only innate immunity, which is also dependent on cellular components such as hemocytes possessing phagocytic and bactericidal activities and on humoral defense molecules such as lectin and lysozyme. In this review, we summarize cellular defense mechanisms in bivalve molluscs focusing on hemocytes: 1) morphology and classification, 2) functional profiles (phagocytosis, encapsulation, wound repair), 3) reactive oxygen species, 4) effects of natural and anthropogenic environmental factors, and 5) stress and signaling pathways.
... In this phase, lysosomal enzymes play a very important role. Mohandas et al. (1985) and Oubella et al. (1994) demonstrated that challenge with bacteria produced a hypersynthesis of lysosomal enzymes by haemocytes. A number of lysosomal enzymes were de-214 Dis Aquat Org 30: 209-215,1997 tected in Ruditapes decussatus haemocytes and the presence of acid phosphatase in the granules suggests that these granules are lysosomes (Lopez et al. in press). ...
Phagocytosis of foreign materials by haemocytes is an important aspect of the internal defence of bivalve molluscs. Two main haemocyte types can be distinguished in the haemolymph of the clam Ruditapes decussatus: granulocytes and hyalinocytes. The ability of clam haemocytes to phagocytose zymosan particles, Vibrio P1 cells and trophozoites of the protistan parasite Perkinsus atlanticus was demonstrated by means of in vitro assays. However, clam haemocytes did not phagocytose P. atlanticus zoospores in the assays. Granulocytes showed the highest phagocytic capacity in each assay. Phagocytic capacity of haemocytes was not significantly affected by clam age. An ultrastructural study of phagocytosis showed the following sequence of events: engulfment of particles by pseudopods, formation of a phagocytic vacuole, fusion of lysosomes with the phagocytic vacuole, and digestion of the particles giving rise to residual bodies that might be discharged.
... Phagocytised particles are generally degraded by lysosomal hydrolytic enzymes within haemocytes . In some cases, lytic enzymes, and lysozyme in particular, may be released into the haemolymph during phagocytosis, thus participating in inactivation of invading microbes (Cheng et al. 1975;Mohandas et al. 1985). Phagocytosis is generally associated with increased oxygen consumption (respiratory burst) and the production of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and intermediate compounds with high bactericidal activity (Pipe 1992). ...
In the last few years, the Manila clam (Tapes philippinarum) has been subjected to intense fishing effort in the Lagoon of Venice owing to their commercial importance. Because of the lack of data concerning fishing-induced immunomodulation in bivalves, the consequences of fishing impact on functional responses of clam haemocytes were investigated. Clams were seasonally collected from September 2000 to July 2001 from three sites: S. Angelo, a free-fishing area, and Chioggia, inside a licensed area for clam culture, in two zones characterised by fishing and non fishing, respectively. Haematocrit, phagocytosis, Neutral Red retention time, and lysozyme and superoxide dismutase activities were evaluated. No clear fishing effect was recorded, whereas a seasonal pattern of the cell para meters analysed was revealed. Alterations in functional responses of haemocytes seem to be more closely dependent on seasonal variations in both environmental parameters and physiological status of clams than on stress caused by fishing, and they may also be related to adaptation strategies of bivalves.
... Several studies have demonstrated that temperature and salinity modi¢cations can a¡ect marine invertebrate defence mechanisms involving haemocytes that contain hydrolytic enzymes implicated with other molecules in pathogen degradation (Gagnaire et al. 2006). These enzymes participate in killing and degrading microorganisms inside and outside of bivalve haemocytes (Cheng & Rodrick 1975; Cheng 1981 Cheng ,1992 Mohandas, Cheng & Bradford 1985; Cajaraville, Pal & Robledo 1995) or modify the surface molecular conformation of pathogenic organisms, therefore favouring phagocytic cells with pathogen recognition (Cheng 1981Cheng , 1983). Based on these antecedents, the aim of this work was to determine the relationship of some environmental conditions with the immunological and physiological parameters of C. gigas. ...
Triploid Crassostrea gigas were cultured during 13 months in Nestier-type oyster trays. The impact of environmental parameters on the physiological and immunological parameters was evaluated. Temperature, salinity and seston were recorded monthly. Seventeen oysters were sampled monthly for immunological and condition index (CI) analyses. Samples were obtained as a haemolymph lysate supernatant (HLS). Protein content was determined using the Bradford method. The activity of hydrolytic enzymes was determined using the API ZYM kit and the lysoplate assay. Seston showed different patterns throughout the cycle. Condition index showed a positive correlation with the protein content of HLS. Protein showed a negative correlation with temperature. Eleven hydrolytic enzymes were detected in samples and higher enzymatic activity corresponded to leucine arylamidase and esterase. Leucine arylamidase and lysozyme activity showed a positive correlation with temperature. Oyster mortality was 28% in our modules and 70% in the oyster farm. Oysters showed low values of CI and haemolymph protein content in summer–autumn when mortalities were observed in the culture system. This finding suggests that these stressed oysters may have insufficient energy to invest in their immune system. It appears that oyster mortality in the culture system resulted from a combination of animal overcrowding, high temperature and low salinity.
... Lysosomal hydrolytic enzymes then degrade phagocytised particles . In some cases, lytic enzymes, and lysozyme in particular, are released into the haemolymph during phagocytosis, thus helping to inactivate invading pathogens (Mohandas et al. 1985). Previous studies have demonstrated that many biotic and abiotic factors can alter haemocyte-dependent defence mechanisms in bivalve molluscs, thus reducing their defence capacities. ...
In the present study, the effects of differing salinities on some important functional responses of haemocytes from the clam,
Chamelea gallina, were investigated. The animals were kept for 7days at 28‰ (hyposalinity), 34‰ (control) and 40‰ salinity (hypersalinity),
and total haemocyte count (THC), haemocyte volume, phagocytosis, lysozyme-like activity (in both haemocyte lysate and cell-free
haemolymph) were measured. The survival-in-air test was also performed. Clams kept at 28‰ showed significantly increased THC
with respect to animals kept at 34 and 40‰. The analysis of haemocyte size frequency distribution highlighted that in clams
kept at 28‰ the haemocyte fraction of about 5μm in diameter and 50–100 femtolitre in volume increased markedly. Conversely,
in animals kept at 40‰ an increase was observed in the haemocyte fraction having about 8–10μm diameter and 400–500 femtolitre
volume. Higher phagocytic activity was recorded in haemocytes from control clams, with respect to that of clams kept at 28
and 34‰. Lysozyme-like activity in haemocyte lysate was shown to increase significantly in animals kept at 28‰ with respect
to that of clams kept at 40‰, whereas enzyme activity in cell-free haemolymph from clams kept at 34‰ was significantly higher
with respect to that of clams maintained at 40‰. A relationship between phagocytosis and lysozyme secretion is suggested.
The resistance to air exposure of clams kept at 28 and 40‰ was shown to decrease significantly; LT50 values fell from 7days in clams kept at 34‰ to 4 and 5days in those kept at 28 and 40‰, respectively. Results demonstrated
that salinity values far from 34‰ affects the functional responses of haemocytes and reduce the resistance of clams to exposure
to air.
... Lysosomal hydrolytic enzymes can then degrade phagocytised particles (Lopez et al., 1997). In some cases, lytic enzymes, and lysozyme in particular, are released into the haemolymph during phagocytosis, thus helping to inactivate invading pathogens (Mohandas et al., 1985). The bivalve immune system also involves the production of reactive oxygen species (ROS) (Pipe, 1992). ...
The effects of anoxia on the immune responses of a commercially important bivalve species, Chamelea gallina, were studied in two different experiments (24-h and 48-h exposure to deoxygenated seawater). After anoxic stress, the capability of clams to recover was also evaluated by maintaining animals for 24 h in aerated seawater. Total haemocyte count (THC), phagocytosis and lysozyme activity (in both haemocyte lysate and cell-free haemolymph) were chosen as cellular biomarkers of exposure to anoxic conditions. Anoxia significantly reduced THC with respect to controls in both experiments. However, clams which recovered from the 24-h anoxia test showed THC values similar to those of controls, whereas in the 48-h test recovery clam THC was similar to that of stressed clams. Significantly decreased phagocytic activity with respect to controls was also observed in both experiments. In the first experiment, haemocytes from recovery clams showed slightly higher phagocytic activity with respect to controls, whereas after 48 h of exposure haemocytes from recovery animals were unable to recover their phagocytic activity. After both 24-h and 48-h anoxia, lysozyme activity was significantly reduced in both haemocyte lysate and cell-free haemolymph. In recovery clams from 24-h anoxia, the lysozyme activity of lysate was significantly lower than in control and stressed clams, whereas in haemolymph it was significantly higher. Conversely, in recovery clams from 48-h anoxia, the enzyme activity of both media decreased significantly with respect to controls. Interestingly, an unexpected additional stress during the 48-h anoxia test was recorded: recovery clams spawned during the recovery phase. To validate the hypothesis that spawning acts as an additional type of stress, the survival-in-air test was also performed. Significant decreases in resistance to air exposure were observed after 48-h anoxia in both stressed and recovery animals. Results show that anoxia strongly affects the functional responses of haemocytes, reducing immunosurveillance in stressed clams. The present study also highlights that additional stress, such as spawning, may further decrease immunosurveillance and reduce the resistance of clams to exposure to air.
... Antimicrobial factors (antimicrobial peptides, lysosomal enzymes, lectins) are synthesized in the hemocytes (Mohandas et al., 1985;Tunkijjanukij and Olafsen, 1998;Mitta et al., 1999Mitta et al., , 2000 where they are engaged in the destruction of bacteria inside the hemocyte and are released into plasma for a systemic antimicrobial response (Cheng, 1986;Cheng et al., 1975;Mitta et al., 1999). These observations agreed with comparison studies between hemocytes or HLS and plasma antibacterial activity that concluded higher activity per unit of protein in hemocytes or HLS (Haug, 2004;Anderson and Beaven, 2001). ...
A screening study of in vitro antibacterial activity was conducted in marine bivalves with economical importance and widespread along the coast of Galicia (NW Spain). Hemocyte lysate supernatant (HLS) and plasma of Mytilus galloprovincialis, Ostrea edulis, Crassostrea gigas, Ruditapes decussatus, Ruditapes philippinarum, and Cerastoderma edule were incubated with Vibrio splendidus and Micrococcus sp. HLS and plasma for all the species demonstrated antibacterial activity, and C. edule had the highest activity per unit of protein in these hemolymph fractions. Significant differences were not found between HLS and plasma activities. Furthermore, antibacterial activity against Micrococcus sp. (Gram-positive) was stronger than against V. splendidus (Gram-negative).
Fluid and its associated mucus from the pallial (mantle) cavity of the ribbed mussel Geukensia demissa (Dwillyn) from Black Rock Harbor, Bridgeport, CT, inhibited the growth of both Gram-positive (Staphylococcus aureus) and Gramnegative (Escherichia coli) bacteria in antimicrobial assays. A significant reduction in size after 24 h was noted in E. coli grown in the presence of ribbed mussel fluid. Mussel-soluble lysozyme levels in the pallial cavity fluid averaged 0.019 + 0.018 relative fluorescence units/μg protein, and no seasonal pattern of lysozyme activity was found (P = 0.522). During the course of the study, copper concentrations ranged from 0.09 to 0.37 ppm and zinc concentrations from 0.17 to 0.66 ppm in the pallial cavity fluid. These values were only slightly higher than the concentrations of these metals found in seawater samples taken at the site, indicating only very low levels of sequestration of heavy metals by G. demissa in the pallial fluid cavity. A comparable study of oysters (Crassostrea virginica) from the same site found lysozyme levels 10 times higher and zinc concentrations two orders of magnitude greater than in that in mussels reported here (Brousseau et al. 2014). These results suggest substantial interspecies variation in profiles of defensive agents involved in antimicrobial activities of marine bivalves and highlight the need for additional studies to characterize these differences. © 2018 National Shellfisheries Association. All rights reserved.
Circulating hemocytes of the snail Biomphalaria glabrata, a major intermediate host for the blood fluke Schistosoma mansoni, represent the primary immune effector cells comprising the host's internal defense system. Within hours of miracidial entry into resistant B. glabrata strains, hemocytes infiltrate around developing sporocysts forming multi-layered cellular capsules that results in larval death, typically within 24–48 h post-infection. Using an in vitro model of hemocyte-sporocyst encapsulation that recapitulates in vivo events, we conducted a comparative proteomic analysis on the responses of hemocytes from inbred B. glabrata strains during the encapsulation of S. mansoni primary sporocysts. This was accomplished by a combination of Laser-capture microdissection (LCM) to isolate sections of hemocyte capsules both in the presence and absence of sporocysts, in conjunction with mass spectrometric analyses to establish protein expression profiles. Comparison of susceptible NMRI snail hemocytes in the presence and absence of sporocysts revealed a dramatic downregulation of proteins in during larval encapsulation, especially those involved in protein/CHO metabolism, immune-related, redox and signaling pathways. One of 4 upregulated proteins was arginase, competitor of nitric oxide synthetase and inhibitor of larval-killing NO production. By contrast, when compared to control capsules, sporocyst-encapsulating hemocytes of resistant BS-90 B. glabrata exhibited a more balanced profile with enhanced expression of shared proteins involved in protein synthesis/processing, immunity, and redox, and unique expression of anti-microbial/anti-parasite proteins. A final comparison of NMRI and BS-90 host hemocyte responses to co-cultured sporocysts demonstrated a decrease or downregulation of 77% of shared proteins by NMRI cells during encapsulation compared to those of the BS-90 strain, including lipopolysaccharide-binding protein, thioredoxin reductase 1 and hemoglobins 1 and 2. Overall, using this in vitro model, results of our proteomic analyses demonstrate striking differences in proteins expressed by susceptible NMRI and resistant BS-90 snail hemocytes to S. mansoni sporocysts during active encapsulation, with NMRI hemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed immune-relevant proteins (e.g., FREP2) compared to BS-90. Our data suggest that snail strain differences in hemocyte protein expression during the encapsulation process account for observed differences in their cytotoxic capacity to interact with and kill sporocysts.
La capacité bioindicatrice des mollusques bivalves vis-à-vis des métaux est actuellement reconnue et utilisée dans le cadre du biomonitoring. La compréhension de cette capacité nécessitait une étude plus fondamentale des voies d'intoxication, de transport et d'élimination des métaux. Une étude anatomique, cytologique et microanalytique des hémocytes, de la glande péricardiale et des reins a montré le rôle des granulocytes dans le transport puis le rôle de détoxication de la glande péricardiale et des reins. Le système lysosomal joue un rôle important. La nature du métal modifie la répartition au sein des organes. Le plomb est bioconcentré en association avec P, S, Ca dans les concrétions des cellules du rein distal. La microanalyse de rayons x a été, à ce titre performante. Une adaptation éventuelle à la présence de micropolluants métalliques a été recherchée par numération des hémocytes, suivi de l'activité enzymatique et des capacités de phagocytose. Il y a tendance à l'augmentation du nombre des hémocytes ; augmentation de l'activité phosphatasique acide mais pas de modification significative de l'activité de phagocytose. Les réponses cytochimiques et biochimiques des hémocytes en présence de métaux peuvent être proposées comme méthodes d'évaluation précoce (7 jours) des effets de la contamination des eaux par les métaux ; le niveau de bioaccumulation étant évalué par la méthode des transplantations seulement après trois semaines
Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods.Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense.No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.
The hemocytes of abalone (Haliotis asinina) were studied by light and electron microscopy in order to describe their main morphological features and to relate these to their role in immune defense. The cells are comprised of two differentiated types: agranulocyte or hyalinocyte and granulocyte. The hyalinocyte is characterized by the presence of several filopodia, a large nucleus with dense chromatin, a moderate amount of cytoplasm, microfilaments, oval and round-shaped mitochondria with rather dense matrix, a considerable amount of rough endoplasmic reticulum, few cytoplasmic granules, coated pits and vesicles, phagocytic vacuoles, and numerous large and small vacuoles. Like the hyalinocyte, the granulocyte possesses similar cytoplasmic organelles but in fewer number, and a peripheral organelle-free zone containing numerous dense granules of various types. The shape of the granules varies from round to oval to elongated forms. Several dense granules exhibit a crystalloid substructure that show close relationship to the plasma membrane. The average size of granulocytes is 9.68 ± 1.12 μm and hyalinocytes is 8.65 ± 0.77 μm.
In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered. © 2015, Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg.
Foreign elements, molecules, or organisms naturally or experimentally introduced into naive molluscs are, although with exceptions, phagocytosed (see reviews by Cheng, 1975, 1981). Of the two categories of hemocytes common to all bivalves, hyalinocytes and granulocytes (Cheng, 1981), the latter in Crassostrea virginica and Mercenaria mercenaria are found to be the most active from the standpoint of phagocytosis (Foley and Cheng, 1975). There are three ways, involving semi-permanent filopodia, classical endocytosis and afunnel-like pseudopodia, by which bacteria are engulfed by hemocytes. The uptake mechanism has been studied at the electron microscope level in Crassostrea gigas (Ruddell, 1971; Feng et al., 1977), C. virginica (Cheng and Cali, 1974; Cheng, 1975), and Mytilus coruscus (Feng et al., 1977). Furthermore, as a rule, such phagocytosed materials, if digestible, are degraded by intracellularly, and some are removed when cells containing such materials migrate to the exterior through epithelial borders (Cheng, 1977a). Since granulocytes are more phagocytic than hyalinocytes, and one of the major differences between these two types of cells is the occurrence of large numbers of cytoplasmic granules in granulocytes, further studies to resolve the nature of these granules revealed that those in C. virginica are electron-lucid vesicles each of which possesses a complex wall (Feng et al., 1971; Cheng and Cali, 1974; Cheng et al., 1974; Cheng, 1975). However, the granules of M. mercenaria granulocytes, which are membrane-bound vesicles containing a homogeneously electron-dense substance (Cheng, 1975; Cheng and Foley, 1975), have subsequently been demonstrated to be true lysosomes (Yoshino and Cheng, 1976). Furthermore, it has been established that when challenged with foreign materials, there is hypersynthesis of certain lysosomal hydrolases within the hemocytes and their subsequent release into serum (Cheng et al., 1975; Cheng and Butler, 1979; Cheng and Mohandas, 1985; Mohandas and Cheng, 1985). These lysosomal hydrolases, which are associated with intracellular degradation (Cheng and Cali, 1974; Cheng et al., 1974), have also been demonstrated to have antimicrobial properties (McDade and Tripp, 1967; Cheng, 1978).
Changes in water temperature and salinity are responsible for a variety of physiological stress responses in aquatic organisms. Stress induced by these factors was recently associated with enhanced reactive oxygen species (ROS) generation, which caused oxidative damage. In the present study, we investigated the time-related effects of changes in water temperature and salinity on mRNA expression and the activities of antioxidant enzymes (SOD and CAT) and lipid peroxidation (LPO) in the gills and digestive glands of the ark shell, Scapharca broughtonii. To investigate physiological responses, hydrogen peroxide (H(2)O(2)), lysozyme activity, aspartate aminotransferase (AspAT), and alanine aminotransferase (AlaAT) were measured in the hemolymph. Water temperature and salinity changes significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. H(2)O(2) concentrations increased significantly in the high-temperature and hyposalinity treatments. LPO, AspAT and AlaAT levels also increased significantly in a time-dependent manner, while lysozyme activity decreased. These results suggest that antioxidant enzymes play important roles in reducing oxidative stress in ark shells exposed to changes in water temperature and salinity.
The overall aim of the symposium is to provide a forum for information exchange between scientists and industry personnel on health management of lobsters. This information exchange will be achieved through the presentation of papers on topics of direct relevance to lobster health management and through panel discussions on issues raised by participants
Hemocytes of the hard clam, Meretrix lusoria (Röding), were studied using light and electron microscopy. On the basis of staining characteristics and morphological criteria, the hemocytes were classified into four major categories: hyalinocytes, larger eosinophilic granulocytes (LEGs), smaller eosinophilic granulocytes (SEGs) and fibrocytes. SEGs were the most numerous components of the hemocytes, while few hyalinocytes were observed under normal conditions in the clam. Morphological characteristics of the granulocytes differed from the granulocytes of marine bivalves that have been reported previously. Granules of LEGs, though, were similar to those of SEGs using light microscopy but were completely different with electron microscopy.
Crassostrea virginica hemocytes, separated from serum and suspended in a maintenance medium, were exposed to the ionophore A23187 + EDTA, A23187 + EGTA, and Escherichia coli + A23187 + EDTA. As controls, identically treated hemocytes were exposed to A23187, EDTA, EGTA, and nonenriched maintenance medium. The activity levels of acid phosphatase in the media associated with the experimental and control sets of hemocytes were assayed at five time intervals postexposure to the various additives. It was determined that A23187 + EDTA stimulated significantly higher levels of enzyme release, with an initial burst at 1 min or earlier, than A23187, EDTA, or nonenriched maintenance medium alone. It was also determined that A23187 + EGTA stimulated significantly less release of acid phosphatase than A23187 + EDTA, but more than A23187, EGTA, or nonenriched maintenance medium individually. In all of the combinations tested, exposure to A23187 + EDTA + E. coli stimulated the greatest release of acid phosphatase from oyster hemocytes. Atomic absorption analyses of the concentrations of Ca2+ and Zn2+ in the maintenance medium and cytosol of all categories of hemocytes revealed that there were rapid and high levels of efflux of Zn2+ from all three sets of experimental hemocytes, especially those exposed to A23187 + EDTA and A23187 + EDTA + E. coli, but only low levels of exocytosis of Ca2+, which were no different between experimental and control groups. This appears to be the first demonstration of the requirement of a chelator (EDTA) in an ionophore-stimulated release of a bioactive molecule from invertebrate cells and of a correlation between the efflux of Zn2+ with ionophore-stimulated secretion of an enzyme.
The occurrence of a number of lysosomal enzymes (Proteases, glycosidases, phosphatases, and esterases) inRuditapes decussatushaemocytes was demonstrated by cytochemical and colorimetric techniques. The levels of 18 enzymes tested monthly varied through the study period (18 months), although they did not conform to a seasonal pattern of variation. No important effect of clam age on enzyme activity levels of haemocytes was detected. In those cytochemical assays in which distinction between granulocytes and hyalinocytes was possible, lysosomal enzymes were only found in granulocytes. Phosphatase was detected inside cytoplasmic granules of granulocytes, suggesting the granules to be lysosomes. NADPH oxidase was not detected in clam haemocytes, which is consistent with the absence of oxidative metabolism coupled with phagocytosis in haemocytes of this clam species. Levels of lysozyme detected inside haemocytes were higher than in serum.
Diseases of shellfish are often classified as either infectious or noninfectious. While little is known about the influence of toxic chemicals on infectious diseases, some information is available on negative effects of xenobiotics on certain cellular and physiological functions, which may lead to disease. Early studies described gross and histopathological alterations caused by high concentrations of different chemicals. Subsequently, finer biological resolutions, including the effects of toxic substances on different tissues, cells, and physiological and biochemical processes in these animals, were obtained. Nevertheless, there are still only primitive perceptions about the exact relationships between toxic chemicals in the environment and the possible diseases they may cause in shellfish. Consistent with modern concerns about the impact of chemical mutagens and/or carcinogens in the aquatic environment, research has been concentrated on neoplastic diseases in shellfish populations from different geographic areas of the world. A common hypothesis, underlying approaches to this research, has been that cancerous diseases in clams, oysters and mussels may be caused by chemical pollutants present in the environment they inhabit. Results which support this hypothesis do not dominate the literature. Some studies of clams and mussels do not preclude a possible causal role for toxic chemicals. However, different studies have shown that shellfish inhabiting exceedingly polluted environments did not have neoplasms, whilst others revealed that neoplastic diseases were present in mussels from essentially pristine areas. Further research is necessary to achieve a more complete understanding of the association between toxic chemicals in the environment and diseases, including neoplasia, in indigenous shellfish populations.
The effects of extracellular products (ECP) and purified proteases from the protozoan parasitePerkinsus marinuson three host defence parameters (haemocyte motility, lysozyme and haemagglutinin) of the eastern oyster,Crassostrea virginica, were investigated. ECP with high proteolytic activities, as well as purified proteases, significantly decreased the random migration of haemocytes through micro-porous filters in Boyden chambers. Stimulation of haemocyte migration byP. marinuscells orP. marinuscell lysate was also dramatically reduced by ECP and purified proteases. Incubation of oyster plasma with ECP and purified proteases caused a significant decrease in lysozyme activity and also appeared to reduce haemagglutinin titres. These data suggest thatP. marinusECP, as well as the proteolytic fraction of the ECP, can modulate some defence parameters of oystersin vitro.
It has been 100 years since the publication of the landmark work of Kellog. Although much has been l e a r n e d about the anatomy and histology of Mercenaria mercenaria since that seminal work, we have a long way to travel on that tortuous pathway known as research. A case in point: although we now know much about the cytology, fine understand the life cycle very well and can spawn animals upon demand throughout the year independent of latitude; yet we have no knowledge concerning the fine structure and functions of follicle and nutritive cells of gonadal acini. In the past 15 years, however, substantial strides have been made in renal cytology, fine structure and general function largely due to the work of M.R Morse and her students. It is expected that investigators will direct concentrated studies of this type to all organ systems and cell types. A Mercenaria genome project should not be in the too distant future. A little more than 50 years ago, Thurlow Nelson stated that the eastern oyster, Crassostrea virginica, was the best known marine animal in the world; I expect that with continued and expanded research on thiscomplex, hearty and commercially impo mercenaria may well become the marine animal about which we have the most scientific information.
This chapter discusses the pests, parasites, diseases, and defense mechanisms of the hard clam, Mercenaria mercenaria. Clams, mussels, scallops, and oysters are harvested commercially for many centuries and have suffered from disease-caused mortalities throughout that time. The study of bivalve parasites, diseases, and defense mechanisms is relatively recent, however, driven largely by epizootic mortalities of oysters in the United States and Europe in the past half century. Investigations show that each molluskan group becomes infected by a similar array of organisms from viruses to copepods, although relatively few cause disease. The distinction between infection and disease is important. Infection refers to the establishment of a foreign organism in host tissues. Disease indicates damage to a body part, organ, or system, which may or may not be caused by an infectious agent, such that the affected organism no longer functions normally. In fact, an infection does not necessarily lead to disease. Many infectious agents are parasites that may cause localized tissue damage, but relatively little overall harm to their hosts.
The effects of tributyltin (TBT, as the chloride) on circulating cells from the clam Tapes philippinarum were investigated, in order to set up quick and reproducible in vitro bioassays to evaluate TBT toxicity in bivalve molluscs. Haemocytes, collected from the adductor muscle of clams, were exposed for 60 min at 25 °C to sublethal concentrations of TBT (0.01, 0.05, 0.1 µM) and the effects on uptake of the vital dye Neutral Red, and both superoxide dismutase (antioxidant enzyme) and lysozyme (bacteriolytic enzyme) activities were spectrophotometrically evaluated. Exposure of haemocytes to 0.05 µM TBT caused a significant increase (P < 0.05) in Neutral Red dye uptake compared with controls, whereas no differences resulted after exposure to 0.01 and 0.1 µM TBT. Enlarged lysosomes were observed in haemocytes exposed to 0.05 µM TBT. Moreover, in haemocytes treated with 0.05 µM and 0.1 µM TBT, superoxide dismutase activity significantly decreased (P < 0.05 and P < 0.01 respectively) with respect to that of controls. A significant decrease in lysozyme activity was also observed in haemocytes exposed to 0.05 (P < 0.01) and 0.1 µM TBT (P < 0.001). These results suggest a relationship between TBT exposure and alterations in functional responses of haemocytes in T. philippinarum. The proposed assays are sensitive, rapid and reproducible. They may be proposed as biomarkers, although their responsiveness needs to be more fully evaluated in haemocytes collected after clam exposure in both laboratory and field conditions. Copyright © 2002 John Wiley & Sons, Ltd.
Large numbers of the soft-shelled clam Mya arenaria from New Bedford Harbor, Massachusetts, exhibit a blood cell disease referred to as hematopoietic neoplasia (HN). Diseased hemocytes differ morphologically from normal hemocytes by their high nucleocytoplasmic ratio. The phagocytic activity, one of the internal defense mechanisms, was compared in the two populations. Surface receptors, essential for recognition during phagocytosis, were demonstrated for both populations using Con A staining. Adherence and uptake were measured after in vitro incubation with yeast. Examination of hemocytes with scanning electron microscopy showed that yeast cells adhered only to the surface of normal hemocytes, and not to diseased hemocytes. Quantitative analysis of adherence and uptake of yeast by hemocytes showed that diseased hemocytes were unable to adhere and ingest yeast. This is thought to be caused by differences in cytoskeletal organization. Finally, the activity of four lysosomal enzymes was used to evaluate intracellular degradation in normal and diseased hemocytes. Histochemical analyses showed diseased hemocytes to have higher than normal acid phosphatase, nonspecific esterases and β-glucuronidase activity. These results indicate that enzyme activity cannot be directly correlated to intracellular degradation. However, it is possible that stress on the lysosomal system may activate certain enzymes.
The morphology, tinctorial properties, ultrastructure and some functions of bivalve haemocytes are reviewed in relation to the simple division of these cells into granular and agranular haemocytes, as suggested by Cheng. Whereas granular haemocytes (granulocytes) form a distinct group, agranular haemocytes are heterogeneous in appearance and ultrastructure. Three types of agranular haemocytes are identified; blast-like cells, basophilic macrophage-like cells, and hyalinocytes. Also the early stages of granulocyte development, and spent granulocytes, may be agranular. The distribution of blast-like cells suggests haematopoiesis may be widespread in connective tissue, with further development of haemocytes in the haemolymph. Consequently, the haemocytes of bivalve haemolymph are less differentiated than vertebrate leucocytes, and their composition may vary greatly between individuals. Not all types occur in each bivalve species; scallops lack granulocytes, and the hyalinocyte is a poorly defined cell type in several groups. There is evidence of functional heterogeneity in granulocytes and macrophage-like cells, and the functions of haemocyte types cannot be reliably extrapolated between species. Brown cells (rhogocytes) are regarded as part of the urinary system overlapping in tissue distribution and some functions.
This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites.
The activity pattern of the lysosomal marker enzyme, acid phosphatase, was studied for 120 hr in the hemolymph of two clam species, Sunetta scripta and Villorita cyprinoides var. cochinensis, exposed to three sublethal concentrations of copper. Fifty specimens of S. scripta were exposed to each of the concentrations of copper (1, 3, and 5 ppm). Fifty specimens of V. cyprinoides var. cochinensis were exposed to 0.15, 0.30 and 0.45 ppm of copper. The enzyme activity was estimated every 24 hr. The results indicate that (1) the activity levels of hemolymph acid phosphatase in clams exposed to sublethal concentrations of copper vary from species to species and are also dependent on the concentration of metal ions used; (2) the metal ion can cause destabilization of the lysosomal membrane and the consequent release of the enzyme into the hemolymph or can trigger hypersynthesis of acid phosphatase, which is subsequently released into the hemolymph; (3) copper ions can inhibit the activity of the enzyme; and (4) depending upon the period of exposure and the concentration of the metal ion, enzyme synthesis can also be adversely affected.
The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme. With more prolonged incubation the majority of the soluble intracellular pool of acid ribonuclease and lysozyme was lost to the extracellular medium. No change in the total content of any of the hydrolases was noted during 180 minutes of incubation in vitro.
The morphological fate of the granules was studied by a histochemical method for acid phosphatase. After the phagocytosis of yeast cell walls there was a disappearance of acid phosphatase-positive granules and an accumulation of reaction product about the ingested particle. Experiments employing macrophages which were supravitally stained with neutral red also demonstrated the loss of neutral red-positive granules and the accumulation of the dye about the yeast cell walls.
These results strongly suggest that lysis of macrophage granules occurs following phagocytosis and that a portion of the granule contents are then resegregated within the newly formed phagocytic vacuole.
Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestallike appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals failed to reveal any corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, which are very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemma invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role of osmotic forces.
Molluscan hemocytes serve a variety of functions. It is known that these cells are involved in wound repair (Pauley and Sparks, 1965; des Voignes and Sparks, 1968; Pauley and Heaton, 1969; Ruddell, 1971b), shell repair (Wagge, 1951, 1955), nutrient digestion and transport (Yonge, 1923, 1926; Takatsuki, 1934a; Yonge and Nicholas, 1940; Zacks and Welsh, 1953; Wagge, 1955; Zacks, 1955; Owen, 1966; Purchon, 1968; Cheng and Cali, 1974; Cheng and Rudo, 1976; Cheng, 1977), excretion (Durham, 1892; Canegallo, 1924; Orton, 1923), and internal defense, i.e., cellular immunity (Stauber, 1950, 1961; Tripp, 1958a; b, 1960; Feng, 1959, 1965; and critical reviews by Cheng, 1967; Cheng and Rifkin, 1970; Cheng, 1979).
The esterase enzymes of the tissues of Atlantic herring (Clupea harengus harengus) were analyzed by starch gel electrophoresis. Four sets of esterase bands were distinguished by their electrophoretic mobility, their relative activity with the two substrates, alpha-naphthyl acetate and alpha-naphthyl butyrate, and their relative concentrations in plasma, liver, and heart tissues. All of the esterases were inhibited by 10−4M solutions of dichlorvos, an organophosphate inhibitor, but none was inhibited by 10−4M eserine sulfate or by 10−4 M EDTA. Polymorphism was noted in all four sets of esterases. Evidence for the genetic control of the fastest migrating set was obtained from population genetic analyses. In this set of esterases, five distinct bands occurred either singly or in pairs. The observed distribution of the three most common bands fits the hypothesis that they are controlled by a set of autosomal alleles. The two rarest bands occurred only in the heterozygous state, as would be expected. Differences in frequencies of two of the genes were detected between herring taken in Western Maine waters and on Georges Bank.
Phagocytosis and related phenomena represent integral features of inflammation in all metazoans. Reviewed herein are the results of studies directed at understanding the role(s) of lysosomal enzymes synthesized and released from circulating hemocytes, especially granulocytes, of gastropods and bivalves as a result of challenge with exogenous, nonself materials. From what is known, most of the mechanisms underlying this inflammation-associated process parallel those of mammalian macrophages; however, immunoglobulins and most probably components of complement are not involved. The required energy for phagocytosis in molluscs appears to be derived from glycolysis alone. Furthermore, nitroblue tetrazolium reduction and the myeloperoxidase-H 2O 2-halide antimicrobial system, both characteristic of mammalian phagocytes, appear to be absent in molluscs. It is concluded that by studying phagocytosis by molluscan hemocytes, a great deal can be learned about the evolution of inflammatory response and its constitutent elements.
The effects of natural infections by rediae of the heterophyid Euhaplorchis californiensis on the digestive gland cells of Cerithidea californica (Gastropoda: Prosobranchiata) were studied through the use of light and electron microscopy. Pathological changes in secretory and digestive cells, including the disruption of digestive vacuoles and cell-cell junctions, the deformation of lumenal microvilli, the lysis of distal plasma membranes, and nuclear polymorphism, usually accompany the migration of larval trematodes into the digestive gland. Degenerating secretory cells are further characterized by Golgi complexes which assume an inflated appearance and a reduction of rough endoplasmic reticulum in the cell's perinuclear region. Finally, an increase in excretory and autophagic activities in the digestive gland cells of infected snails may be considered possible mechanisms by which the host is able to compensate for adverse metabolic or nutritional stresses of trematode parasitism.
Amine-citrate buffer systems for pH control in starch gel electrophoresis gave good resolution of some dehydrogenase isozymes. The pK's of three new amine buffers, N-(3-aminopropyl)-morpholine, pK2 25 C, 6.12; N-(3-aminopropyl)-diethanolamine, pK2 25 C, 6.90; and 1,3-bis(dimethylamino)-2-propanol, pK2 25 C, 7.55, were determined at 5 C intervals in the range 10–40 C. These compounds, together with N, N-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bis-Tris) and tris-(hydroxymethyl)-methylamine(Tris), provide a series of amine buffers with pK's at 0.5 unit intervals in the pH range 6.1–8.1.
An aqueous suspension of a granulosis virus (GV) from Plodia interpunctella (Hübner) controlled P. interpunctella in stored inshell 'Nonpareil' almonds for 134 days. As a result, feeding damage to the treated nuts was substantially reduced, and the percentage of rejects was decreased by as much as 88%. Virus suspension stored at −80°C for 18 months lost no activity.
Three of 5 native populations and a laboratory strain of Plodia interpunctella (Hübner) were found resistant to malathion. A susceptible population stressed with malathion showed a 110-fold increase in resistance in 5 generations. Reciprocal crosses of malathion-resistant and malathion-susceptible Indian meal moths showed that transport of resistant insects into an area where only susceptible moths are found may cause an increase in resistant moths in that area.
Oyster hemolymph lyses suspensions of some gram-positive bacteria and degrades cell walls isolated from Micrococcus lysodeikticus with the release of amino and reducing sugars. This indicates that the bacteriolytic substance in oyster hemolymph is lysozyme.
The total serum protein concentrations and levels of aminopeptidase and lysozyme activities in the sera of the gastropod Biomphalaria glabrata have been determined. The groups of snails from which hemolymph samples were taken for study included (1) untampered controls, (2) sham-injected snails, (3) heat-killed Bacillus megaterium-injected, and (4) live B. megaterium-injected ones. Our results indicate that there are significant elevations in the levels of aminopeptidase activity in 2 hr in the sera of snails that had been sham-, dead bacteria-, and liver bacteria-injected. The levels of lysozyme activity were not altered in sham-, dead bacteria-, and live bacteria-injected snails. This is contrary to an earlier finding (T. C. Cheng, M. J. Chorney, and T. P. Yoshino, 1977. J. Invertebr. Pathol., 29, 170–174), and the difference is believed to be due to the age of the snails employed. Comparisons of total serum proteins have revealed that the concentration in snails injected with live B. megaterium is significantly higher than in sham-injected ones. This may be due to increase of some yet undetermined serum protein fraction.
The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.
The hemolymph cells of Mercenaria mercenaria were studied with the transmission electron microscope. Three morphological types of cells, granulocytes, hyalinocytes, and fibrocytes, are distinguishable and their fine structural characteristics are described. However, as a result of analyzing the fine structural features of the so-called fibrocytes of M. mercenaria, i.e., the inclusion of large aggregates of glycogen granules in their cytoplasm and the occurrence of primary phagosomes enclosing partially degraded exogenous material and digestive lamellae, it is suggested that fibrocytes are actually granulocytes which are at the terminal phase of their physiologic cycle relative to the degradation of phagocytized nonself materials. The cytoplasmic granules of M. mercenaria granulocytes are structurally different from those of Crassostrea virginica in that they are delimited by a unit membrane, rather than by a complex wall, and include a homogenously electron-dense material. Lipidlike droplets are reported from both granulocytes and hyalinocytes of M. mercenaria for the first time.
Human PMN release lysosomal enzymes (ß-glucuronidase, acid phosphatase) when exposed to immune complexes, but do not release cytoplasmic LDH. The cells remain viable, and failure of LDH to appear in supernatants is not due to selective absorption or inactivation. Release of enzymes is not due to platelet contamination and is only partially enhanced by fresh serum. The selective release of lysosomal enzymes after uptake of complexes resembles that induced by inert particles of zymosan, and can be distinguished from the concurrent release of all enzymes after cell death induced by membrane-lytic crystals of MSU. Uptake of complexes, zymosan, or MSU particles is accompanied by concomitant increases in C-1 oxidation of glucose. Although MSU-induced damage can be retarded by the presence of Tris buffer, immune complexes and zymosan selectively release lysosomal hydrolases in the presence or absence of Tris buffer.
Agents which elevate the level, within cells, of cAMP (PGE1, theophylline, 2-CA) and cAMP itself inhibit the selective extrusion of acid hydrolases from leukocytes without affecting the viability of cells. Leukocytes may respond to immune particles by regurgitating a portion of their lysosomal hydrolases during phagocytosis.
Levels of lysozyme activity have been determined in the serum and cells of untreated Biomphalaria glabrata and in snails that had been challenged with heat-killed Bacillus megaterium and water at 1, 2, and 4 hr postinjection. Lysozyme activities have also been ascertained in sham-injected snails at 1, 2, and 4 hr postchallenge. Our results indicate significant alterations in the serum lysozyme activity levels at 2 and 4 hr postchallenge with bacteria and at 1 hr postinjection of water. Also, there is a significant increase in cell lysozyme activity at 1 hr postchallenge with B. megaterium. It is concluded that lysozyme is released from phagocytes into serum as a result of challenge with B. megaterium. Although the exact role of the released enzyme is uncertain, it is hypothesized that it may serve as a humoral defense molecule.
Previously reported results on natural populations of brown trout (Salmo trutta L.) urged further studies using additional electrophoretically detectable loci. Samples of brain, eye, heart, kidney, liver, and muscle from approximately 50 specimens from each of two populations and a large number of muscle samples from several additional populations were examined. Electrophoretic and staining methods for the 37 enzymes studied are given in detail. Of a total of 69 loci detected, 54 loci were considered usable in population genetics screenings. The expression of the loci coding for these enzymes is described and interpreted. The results presented will serve as a basis for a more detailed examination of genetic variation in brown trout populations.
Many delayed-type reactions contain large infiltrates of basophils whose function is unknown. We have studied these cutaneous basophil hypersensitivity (CBH) reactions in guinea-pigs to ascertain whether basophils that are recruited to delayed reaction sites could be triggered for immediate reactivity. We compared 24 h CBH reactions with nearby skin for immediate hypersensitivity by challenging each site with small amounts of antigen. CBH sites had augmented immediate increases in vascular permeability detected by extravasation of Evan's blue dye. The ability to elicit this augmented anaphylactic phenomenon correlated with the local presence of basophils, and light microscopy at CBH reactions 15 min after antigen challenge showed a 50% decline in basophil counts. Electron microscopy showed that progressive anaphylactic-type degranulation of local basophils occurred within minutes following reintroduction of antigen. There was fusion of vacuoles containing granules, exocytosis of granules, and dissolution of granules, without ultrastructural disruption of cellular integrity. These results establish that basophils in CBH reactions can be triggered with soluble antigen to undergo anaphylactic degranulation, with the immediate release of vasoactive mediators. We have termed this phenomenon 'cutaneous basophil anaphylaxis'. Thus, one function of basophils at sites of delayed hypersensitivity may be to provide the potential for augmented, local, immediate anaphylactic reactivity.
The levels of lipase activity in both the cellular and serum constituents of the hemolymph of Biomphalaria glabrata that had been challenged in vitro to beat-killed and sonicated Bacillus megaterium as well as samples challenged with live B. megaterium were ascertained. There were no significant alterations in the levels of enzyme activity in both cells and serum of the samples that had been challenged with sonicated bacteria; however, there was a signficant elevation in the enzyme activity associated with both the cells and serum of hemolymph that had been challenged with live bacteria. It has been concluded that live B. megaterium can stimulate hypersynthesis of lipase, a lysosomal enzyme, in phagocytes of B. glabrata and that this enzyme subsequently is released into serum. Consequently, the hydrolysis of lipid constituents of bacteria could theoretically occur in serum as well as within phagocytes.
The loss of cytoplasmic granules, which have been shown to be lysosomes, from granulocytes of Mercenaria mercenaria during the in vitro phagocytosis of Bacillus megaterium has been demonstrated semiquantitatively. This process, designated as degranulation, represents the morphological basis of the release of lysosomal enzymes from granulocytes into serum as- sociated with phagocytosis. In addition to degranulation associated with phagocytosis, there is rearrangement of lysosomes, the appearance of large vacuolated areas in the cytoplasm, con- traction of cell margins, and the appearance of atypically large granules in granulocytes of M. mercenaria.
The levels of aminopeptidase activity in the serum and hemolymph cells of the American oyster, Crassostrea virginica, have been determined to be 2.89 ± 2.25 and 0.62 ± 0.39 Sigma units/ml, respectively. Low levels of aminopeptidase activity have been demonstrated cytochemically within cytoplasmic granules, i.e., secondary phagosomes, of circulating hemolymph cells. Exposure of whole hemolymph to heat-killed Bacillus megaterium or to sterile sea water results in a significant increase in cellular aminopeptidase activity, with the level of activity being the highest in cells that had been exposed to bacteria. The level of aminopeptidase activity in serum is unaltered in similarly challenged whole hemolymph. It is concluded that aminopeptidase synthesized in cells during phagocytosis or as a result of stimulation by exposure to sea water is not released into the serum but is retained intracellularly. Our studies suggest that it is the intracellular and not the serum aminopeptidases whith are of primary importance in the degradation of B. megaterium.
Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.
Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.
1.1. The activities of lysozyme, acid and alkaline phosphatases, β-glucuronidase, amylase, lipase, glutamic-oxalacetic transaminase, and glutamic-pyruvic transaminase in the whole hemolymph and 4000 g pellets and supernatants of Crassostrea virginica and Mercenaria mercenaria were assayed. All of these enzymes, except for amylase, occurred in whole hemolymph as well as in the fractions of both species of molluscs.2.2. Amylase only occurred in the whole hemolymph and serum of C. virginica. Since this mollusc possesses a crystalline style, the amylase is believed to have originated from this structure.3.3. It is postulated that the lysosomal enzymes detected in the serum of both species of molluscs had been released from certain hemolymph cells and may play a role in destroying certain invading organisms.
Quantitative determinations of lipase activity in the sera and hemolymph cell homogenates of Mya arenaria that had been challenged with heat-killed Bacillus megaterium and those of control clams have revealed that there is an increase in both fractions of the hemolymph during phagocytosis.The occurrence of lipase in serum and cells suggests that suitable substrates could be degraded by hydrolysis extracellularly as well as within phagocytes.
Acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase) activity within electron-opaque, membrane-bound vesicles of Mercenaria mercenaria granulocytes has been localized by employing cytochemistry at the light and electron microscope levels. These vesicles can now be considered lysosomes. They presumably function, at least in part, as storage organelles for acid hydrolases, and are therefore analogous to the granules in mammalian polymorphonuclear and monocytic leucocytes. Lysosomes containing acid phosphatase are probably the sources of this enzyme found in cellular and serum fractions of the hemolymph of M. mercenaria, although the mechanism for enzyme release remains uncertain.
Histochemical studies of the digestive gland of Lymnaea truncatula have revealed features of the normal digestive processes in which at least 3 cell types are involved: digestive cells, mucus cells and basiphil cells. An intracellular vacuolar digestive system containing lysosomal enzymes appears to operate in the digestive cells. Parasitization by Fasciola hepatica evokes considerable structural and histochemical changes in the host digestive gland. There is, in general, increased cellular vacuolation together with increased lysosomal enzyme activity. The overall effect appears to be an increase in intracellular digestive processes, including autolysis, and it is suggested that this may be a consequence of the host attempting to maintain its nutritional requirements under parasitic stress. Starvation of un-infected specimens induces structural and histochemical changes which closely resemble the effects of parasitization.
Lysozyme activity has been demonstrated in the hemolymph of the soft-shelled clam, Mya arenaria. When whole hemolymph is centrifuged at 4000 and 10,000 x g and each constituent is assayed, lysozyme activity is found to be greater in the two supernatants than in the corresponding pellets.The lysozyme from M. arenaria hemolymph is salt dependent, relatively heat stabile, very sensitive to alterations in ionic concentration and the presence of heavy metals, and has an optimal pH of 5.0 when 0.1 M glycylglycine, 0.1 M imidazole, or 0.1 M phosphate buffers are employed but an optimal pH of 4.5 when 0.1 M Tris-HCl buffer is used.A Hill plot of the data resulting from salt reactivation studies indicates that the lysozyme in M. arenaria hemolymph includes at least 2.0 interacting binding sites for NaCl and KCl.When tested against a number of bacteria, the lysozyme is most active against Micrococcus lysodeitikus and Bacillus megaterium. It is less active against Proteus vulgaris, Salmonella pullorum, Shigella sonn...
This is a good review with many references. The subject matter is classified as follows: definitions of endocytosis (phagocytosis and pinocytosis); and general considerations of the reticuloendothelial system. A chapter called Functional activities of cell organelles in endocytosis is divided in 4 subchapters: attachment to the cell surface; engulfment (ingestion or internalization); interaction of phagosomes and lysosomes; and other organelles. One page deals with phagocytosis in pathologic conditions. It is followed by a chapter called General considerations: effect of external agents on phagocytosis; janitorial versus kamikaze phagocytosis; role of phagocytosis in the immune response; and other examples of endocytosis.
Lysozyme activity has been demonstrated in both the supernatant and pellet fractions of whole hemolymph of the American oyster, Crassostrea virginica, subjected to centrifugation at 4000 and 10,000 × g. In each case the enzyme activity is greater in the supernatant than in the pellet.The lytic activity of the molluscan lysozyme on Micrococcus lysodeikticus, like that of egg-white lysozyme, is salt dependent, is relatively heat stable, and is very sensitive to changes in ionic concentration. The optimal pH of the molluscan enzyme, however, ranges from 5.0 to 5.5, depending on the buffer employed.When tested against a number of bacteria, the oyster lysozyme has been found to be active against not only M. lysodeikticus but also Bacillus subtilis, B. megaterium, Escherichia coli, Gaffkya tetragena, Salmonella pullorum, and Shigella sonnei, although it is less active against the last four mentioned. It is not active against Staphylococcus aureus.It is postulated that the lysozyme in the serum of C. virginica has its origin in cytoplasmic phagosomes of granulocytes and is released when these organelles become ruptured.
Anaphylactic degranulation of guinea pig basophilic leukocytes, induced in vitro either with Concanavalin A or sheep serum (antigen), was resolved by transmission electron microscopy into two phases: (1) fusion of cytoplasmic granule membranes to form degranulation sacs communicating with the extracellular space by narrow pores and (2) resolution of degranulation sacs with concomitant granule matrix extrusion. Fusion of granule membranes occurred in the absence of obvious alterations of cytoplasmic filaments or microtubules but was preceded by a rapid increase in the number of 50- to 70-nm. cytoplasmic vesicles, a process evident 1 minute after exposure to lectin. By 5 minutes and at later intervals up to 20 minutes, as individual granule membranes fused to form degranulation sacs, vesicle frequency plunged to values one-half or less of control levels. Cytoplasmic vesicles were apparently incorporated into degranulation sacs and may have had a role in joining together the membranes of adjacent granules. Histamine release, detected at 5 minutes and maximal at 20 minutes, occurred at times when communications between degranulations sacs and the extracellular space were so narrow as to retain most recognizable granule matrix material. Resolution of degranulation sacs proceeded over a period of a day in culture and, in Concanavalin A-induced anaphylaxis, was sometimes incomplete even after 36 hours. During this phase, the frequency of cytoplasmic vesicles returned to normal or supernormal values, and the thin cytoplasmic processes forming the walls of degranulation sacs developed prominent, longitudinally disposed cytoplasmic filaments and ultimately retracted into the main cell body, depositing the membrane-free cytoplasmic granule matrix material outside the perimeter of the cell. Guinea pig basophil anaphylactic degranulation thus differs morphologically and kinetically from mast cell and basophil degranulation in other species in which granule membrane fusion and granule matrix extrusion occur nearly stimultaneously and are complete within minutes. The guinea pig basophil provides a useful model for dissociating these two intrinsic components of the degranulation process.
The fetus can be considered an allograft with up to one-half of its MHC antigens being potentially recognized by the mother as foreign. This study compares expression of OKT3, OKT4, OKT8, Kappa, Lambda and Ia antigens on lymphocytes in the peripheral blood of normal non-pregnant women, normal pregnant women, patients who are chronic spontaneous aborters and pregnant insulin-dependent diabetic women. Monoclonal antibodies and cytofluorometric analyses were used for these determinations. There were no significant differences (P = 0.01) between these groups for T-cell markers. A statistically significant (P = 0.001) increased ratio of cells bearing surface immunoglobulin to those expressing Ia antigen (K&L/Ia) was observed between normal non-pregnant controls and women with a history of chronic spontaneous abortion. It is concluded that T-lymphocytes in the peripheral blood do not demonstrate a phenotypic abnormality that would account for the non-rejection of the fetal allograft; however, women with chronic spontaneous abortion may have abnormal B-cell differentiation or T-cell activation that mediates chronic spontaneous abortion.
Cutaneous lesions elicited in guinea pigs by primary and secondary feeding populations of the argasid tick, Ornithodorus tartakovskyi, were analyzed by light and electron microscopy. Small clusters of basophils appeared at primary bite sites within 24 hr of tick attachment, and by 72 hr constituted approximately 11% of the total leukocytes. Secondary feeding sites exhibited an augmented cellular infiltrate that was dominated by basophils at all times (48-56% of total cells). Eosinophil proliferation was minimal, however, and the remaining cells were of the mononuclear type. Despite mounting a strong cutaneous basophil response of the kind that mediates immune rejection of prolonged-feeding ixodid ticks, the guinea pigs showed no resistance to the fast-feeding Argasidae. It is suggested that argasid ticks probably complete their blood meal prior to basophil arrival at the bite site. Electron microscopy indicated that the number of epidermal Langerhans cells increased with time in both primary and secondary lesions; these cells were more numerous in challenge infections however, and seemed also to occur in the dermis. Basophils at secondary bite sites exhibited three kinds of structural alterations classified as: (1) piecemeal alterations--involving a vesicular degranulation mechanism; (2) an anaphylactic-type of alteration--involving single or compound exocytosis of whole granules; and (3) cytotoxic alterations culminating in complete disintegration. The majority of basophils in 72 hr secondary lesions exhibited cytotoxic alterations. It is suggested that such changes result from contact with tick-derived toxins or enzymes.
Electron microscopy has been used to monitor cellular activity in dermal lesions elicited by larval Rhipicephalus appendiculatus feeding on actively sensitised guinea pigs and recipients of immune serum. The early primary response is characterised by mononuclear cells, many of which appear to be activated fibroblasts. Collagen deposition is enhanced as the reaction progresses. Granulocytes of all types appear in the lesion between 18 and 96 hr but they show no evidence of degranulation. Free, membrane-bounded eosinophil and basophil secretion granules may, however, be identified in the dermis at day 5 or 6, but they seem to be liberated as a consequence of cellular disruption, rather than active degranulation. Some feeding sites resume a normal morphology by day 7. Lesions induced in actively sensitised hosts by a secondary feeding tick population are dominated by basophils. These cells begin to infiltrate the dermis within 6 hr and they show evidence of anaphylactic degranulation at 12 hr. Maximal release of membrane-free secretion granules occurs at about 18 hr post-attachment, at which time eosinophils become prominent. Degranulating basophils show a reduction in numbers from 24 to 96 hr, and phagocytic macrophages ingest residual granules and cellular debris. Guinea pigs sensitised with immune serum and subjected to challenge exhibit lesions similar to but less dramatic than those of actively sensitised and challenged animals. Anaphylactic degranulation of basophils occurs both in the dermis and within blood vessels. The immunological consequences of these events are discussed in relation to other models of cutaneous basophil hypersensitivity.
The estab-lishment of cell lines from imaginal wing discs of Spodopteru~tcgiperdrl and Plodia it~~erptmctellrr
- Lynn D E H And Obeklander
LYNN. D. E.. AND OBEKLANDER. H. 1983. The estab-lishment of cell lines from imaginal wing discs of Spodopteru~tcgiperdrl and Plodia it~~erptmctellrr. J. Insect Physiol.. 29, 591-596.
Separation and visualization of enzymes on gels Chro-matogrdphic & Electrophoretic Techniques Plodia iwierpwzctella and Cadra c,crlctella resistance in strains to malathion and synergized pyrethrins
- Texas M J Siciliano
- Shaw C R L And
- L L Mcdonald
- L M Redlingek
- R D And Jones
Itt " Studies in Genetics VI. " pp. 49-90. Univ. Texas Publ. 7103, Austin. Texas. SICILIANO. M. J., AND SHAW. C. R. 1976. Separation and visualization of enzymes on gels. In " Chro-matogrdphic & Electrophoretic Techniques. Vol. 2, Zone Electrophoresis " (I. Smith, ed.). pp. 185-209. William Heinemann Medical Books. London. ZETTLER. J. L., MCDONALD, L. L.. REDLINGEK, L. M.. AND JONES. R. D. 1973. Plodia iwierpwzctella and Cadra c,crlctella resistance in strains to malathion and synergized pyrethrins. J. Eron. Ent~tn~~l., 66, 1049-10.50.
The role of hemocytic hydrolases in the defense of molluscs against invading parasites
- Cheng
Studies on Hemolymph Cells of Marine Pelecypods
- Foley