The lysozyme of the plaice Pleuronectes platessa L.

Lysozyme from the serum of the plaice, Pleuronectes platessa L., has been purified 78-fold with chitin coated cellulose.

• 1.2. Further purification on CM-cellulose yielded a single band on acrylamide electrophoresis, exhibiting lysozyme activity.
• 2.3. The quantitative amino acid composition of plaice serum lysozyme is reported.
• 3.4. The mol. wt is identical with hen egg white lysozyme.
• 4.5. A method is described for identifying fractions with lysozyme activity in polyacrylamide gels.

     

A trypanosome from the plaice, Pleuronectes platessa L.

The results of a 12 month study of Trypanosoma platessae Lebailly (1904), a haemoflagellate parasitising the plaice, Pleuronectes platessa L., are presented. The morphological features of the trypanosome are described, with experimental investigations of the host specificity of T, platessae . The flagellate occurred at low parasitaemias in wild plaice with a seasonal variation in the numbers of infected hosts, the percentage of parasitised fish reaching a maximum of 20 % in March and declining in the summer months.
Attempts to establish T. platessae in the laboratory, by means of passage of infected blood, gave inconsistent results. Elevated levels of β-globulins were detected in the sera of T. platessae infected plaice, and it is suggested that this may be associated with antibody secretion to the parasite.     

Aspects of leucocyte migration in the plaice, Pleuronectes platessa L.

Histological studies on plaice tissues revealed mononuclear phagocytes emigrating through capillary endothelium in response to bloodborne foreign erythrocytes, with a >50% increase in circulating leucocytes within 2 h. Despite this rapid influx of leucocytes, cellular accumulation during peritoneal inflammatory responses was significantly slower than in mammals. In vitro studies with chemotaxis chambers revealed that random leucocyte migration was enhanced in the presence of endogenous chemostimulatory substances (inflammatory exudate fluid and endotoxin-activated plaice serum) although directional migration was not demonstrated. It is suggested that, compared with mammals, the slower accumulation of teleostean leucocytes at sites of injury is not due to a locomotory defect but possibly results from lower levels of endogenous chemoattractants being generated.     

The immunohistochemical localization of lysozyme in plaice (Pleuronectes platessa L.) tissues

The location of lysozyme in fresh plaice ( Pleuronectes platessa L.) tissues was studied using an immunofluorescent technique. An antiserum to a homogeneous sample of plaice serum lysozyme was prepared in rabbits and was shown to be monospecific by immunodiffusion and inhibition studies. This preparation, used in conjunction with fluorescent labelled sheep anti-rabbit immunoglobulin, demonstrated lysozyme in plaice neutrophils, peritoneal macrophages, gill cartilage and epithelium, spleen, kidney, skin and mucus. The distribution of lysozyme in the skin and secreted mucus was affected by the method of killing the fish.     

Seasonal changes in thyroidal status in the plaice, Pleuronectes platessa L.

A chemically specific analysis for plasma thyroid hormones has been used to disclose a seasonal bimodality in the concentrations of thyroxine (T₄) and triiodothyronine (T₃) in the plaice; maxima in both hormone levels occur in winter and in summer. The ratio of T₄ concentrations to T₃ varies seasonally being at a minimum in summer. A highly significant correlation of T₄ plasma levels with the landings per unit fishing effort for the same area has been observed.     

Metabolic and immunological effects of endotoxin in the plaice, Pleuronectes platessa L.

In an effort to identify factors contributing to the resistance of fish to endotoxin toxicity, the metabolic effects of an intraperitoneal injection of a Boivin preparation of E. coli lipopoly-saccharide (LPS) were studied in plaice over 4 days. Significant changes were found in serum concentrations of glucose, cortisol and non-esterified fatty acids but not in total or free cholesterol. The effect of injected LPS on the phagocytic capacity of kidney neutrophils was examined because of the possible use of LPS to promote non-specific immunity. There was no enhancement of the chemiluminescent response to the phagocytosis of unopsonized bacteria by neutrophils from plaice 24 h after LPS treatment.     

Seasonal changes in serum glucose and condition of the plaice, Pleuronectes platessa L.

Monthly sampling of plaice caught off the north-east coast of Scotland between February 1983 and May 1984 revealed a seasonal increase in condition factor, hepatosomatic index and serum glucose, which appeared to be related to the period of active feeding. The spleen indices followed a similar seasonal increase, although this was not statistically significant, while the kidney indices showed little change over the 16 months. The peak for the gonadosomatic index was in February/March 1984, but among these plaice (aged 4–5 years) there were still large numbers of immature males and females and there was no increase in 1983. Apart from the hepatosomatic index for January 1984, there was no significant difference between male and female plaice.     

Seasonal changes in serum lipid composition of the plaice, Pleuronectes platessa L.

Seasonal changes in the major lipid classes of plaice serum were examined in fish caught off the north-east coast of Scotland. Both male and female plaice were sampled monthly between June 1983 and May 1984. Total lipid, total cholesterol, free cholesterol, triglycerides, phospholipids and non-esterified fatty acids were measured using commercial kits validated against standard reference methods. There were no significant differences in lipid levels between male and female fish and, although there were real differences between months for each lipid class, there was no regular pattern over the 12-month period. All the lipid class values, however, reached a maximum in September, which could be related to the increase in feeding after spawning. The smaller peak in February could be associated with the mobilization of lipid reserves for gonadal development and to overcome the effect of starvation during spawning.     

Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice, Pleuronectes platessa L.

The conclusion from two in vivo experiments is that a significant proportion of the lactic acid, normally formed by glycolysis from glycogen and held in the muscle cells following exhausting exercise of the anaerobic swimming muscle of the teleost fish Pleuronectes platessa L, is converted by gluconeogenesis to form glycogen in the recovering muscle.
In the first experiment a technique for measurement of [³H]glucose turnover in the plaice was developed and applied to measure turnover in resting and exhausted fish. It is concluded that insufficient glucose was moved through the circulation to account for the rate of glycogen formation observed in the recovering exhausted muscle.
In the second experiment, an intramuscular injection of [¹⁴C]lactate to exhausted fish revealed a direct uptake of [¹⁴C]lactate by the recovering muscle cells, and the incorporation of substantial proportions of lactate into the restored glycogen. Simultaneous use of [³H]-mannitol allowed measurement of the isotope distribution between extra- and intracellular spaces.     

Studies on the skin of plaice (Pleuronectes platessa L.)

Eighty-nine O-group plaice from a natural population were exposed at 15°C to heavy infection by Cryptocotyle lingua cercariae. Subsequently 45 fish were retained at 15°C, whilst 44 were held at 5°C. Both groups were sampled by killing individual fish at intervals of 6,18,42 h and daily thereafter up to 710 h. Entire fish were fixed immediately in formol saline, transversely sectioned and stained by H & E, PAS, PAS-diastase, JSDB 109, Picro-Mallory, Masson's trichrome, Gram-Weigert and Alcian blue. Histopathological observations showed: (a) epidermal lesions associated with encysted metacercariae in adjacent tissues; (b) myofibrillar necrosis associated with bacteria possibly introduced by the parasite; and (c) a reactive swelling of the intermuscular septa. The progressive development of the parasite cyst and host capsule is described. Development of both was markedly inhibited at the lower temperature, but the inflammatory response at either temperature was slight. This may be evidence of a long-standing host-parasite relationship which has evolved to an advanced state of adaptation on the part of the parasite and tolerance on the part of the host.     

Studies on the skin of plaice (Pleuronectes platessa L.)

Histological and ultrastructural examinations were carried out on the integuments of larval plaice from hatching to 60 days. The skin of the newly hatched larva consisted of a delicate epidermis overlying a fluid filled dermal space. As the fish matured this became thicker, the fibrous dermis developed and the mitochondrion rich 'chloride cells' degenerated. Around the 42nd day the eosinophil granule cell appeared in the basal layers of the epidermis and by the sixtieth day the epidermis could be considered fully developed.     

Gastric evacuation in plaice, Pleuronectes platessa L.: effects of temperature and fish size

Using a volume dependent model of gastric evacuation, the effects of temperature and fish size were examined. Rates of gastric evacuation were unaffected by fish size but increased with increasing temperature. The relationship between maximum stomach volume and fish weight was found to be a linear one. From information of gastric evacuation rates and stomach volume, the amount of food evacuated from the stomach per day was calculated for different size classes of fish. Daily food evacuation increased in proportion to body weight to the power 0·68. Assuming these methods give a crude estimate of daily food intake, the results are discussed in relation to published work on food intake in fishes.     

Peritoneal inflammatory cells in plaice, Pleuronectes platessa L.: effects of stress and endotoxin

When plaice were injected intraperitoneally with either oyster glycogen or live Vibrio alginolyticus an acute cellular inflammatory response was observed. The duration of these responses, 7 and 15 days respectively, exceeded the time course of the mammalian cellular inflammatory reaction. Peak leucocyte numbers were found at 2–3 days and neutrophils, which were phagocytic, were more numerous than macrophages. Although the increase in macrophage numbers was less marked, these cells appeared more actively phagocytic than neutrophils. Cortisol injections and environmentally-induced stress caused a significant reduction in the extent of inflammatory cell infiltrates, while endotoxin significantly enhanced the response.     

Gastric evacuation in plaice, Pleuronectes platessa L.: effects of temperature and meal size

The pattern of gastric evacuation in the plaice can be described by a single curve of curvilinear form, relating the amount of food remaining in the stomach and the post-prandial time. This curve can be transformed to linear form, conforming to a volume dependent model of evacuation. This allows the calculation of the time for complete evacuation of the stomach. The shape of the gastric evacuation curve is unchanged by the size of the meal fed. With increase in temperature however, the form of the curve does change, resulting in shorter times for complete evacuation.     

Studies on the skin of plaice (Pleuronectes platessa L.)

A histological, histochemical and ultrastructural examination of the skin of wild and cultured plaice was carried out, using fish from each year class from 0+ to 4+. The skin was shown to be similar in general structure to that of other teleosts but a previously undescribed cell, designated the Eosinophilic Granular Cell, a dendritic secretory cell found throughout the basal layers of the epidermis, is described. It was fixed only by formalin or dichromate, and contained numerous acidophilic granules. Melanin-bearing macrophages were observed migrating through the epithelium, but no DOPA or tyrosinase positive cells were observed by the methods used. Mast cells were very common in the dermis but were only demonstrable by special techniques. The melanophore and guano-phore systems are described and although no melanophores or melanocytes were found in the unpigmented areas of partially pigmented hatchery-reared fish, the integrity of the guanophore system was complete in such fish.     

Precipitating activity in the sera of plaice, Pleuronectes platessa L. to a helminth antigen

An antigen abstract of Proleptus obtusus an intestinal nematode of the common dogfish has been shown to precipitate with a serum component of plaice. As the parasite has never been shown to infect plaice it is suggested that perhaps the serum component might be C–reactive protein (CRP). However, characterization of the component revealed that it was specific immunoglobulin analagous to antibody of the IgM class. It is suggested the helminth antigen involved is cross–reacting with a common antigen to which the plaice is normally exposed.     

Monoaminergic nerves in the skin of plaice Pleuronectes platessa (L.)

1. The fluorescent histochemical technique of Falck and Hillarp was applied to plaice skin. The presence of monoaminergic nerve terminals, containing predominantly stores of adrenaline, forming a plexus in and around the melanophore layer was demonstrated. 2. Such stores were enriched by noradrenaline in the presence of monoamine oxidase inhibitor, unaffected by spinal section, depleted by spinal nerve section or ligatures and abolished by reserpine. 3. The observations support the view that teleost sympathetic melanophore aggregating nerves are truly adrenergic.     

Stress and water balance in the plaice Pleuronectes platessa

Summary Drinking and branchial water permeability have been measured in plaice, and the effect of handling stress on these and on water balance is reported. Stress markedly disturbs water balance, with a weight loss of 1.6% over 7 h, and a recovery period exceeding 19 h. Depression of the normal rate of ingestion accounts for about 14% of this imbalance. Stress-induced urination is not a significant factor. Branchial diffusional water permeability is more than trebled in response to handling, but a measure of accommodation is seen with repeated handling. The ratio of osmotic to diffusional branchial water permeability is about 2.5 and is not appreciably affected by stress. Oxgen uptake is enhanced in response to stress, and this is discussed in relation to the effects of stress on water balance.     

New microsatellites primers for plaice,Pleuronectes platessa L. (Teleostei: Pleuronectidae)

Seven microsatellite loci were developed for Pleuronectes platessa, a commercially important flatfish found throughout northern European waters. The number of alleles per locus ranged from 11 to 33 and heterozygosities from 0.74 to 0.96. Cross‐reactivity was tested against six other flatfish species. Significant amplification was found in all species from one to all seven of the loci.