Investigations of phagocytosis concerning the immunological defence mechanism of Mytilus edulis using a sublethal luminescent bacterial assay (Photobacterium phosphoreum)

• 1.1. A simple method for the determination of phagocytosis activity using mussel hemocytes by measuring the bioluminescence is presented.
• 2.2. The immunological defence activity based on phagocytosis is measured and quantified by a luminescent bacterial assay with Photobacterium phosphoreum.
• 3.3. The measuring system allows us to establish the stress of the immunological defence mechanism of organisms exposed to chemicals and polluted rivers or sewage. Results with reference substances and the phagocytosis indices of exposed mussels from Norwegian aquaculture plants compared to those of mussels from the German Wadden Sea are given as examples.

     

Comparative study of predatory responses in blue mussels (Mytilus edulis L.) produced in suspended long line cultures or collected from natural bottom mussel beds

Blue mussels (Mytilus edulis L.) are a valuable resource for commercial shellfish production and may also have uses as a tool in habitat improvement, because mussel beds can increase habitat diversity and complexity. A prerequisite for both commercial mussel production and habitat improvement is the availability of seed mussels collected with minimum impact on the benthic ecosystem. To examine whether mussels collected in suspended cultures can be used for bottom culture production and as tool in habitat improvement, the differences in predatory defence responses between suspended and bottom mussels exposed to the predatory shore crab (Carcinus maenas L.) were tested in laboratory experiments and in the field. Predatory defence responses (byssal attachment and aggregation) and morphological traits were tested in laboratory, while growth and mortality were examined in field experiments. Suspended mussels had an active response in relation to the predator by developing a significantly firmer attachment to the substrate and a closer aggregated structure. Bottom mussels had a passive strategy by having a thicker shell and larger relative size of the adductor muscle. In a field experiment mussels originated from suspended cultures had a higher length increment and lower mortality when compared to bottom mussels. It is concluded that suspended mussels potentially are an alternative resource to bottom culture and can be used in habitat improvement of mussel beds, but that the use of suspended mussels has to be tested further in large-scale field experiments.     

Effects of seawater acidification and salinity alterations on metabolic,osmoregulation and oxidative stress markers in Mytilus galloprovincialis

The impacts of seawater acidification and salinity shifts on metabolism, energy reserves, and oxidative status of mussels have been largely neglected. With the aim to increase the current knowledge for the mussel Mytilus galloprovincialis a 28-day chronic test was conducted during which mussels were exposed to two pH (7.8 and 7.3; both at control salinity 28) and three salinity (14, 28 and 35, at control pH, 7.8) levels. After exposure to different conditions, mussels electron transport system activity, energy reserves (protein and glycogen content) carbonic anhydrase activity, antioxidant defences and cellular damage were measured. Results obtained showed that mussels exposed to seawater acidification presented decreased metabolic capacity that may have induced lower energy expenditure (observed in higher glycogen, protein and lipids content at this condition). Low pH condition induced the increase of carbonic anhydrase activity that was related to acid-base balance, while no significant activation of antioxidant defence mechanisms was observed resulting in higher LPO. Regarding the impacts of salinity, the present study showed that at the highest salinity (35) mussels presented lower metabolic activity (also related to lower energetic expenditure) and an opposite response was observed at salinity 14. Carbonic anhydrase slightly increased at stressful salinity conditions, a mechanism of homeostasis maintenance. Lower metabolic activity at the highest salinity, probably related to valves closure, helped to mitigate the increase of LPO in this condition. At low salinity (14), despite an increase of antioxidant enzymes activity, LPO increased, probably as a result of ROS overproduction from higher electron transport system activity. The present findings demonstrated that Mytilus galloprovincialis oxidative status and metabolic capacity were negatively affected by low pH and salinity changes, with alterations that may lead to physiological impairments namely on mussels reproductive output, growth performance and resistance to disease, with ecological and economic implications.Indicators: Physiological and biochemical changes in Mytilus galloprovincialis in response to low pH and salinity changes     

The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea

The activity of the antioxidant defence enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSH-Px, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2) and the phase II biotransformation enzyme glutathione-S-transferase (GST, EC 2.5.1.18) in whole mussels (Mytilus galloprovincialis) were studied. The mussels were collected in winter and in spring at two localities in the Adriatic Sea: Bar Port and Tivat Bay. Our results show that the activities of SOD, GSH-Px and GST were seasonally dependent with higher activities in winter. GR activity was also higher in winter, but only in mussels from Bar Port. In mussels from Tivat Bay, GR activity was lower in winter compared to spring. In addition, a decrease in CAT activity in mussels from Bar Port compared to those from Tivat Bay was found. It can be concluded that seasonal variations should be incorporated into interpretation of biomonitoring studies in mussels.     

Bactericidal properties of neutrophils from peripheral blood (PMN)of patients with Lyme borreliosis

In recent years in Poland, the interest has increased in studies about tick borne diseases, mainly Lyme borreliosis. Immune response and genotype of pathogen play an important role in the course of this disease. Phagocytic cells, especially PMN are dominant in defence mechanisms against bacterial infections. The main feature of PMN is their ability to destroy pathogenic microorganisms by phagocytosis. The aim of this study was to estimate the phagocytic activity of PMN connected with intracellular respiratory burst in patients with Lyme borreliosis. The PMN activity tests completed were: phagocytosis, spontaneous and reduced of nitrotetralizate blue test (NBT). Decreased phagocytic activity and oxygen metabolism of PMN from patients with borreliosis in comparison with values of controls were found. Normalization of these parameters after treatment was observed. Changed phagocytic activity connected with intracellular oxygen metabolism during the course of therapy was the main observation. Depression of phagocytic activity of PMN connected with oxygen metabolism can influence defence reactions in patients with Lyme borreliosis. It is suggested that changes observed are acquired and associated with Borrelia burgdorferi presence.     

Oxidative effects of inorganic and organic contaminants on haemolymph of mussels

We applied a newly-established method in haemolymph of mussels, Mytilus galloprovincialis, exposed to different concentrations of heavy metals, such as zinc and cadmium and organic pollutants, such as PAHs and lindane, for the detection of total antioxidant capacity (TAC). The susceptibility of exposed mussels was increased in relation to oxidative stress induced by contaminants tested. Oxidative modifications of proteins were estimated by measuring protein carbonyl content (PCC) and malondialdehyde levels (MDA). For PCC measurement, a highly sensitive and accurate ELISA method, which requires only 5 µg of protein, was used. The significant increase of PCC and MDA in haemolymph of exposed mussels reinforces its role as biomarkers of oxidative stress. Significant correlation of TAC assay, PCC and MDA was conducted in order to evaluate the utility of PCC and TAC assay, used in the present study, as tools for determining oxidative effects of pollutants in mussels. The results reinforce the application of PCC method as useful tool for the determination of PCC alterations in haemolymph of mussels exposed to different levels of contaminants. In addition, the TAC method gives encouraging results, concerning its ability to predict antioxidant efficiency in haemolymph of mussels exposed to inorganic and organic contaminants.     

Impaired defense mechanisms in bay mussels, Mytilus edulis, with hemic neoplasia

Immunocompetence of bay mussels, Mytilus edulis, with hemic neoplasia was investigated with an in vitro yeast phagocytosis assay and by in vivo clearance from the blood of injected Cytophaga sp. bacteria. The yeast phagocytosis assay was conducted with hemocytes maintained in 90% plasma. Neoplastic hemocytes, characterized by enlarged nuclei and scant cytoplasm, failed to phagocytose yeast cells. In contrast, greater than 90% of hemocytes from unaffected animals and morphologically normal hemocytes from mussels with the disease phagocytosed yeast. Substitution of normal plasma with that from a mussel with advanced disease (essentially 100% neoplastic hemocytes) did not affect the phagocytic capability of normal hemocytes. Conversely, normal plasma did not enhance the phagocytic capabilities of neoplastic cells. Mussels with advanced disease showed reduced bacterial clearance; control or lightly affected mussels (less than 11% neoplastic hemocytes) cleared greater than 90% of injected bacteria in 4 hr, while mussels with advanced disease cleared 44-83%. These experiments indicate that mussels with advanced hemic neoplasia have compromised defense systems. This may account for the reported mortality in mussels and other bivalve molluscs with hemic neoplasia.     

Effects of PCB 126 and cadmium on the anaerobic metabolism of the mussel Mytilus edulis L.

Exposure of mussels to PCB 126 and/or cadmium resulted in elevated anaerobic metabolic activity during 48 hr of aerial exposure. In mussels exposed to PCB 126 or cadmium this was achieved by an increased opine production. The largest increase was observed in PCB exposed mussels. In mussels that were exposed to PCB 126 and cadmium simultaneously the production of both succinate and opines had increased. Simultaneous exposure to PCB 126 and cadmium had a more negative effect on the aerial survival time than exposure to PCB 126 alone, whereas exposure to cadmium alone did not have a significant effect on the tolerance to aerial exposure.     

Phagocytosis by Lymnaea stagnalis haemocytes: a potential role for phosphatidylinositol 3-kinase but not protein kinase A

The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, significantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 microM LY294002 reducing internalization of fluorescent-conjugated Escherichia coli by 62% (P < or = 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without effect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs.     

Effects of sea lice (Lepeophtheirus salmonis Kröyer, 1837) infestation on macrophage functions in Atlantic salmon (Salmo salar L.)

Experiments were conducted to determine the effects of sea lice, Lepeophtheirus salmonis, on non-specific defence mechanisms in Atlantic salmon, Salmo salar, by experimentally infesting hatchery-reared 1 and 2 year old post-smolts, S1 and S2, with laboratory grown infective copepodids at moderate to high infection intensities ranging from 15-285 lice per fish. The effects of sea lice-induced stress were investigated by measuring the blood levels of cortisol and glucose as indicators of primary and secondary stress responses, and by changes in macrophage respiratory burst activity and phagocytosis as indicators of tertiary stress responses as well as non-specific defence mechanisms. Fish were sampled prior to sea lice infestation at day 0 and at days 3, 7, 14 and 21 post-infestation. Sea lice were at copepodid stage at day 3, at chalimus stages at days 7 and 14, and at pre-adult stage at day 21. Blood levels of cortisol and glucose were found to be significantly increased at day 21 in fish-infested with the highest levels. Macrophage respiratory burst and phagocytic activities were found to be significantly decreased only at day 21. These results indicate that sea lice do not suppress host defence mechanisms during the earlier stages of infestation. They do have effects on the development of chronic stress and on the host non-specific defence mechanisms soon after the lice reach the pre-adult stage.     

The influence of temperature and dose on antibacterial peptide response against lipopolysaccharide in the blue mussel,Mytilus edulis

Blue mussels (Mytilus edulis) were inoculated with two different doses of lipopolysaccharides (LPS) or phosphate-saline (PS) buffer under different temperature conditions (6 and 20 degrees C). The activity of the antibacterial peptide fraction, purified through reverse phase chromatography from mussel haemolyph, was compared at different time intervals after the inoculation. The activity was determined as the minimal peptide concentration that inhibited growth of the Gram-negative bacteria Escherichia coli D21, by using radial diffusion assay. The antibacterial activity for mussels inoculated with LPS changed over time, both at 6 and 20 degrees C, but those inoculated with PS-buffer did not. The response was enhanced within a time course of 3h. The higher temperature did increase the inhibitory activity and made the mussel respond at an earlier stage, in comparison to that at 6 degrees C. At 20 degrees C, mussels inoculated with 10 microg of LPS responded faster than those inoculated with 0.1 microg of LPS. In addition, cytotoxic effects of LPS on mussel haemocytes were investigated in vitro, using a colorimetric assay. The survival index (SI%) for haemocytes decreased with 76% at 6 degrees C but increased with 100% at 20 degrees C, irrespective of the dose of LPS. This indicated that LPS did not influence the viability of the haemocytes but the high temperature increased their metabolic state. Likely, antibacterial response was provoked by LPS in a dose-dependent manner and favoured by higher metabolic state of the haemocytes, elicited at higher temperature. These results provide important considerations for variability in the internal defence of mussels and consequently, also the retention of viable human pathogens in mussels.     

The effect of salinity change on the heart rate of Mytilus edulis specimens from different ecological zones

The heart rate of specimens of Mytilus edulis (L.) both from the sublittoral and littoral zones exposed to normal and altered salinity was investigated in a long-term experiment. The heart rate was monitored by a non-invasive method for nine days. The heart rate of sublittoral mussels was higher than that of littoral ones. This suggests a higher level of metabolic activity in sublittoral mussels. When exposed to moderate hyposalinity (15 g l⁻¹) M. edulis from both zones showed a significant decrease in the heart rate with respect to the control salinity (25 g l⁻¹), but sublittoral mussels had a more prominent bradycardia. The heart beat quickly accelerated in all organisms when they were returned to the control salinity medium. Throughout the experiment, heart contractions halted with distinct periodicity in all mussels.     

Effect of anthropogenic feeding regimes on activity rhythms of laboratory mussels exposed to natural light

Anthropogenic disturbance may affect animal behaviour and should generally be minimised. We examined how anthropogenic disturbance (24 h food deprivation) affected circadian rhythms in laboratory mussels Mytilus edulis exposed to natural light in the absence of tides. Repeated measures data were collected on mussel gape angle, exhalant pumping and valve adduction using a Hall sensor system over eight consecutive 24 h periods when exposed to two feeding conditions after 24 h food deprivation. Mussels (fed once per day at either midday or midnight) exposed to natural light showed a clear day–night rhythm with increased nocturnal activity: significantly greater gape angle, increased exhalant pumping and had significantly higher valve adduction rates. However, circadian rhythms were less clear directly after anthropogenic food deprivation, in terms of the circadian rhythm in gape angle becoming significantly more apparent over the following days. Unlike mussels fed at midnight, those fed at midday displayed no significant change in gape angle from the hour before to the hour after they were fed, i.e. mussels given food at midday reacted to this food less than mussels fed at midnight. We suggest that independent of feeding time, laboratory mussels exposed to natural light and free from anthropogenic disturbance increase feeding activity at night because their circadian rhythms are strongly influenced by light levels. This study emphasises that the behaviour of animals in the laboratory and in the wild can be altered by anthropogenic disturbances such as vibrations caused by experimental setups and artificial illumination at night.     

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.     

Factors organizing rocky intertidal communities of New England: Herbivory and predation in sheltered bays

In New England, U.S.A., shores exposed to severe wave action are dominated by the common blue mussel Mytilus edulis L. while moderately protected areas are covered with perennial algae. It is thought that algae are limited by mussels which are a superior competitor. Because the effectiveness of predators is inhibited by wave activity, it is assumed that the rate of predation, which varies across this environmental gradient, accounts for the observed distribution of mussels and algae.Shores along sheltered bays appear to be an exception to this pattern and this study addresses some of the possible causes. In New England bays, mussels and barnacles Semibalanus balanoides (L.) are the most common organisms on the solid surfaces in the lower intertidal zone. Perennial macroalgae, such as Chondrus crispas Stackhouse and Fucus vesiculosus L., are rare. The distribution and abundance of species differs from that on moderately protected shores and is similar to very exposed shores which are dominated by mussels and barnacles.Herbivory by the common periwinkle Littorina littorea (L.) limits the abundance of F. vesiculosus and indirectly affects the success of mussels. During 4 years of experimental manipulations, F. vesiculosus rarely recruited in the presence of periwinkles but dominated experimental surfaces if periwinkles were excluded. When experimental surfaces with F. vesiculosus, which had been protected from herbivory for > 1 year, were exposed to natural conditions, herbivores cleared most of the surfaces within several months. Recruitment by barnacles and mussels was higher when periwinkles were excluded. However, the effect of periwinkles on mussels was indirect; the snails reduced barnacle success and thus reduced mussel recruitment which was enhanced by the surface irregularities provided by barnacles.The occurrence of mussels in sheltered bays is not due to a lack of predators. Predators were commonly seen at all sites. Most mussels on experimental surfaces were removed <4 wk when surfaces were exposed to natural levels of predation. Experiments do not provide an explanation for the occurrence of mussels, although the enhancement of mussel recruitment by barnacles suggests that the availability of settlement sites may be important.     

Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels.

1. The seasonal variations in the level of antioxidant compounds (glutathione (GSH), vitamin E, carotenoids) and in the activity of antioxidant enzymes, superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), GSH-peroxidase (EC 1.11.1.9) in the digestive gland of mussels (Mytilus sp.) were evaluated. The lipid peroxidation process was also measured by determining the tissue concentration of malondialdehyde (MDA). 2. The physiological fluctuations of the antioxidant defence systems were inversely related to the accumulation of lipid peroxidation products (MDA) in the tissue. The observed seasonal variations are presumably related to the changing metabolic status of the animals, itself dependent on such factors as gonad ripening and food availability. 3. In particular, the obtained data indicate that a reduction of the antioxidant defence systems, occurring during winter, could be directly responsible for an enhanced susceptibility of mussels tissues to oxidative stress, as indicated by the high MDA concentration observed in this period.     

Relevance of mytilid shell microtopographies for fouling defence – a global comparison

Prevention of epibiosis is of vital importance for most aquatic organisms, which can have consequences for their ability to invade new areas. Surface microtopography of the shell periostracum has been shown to have antifouling properties for mytilid mussels, and the topography shows regional differences. This article examines whether an optimal shell design exists and evaluates the degree to which shell microstructure is matched with the properties of the local fouling community. Biomimics of four mytilid species from different regional provenances were exposed at eight different sites in both northern and southern hemispheres. Tendencies of the microtopography to both inhibit and facilitate fouling were detected after 3 and 6 weeks of immersion. However, on a global scale, all microtopographies failed to prevent fouling in a consistent manner when exposed to various fouling communities and when decoupled from other shell properties. It is therefore suggested that the recently discovered chemical anti-microfouling properties of the periostracum complement the anti-macrofouling defence offered by shell microtopography.     

Spleen volume varies with colony size and parasite load in a colonial bird

Comparisons across bird species have indicated that those more exposed to parasites and pathogens invest more in immunological defence, as measured by spleen size. We investigated how spleen volume varied with colony size, parasite load and an individual's colony-size history in the cliff swallow, Petrochelidon pyrrhonota, a colonial passerine bird of North America. We used a sample of over 1700 birds that had all died during a period of inclement weather in 1996. We experimentally manipulated ectoparasitism by fumigating nests in some colonies prior to the bad weather. Birds from parasite-free colonies had significantly smaller spleens than those from naturally infested sites; spleen volume did not differ between the sexes and did not vary with age. Mean spleen volume increased significantly with the colony size at a site prior to the bad weather in 1996 and at the site in 1995, both measures of colony size being indices of ectoparasitism at a site. An individual's history of breeding-colony size (defined as the average colony size it had occupied in years prior to 1996) had no association with its spleen size. The results are consistent with parasite-induced splenomegaly whenever birds are exposed to large numbers of ectoparasites. The results do not support spleen size as being a signal of differential life-history investment in immunological defence among individuals and thus run counter to interpretations from recent cross-species comparisons.     

Quantification of phagocytosis in human neutrophils by flow cytometry.

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.