Frank E. Friedl

Factors affecting in vitro phagocytosis by oyster hemocytes

Abstract Hemocytes withdrawn from the pericardial cavity of the American oyster, Crassostrea virginica , were settled onto monolayers of immobilized fluorescent polystyrene microspheres (beads) in culture flasks. Phagocytosis was quantitated by fluorescence microscopy and flow cytometry after enzymatic detachment of the cells from unphagocytized beads and fixation of the cells. Unattached beads were removed by settling the mixture through a discontinuous sucrose gradient. Internalization of the beads following this procedure was verified by transmission electron microscopy. Phagocytosis was evident within 15 min after addition of the cells to the monolayer. Flow cytometry showed distinct cell populations corresponding to cells containing one to five or more beads indicating that multiple particle phagocytosis occurs rapidly after contact between cells and particles. Hemocytes incubated with beads at various temperatures showed maximum phagocytic activity over a wide range (10°–37°C) with inhibition occurring below 8°C. Inhibition of cell attachment to the bead monolayer was evident at 0° and 4°C and is probably a major cause of the decreased amount of phagocytosis observed at low temperatures. Treatment of the cells with cytochalasin B prior to incubation inhibited phagocytosis. Incubation of the cells with beads under atmospheres of pure nitrogen or helium produced no inhibition of phagocytosis. These data suggest that the hemocytes of the American oyster are able to phagocytize particles rapidly at extremes of the range of biological temperatures and under completely anaerobic conditions.

Nitrogen excretion by the fresh water pulmonate snail, Lymnaea stagnalis jugularis say☆

1. 1. The average per cent partition of excretory nitrogen produced into an ambient medium by Lymnaea stagnalis was found to be approximately: ammonia, 50; urea, 30; unidentified residual N, 20. Of the residual, about one-quarter appears to be volatile. 2. 2. The endogenous nature of the ammonia and urea produced is supported by their appearance in the presence of both penicillin and streptomycin as bacteriostatic agents.

Cytometric investigations on hemocytes of the American oyster, Crassostrea virginica

Pericardial hemolymph was obtained from American Oysters (Crassostrea virginica) and the hemocytes characterized by flow cytometry. The cells were found to have a broad unimodal size distribution with a median diameter of 7 micrometers. Total protein measured by flow cytometric fluorescence of dansylated cells also revealed a broad unimodal distribution similar to that obtained for size. The proportion of hemocytes in each stage of the cell cycle was measured using DNA-specific DAPI fluorescence. Histograms showed a single peak representing the G(0)/G(1) population. There was no evidence of S or G(2)+M phases of the cell cycle, nor was polyploidy seen. The forward and orthogonal light scatter of fixed hemocytes showed no evidence of sub-populations on the basis of cytoplasmic granularity. Thus, in terms of these parameters, oyster hemocytes appear to represent a single population exhibiting graded cellular differences.

Atrial natriuretic peptides in the heart and hemolymph of the oyster, Crassostrea virginica: a comparison with vertebrates.

1. The content of atrial natriuretic peptides (ANPs) in the auricles of oysters, Crassostrea virginica, was significantly (P < 0.01) greater than in their ventricles. 2. High-performance gel permeation chromatography (HP-GPC) followed by ANF radioimmunoassay revealed two peaks in both oyster and vertebrate (rat) hearts--a major peak where the 12.6-14 kDa ANF prohormone elutes and a smaller peak where the pure human form of ANF elutes. 3. HP-GPC evaluation followed by proANF 31-67 radioimmunoassay revealed only an ANF-like prohormone while HP-GPC followed by proANF 1-30 radioimmunoassay revealed the ANF prohormone and a proANF 1-30-like peptide in oyster and rat hearts. 4. ANPs concentrations in hemolymph were 940 +/- 129, 225 +/- 25, and 100 +/- 10 pg/ml by the proANF 1-30, proANF 31-67, and ANF radioimmunoassays, respectively. 5. Atrial natriuretic-like peptides are present in the oyster heart in molecular species similar to vertebrate species and these peptides are also present in hemolymph.

Dermal Myiasis of the Eyelid

A case report describes the clinical findings of dermal myiasis of the upper eyelid in a 6-year-old girl from South America. Dermal myiasis due to Dermatobia hominis can simulate a common furuncle and is known to occur in world travelers.

Ureogenesis in the snail Lymnaea stagnalis jugularis

Abstract 1. Arginase activity (EC 3.5.3.1) in homogenates of Lymnaea stagnalis digestive gland has been re-examined and further characterized. 2. Ornithine transcarbamylase (EC 2.1.3.3) was demonstrable in CTB homogenates of digestive using glycylglycine buffer at pH 8·3. 3. Metabolism of citrulline to arginine, characteristic of tissues with urea cycle activities, could not be demonstrated in vitro uisng digestive gland homogenates in a system similar to the “overall” system of Ratner less ATP-generating components.

The production of externally measurable ammonia and urea in the snail, Lymnaea stagnalis jugularis say☆

Abstract 1. 1. Production of ammonia and urea by snails isolated singly for 24 hr at 25°C has been measured. In average values, snails weighing 225, 479, 1134 and 2480 mg (whole weight) have been found to produce 1·0, 1·2, 4·2 and 6·0 μmoles of ammonia respectively. Snails weighing 203, 479, 1134 and 2480 mg likewise produced 0·3, 0·4, 0·95 and 1·7 μmoles of urea in the same period. 2. 2. The rates of production (μmoles/g whole wt. per hr) of both ammonia and urea appear to be relatively constant over the approximate twelve-fold weight range indicated above (ammonia, 0·1–0·2 μmoles/g per hr; urea 0·03–0·06 μmole/g per hr.).

Hemolymph glucose in the freshwater pulmonate snail Lymnaea stagnalis: Basal values and an effect of ingested carbohydrate☆

Abstract 1. 1. In the freshwater pulmonate gastropod Lymnaea stagnalis jugularis Say, a basal level of hemolymph glucose for nonfeeding animals appears to be about 30 μg/ml (3 mg %). 2. 2. Elevated levels of hemolymph glucose can be induced by starch feeding with subsequent return to the basal level when food is withheld.

Alteration of environmental salinity modulates atrial natriuretic peptide concentrations in the gills of the oyster, Crassostrea virginica

Abstract Because gills are frequently important in ion transport and osmoregulation, the present investigation was designed to determine if atrial natriuretic peptides (putative osmoregulatory peptides) are present within the gills of a representative euryhaline osmoconforming invertebrate. Utilizing three radioimmunoassays devised to amino acids 1–30, 31–67, and 99–126 of the 126 amino acid prohormone, the gills of 72 eastern oysters, Crassostrea virginica were examined and each found to contain atrial natriuretic peptides, whose content was approximately one-seventh of that within the heart of the oyster. In high salinity (32 parts per thousand, ppt) the content of atrial natriuretic peptides recognized by each of the three radioimmunoassays was lower at 3 days (P

Uptake and tissue distribution of abiotic particles from the alimentary tract of the American oyster: A simulation of intracellular parasitism☆

Abstract Windows were cut in the left valves of American oysters, Crassostrea virginica, to access the mouth. A suspension of 2.16-μm-diameter fluorescent polystyrene beads were introduced into their stomachs by intubation and the windows were sealed. Stomach contents were sampled and vibratome cross sections were made at various time intervals postintubation. The locations of the beads in the tissues were mapped using an image analysis system. Hemocytes containing phagocytized beads were found in the lumen of the stomach shortly after intubation. Hemocytes with internalized beads were clustered in the tissues around the gut, in the mantle cavity, and in the palps at 2 hr postintubation. At 2 and 5 days after intubation the intracellular beads were randomly scattered in the tissues and not necessarily associated with the digestive tract. The number of beads present in the tissues diminished with time but some remained in the tissues 10 days after intubation. The polystyrene beads simulate to some extent parasites, which are phagocytized but are not destroyed by the hemocytes. These data show that indigestible particles carried in the phagosomes of hemocytes can enter the tissues from the stomach. The phagocytized particles are gradually eliminated from the tissues, but a number remain residual in the tissues within hemocytes for more than 10 days.