Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Infection of the European chafer,Amphimallon majalis,by Bacillus popilliae: Ultrastructure

Electron microscopical studies indicate that midgut alterations observed in larval Amphimallon majalis during Bacillus popilliae invasion were due to infection of epithelium, wound repair, and hemocytic encapsulation. Hemocytes bearing angular cytoplasmic granules formed the capsule which adhered to the basal lamina subjacent to the mesenteronic lesion. Exocytosis of hemocyte granules into bacilli-containing endocytic invaginations of plasma membranes and vacuoles was observed. These hemocytes as well as mesenteronic cells exhibited bactericidal capabilities.     

Hemocyte classification and differential counts in the dungeness crab, Cancer magister

The primary purposes of this research were to describe and classify the circulating hemocytes of Cancer magister and devise a method for making differential hemocyte counts for crustaceans. C. magister hemocytes were classified using two simple criteria: the presence or absence of cytoplasmic granules and staining characteristics of the granules, if present. Hyalinocytes (HC) were devoid of granules, intermediate granulocytes (IG) contained basophilic granules or a mixture of basophilic and acidophilic granules, and eosinophilic granulocytes (EG) contained large, acidophilic granules. Hemocyte renewal and a hypothetical maturation sequence of C. magister hemocytes are described and discussed. Differential counts revealed that granulocytes were more abundant than hyalinocytes. For 22 crabs, the mean percentage (and range) of each hemocyte class was: IG, 65.97 (57.50–73.80); EG, 17.76 (4.70–26.47); and HC, 16.25 (3.40–34.67). After additional data are collected and analyzed, the routine use of differential counts may prove to be a valuable method for monitoring the status and health of C. magister and perhaps other crustaceans as well.     

Composition of cellulin,the unique chitin-glucan granules of the fungus,Apodachlya sp.

Cellulin granules, the polysaccharide inclusions found uniquely in oomycetous fungi of the order Leptomitales, were isolated from Apodachlya sp. The granules were prepared free of cell wall and cytoplasmic contaminants. Biochemical analyses and X-ray diffraction studies demonstrated that the granules were composed of 60% chitin and 39% glucan consisting of β-1,3-and β-1,6-linked glucose units. A protein content of only 0.1% was attributed to an insignificant amount of cytoplasmic contamination. Isolated granules and those in situ showed no apparent differences in their microscopic form.     

Dynamics in the size structure of Skeletonema costatum (Greville) Cleve under conditions of reduced photosynthetically available radiation in a dredged tropical estuary

This study investigates the size-fractionated productivity and chlorophyll a concentrations in Ponggol estuary, a heavily dredged, light-limited and eutrophic tropical estuary located on the northeastern coast of Singapore. A 90% reduction in the photosynthetically available radiation (PAR) was seen in the subsurface waters of the dredged stations, when compared to an average reduction of about 75% in the subsurface waters of the un-dredged station. High phytoplankton production rates and chlorophyll a concentrations were recorded in the surface waters, with a significant reduction in the subsurface waters, especially at the two dredged stations. Out of the four size-classes of phytoplankton (0.2-2 μm, 2-20 μm, 20-200 μm and >200 μm) investigated, the relative dominance of the smaller size class of 2-20 μm over the 20-200 μm size class shifted depending on the availability of PAR. The size class 2-20 μm was observed to contribute up to 60 and 57% of the total production, respectively, in the surface waters of the un-dredged and dredged stations of the estuary. A relatively major contribution of 49% came from the cells of the small size-fraction of 2-20 μm in the subsurface waters of the un-dredged station. On the contrary, cells of the 20-200 μm size-class contributed up to 58% in the subsurface waters of the dredged station. Microscopic examination of the phytoplankton cells sampled showed morphological differences in the cells of the phytoplankton species Skeletonema costatum with some cells being distinctly larger in size than others. The larger cells were predominant in the dredged subsurface waters. In situ mesocosms with treatments exposed to high light irradiation registered a significant contribution by the smaller size-class of 2-20 μm, in contrast to the larger 20-200 μm dominating in the two treatments subjected to low light conditions. Based on the observations in this study and a review of the literature, it is hypothesized that the cells of S. costatum may actually be of two genetically different strains, whose relative dominance in the environment may be controlled by the quantum of available light. Thus, this shift in the relative dominance of one size fraction over the other is a response to altered PAR levels as the result of dredging.     

Block-Surface Staining for Differentiation of Starch and Cell Walls in Wheat Endosperm

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starch granule distribution in cells of wheat [Triticum aestivum L.] caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 μm³ to 22,000 μm³ with approximately 90% of the granules measuring less than 752 μm³ (ca. 11 μm in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells.     

罗氏沼虾与克氏原螯虾血细胞的比较研究

对罗氏沼虾与克氏原螫虾血细胞的分类与组成进行了染色观察比较研究。根据染色后光镜下血细胞的核质比、颗粒的大小和数量等来对血细胞进行分类,罗氏沼虾与克氏原螯虾的血细胞均可分为透明细胞、半颗粒细胞和颗粒细胞三类;其血细胞浓度分别为1.02±0.21×10⁷ Ind·ml^-1和0.85±0.15×10⁷Ind·ml^-1。2种虾血细胞的颗粒形态存在显著差异;透明细胞、半颗粒细胞和颗粒细胞占血细胞总量的百分比在罗氏沼虾为21.3±6.3%,45.7±2.5%,33.0±6.8%:在克氏原螯虾为12.0±5.8%、49.5±5.1%和38.5±9.5%。     

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.Key words: Carcinus aestuarii, crab, haemocytes, light microscopy, cytochemical assays, morphological characterisation.     

Two New Species of Eimeria from Colubrid Snakes in Iowa*

SYNOPSIS. Oocysts of Eimeria natricis sp. n. and Eimeria sipedon sp. n. were isolated from 2 Northern Water Snakes, Natrix sipedon sipedon (Linnaeus), in Iowa. Oocysts of E. natricis were elongate, slightly ovoid, averaging in size 32.3 × 17.5 μm; sporocysts averaged 15.7 × 8.2 μm. Oocysts of E. sipedon were broadly ovoid averaging in size 18.0 × 15.9 μm; sporocysts averaged 12.0 × 7.4 μm. In both species, sporocyst residua were present, but micropyles, polar granules and oocyst residua were lacking.     

A comparative ultrastructural study of blood cells from nine insect orders

Summary An ultrastructual study of hemocytes from 9 different insect orders has led to the identification of 8 cell types: (1) Plasmatocytes, whose cytoplasm is filled with small dense lysosomes and large heterogeneous structures, are phagocytic cells. (2) Granulocytes, filled with uniformly electron dense granules, are involved in capsule formation. (3) Coagulocytes, which contain granules and structured globules and which possess a well developed RER, are involved in phagocytosis. (4) Spherule cells are filled with large spherical inclusions. (5) Oenocytoids are large cells with few cytoplasmic organelles. These 5 hemocyte types represent the majority of insect blood cells. (6) Prohemocytes, blastic cells which are one of the stem cells of hemocytes, are very few in number in each species investigated. (7) Thrombocytoids and (8) Prodocytes are restricted to a small number of insect species.The ultrastructural characteristics of these hemocyte types are discussed.     

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

Hemocytes of the palaemonids Macrobrachium rosenbergiiand M. acanthurus,and of the Penaeid Penaeus paulensis

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages. J. Morphol. 236:209–221, 1998. © 1998 Wiley-Liss, Inc.     

The fine structure of the diapause-regulator cell in the suboesophageal ganglion in the silkworm, Bombyx mori

In the suboesophageal ganglion of Bombyx mori the diapause-regulator producing cells which may give information to the diapause-factor cells were found by means of electron microscopy.The diapause-regulator producing cells had larger granules (200 to 500 mμ dia.) than did the diapause-factor cells which were partially surrounded by the former. Highly electron-dense material of lysosome in the diapause regulator producing cells was observed in the diapause-egg producer, but such lysosomes were not present in the non-diapause-egg producer. It was found that many cytoplasmic granules fuse with lysosome, and smaller granules arise from lysosomes. Some implications regarding the diapause-factor cell and the diapause regulator producing cell are discussed.     

Studies on the Minimum Reproducible Unit of Staphylococcal L-Forms

The minimum size of a reproducible unit of staphylococcal L-forms was determined by filtration and electron microscopic methods. Ultrathin sections of an induced strain of Staphylococcal L-forms (STA-EMT-1) in liquid medium revealed several types of structures, all of which were bound by a single membrane and most of which possessed ribosome-like granules. Many of the small granules were less than 0.3 μm and were attached to the membrane of the large bodies. Using a serial filtration method, it was observed that viable L-forms were still detected in 0.22 μm filtrate, but the viable cell count of L-forms decreased in number with the decrease in pore size of membrane filters. A fractionation technique, using L-forms filtered through a membrane filter with a 0.45 μm pore size, revealed that there were three classes of small bodies but only the first class with ribosome-like granules over approximately 0.2 μm in diameter seems to be able to reproduce.     

STUDIES ON DROSOPHILA EMBRYONIC CELLS IN VITRO I. CHARACTERISTICS OF CELL TYPES IN CULTURE *

Embryonic cells from Drosophila melanogaster were cultured in medium K-17 supplemented with 0.1 mg/ml of fetuin and 15% fetal bovine serum, and the behavior of embryonic cells, the characteristics of some cell types and the maintenance of the phenotypic properties of the cells were examined. Embryos at the stage with a saclike midgut (12 hr eggs) were found to be the best materials for cultivation. Muscle cells, epithelial cells, fibroblastic cells and small cells were observed as distinct migrating cell types. Muscle cells pulsated synchrónously when two or more cells made contact with each other and this pulsation continued for more than six weeks under the culture conditions employed. Epithelial cells matured with increase in size and in the number of cytoplasmic granules. Fibroblastic cells had conspicuous motile activity. Small cells were peculiar for their size of about 3 μm diameter and formed groups of about forty to fifty cells. Their characteristic shape and size suggested that they were imaginal disc cells. Cellular spheres, which consisted of epithelial cells and fibroblastic cells, and nerve fibers of nerve cells were also observed. Some mitotic figures, suggesting the occurrence of cell division, were observed in cultures of epithelial cells.     

Poly(3-Hydroxybutyrate) Granules at the Early Stages of Formation Are Localized Close to the Cytoplasmic Membrane in Caryophanon latum

Localization of newly synthesized poly(3hydroxybutyrate) (PHB) granules was determined by confocal laser scanning fluorescence microscopy of Nile red-stained cells and by transmission electron microscopy (TEM). PHB granules of Nile red-stained living cells of Caryophanon latum at the early stages of PHB accumulation were frequently found at or close to the cytoplasmic membrane. TEM analysis of the same culture revealed electron-translucent globular structures resembling PHB granules that were nonrandomly distributed in the cell lumen but were frequently found at or close to the cytoplasmic membrane. Immunogold labeling using PHB-specific antiserum confirmed that the electron-translucent structures represented PHB granules. Electron microscopy examination of PHB granules after cell lysis revealed that PHB granules were often associated with membrane vesicles. Nonrandom localization of PHB granules was also found in Beijerinckia indica. Cells of this species harbored one PHB granule at each cell pole. Our results show that newly synthesized PHB granules often are close to or even in physical contact with the cytoplasmic membrane. Possible explanations for this unexpected finding and a hypothetical model of PHB granule formation in C. latum are discussed.     

Temporal variability in egg production rates of Acartia tonsa Dana in Long Island Sound

The relationship between week-by-week variations in the in situ egg production rates of Acartia tonsa Dana and changes in chlorophyll concentration in several size fractions was investigated by incubating adult females in natural sea water for 24-h periods. Our results indicate that the egg production of A. tonsa in Long Island Sound was better related to the 10 μm chlorophyll size fraction than to the total chlorophyll concentration. The < 10 μm size fraction comprised the greatest percentage of the chlorophyll during July and August when the water column was stratified. Egg production rates were lowest (8.7 eggs · female⁻¹ · day⁻¹) in early August when less than 0.5 μg chlorophyll 1 ⁻¹ was observed in the 10 μm chlorophyll a size fraction. Following destratification in late August, the “fall” diatom bloom occurred and egg production rates increased to the maximum observed rate of 56.6 eggs · female⁻¹ · day⁻¹. At this time, the concentration of the 10μm chlorophyll size fraction was 5.5 μg 1⁻¹. Maximum egg production rates were observed at chlorophyll concentrations as low as 0.8 μg 1⁻¹ in the 10 μm size fraction.     

The role of cotyledon cell structure during in vitro digestion of starch in navy beans

Studies on the physico-chemical, microstructural characteristics and in vitro (under simulated gastric and small intestine conditions) starch digestibility of navy beans were carried out. The microstructure of raw and cooked beans observed through scanning electron microscopy (SEM) showed the presence of hexagonal or angular shaped cotyledon cells (50-100 μm size) containing starch granules with a size ranging between 10 and 50 μm. The extent of starch hydrolysis (%) after 120 min of in vitro gastro-intestinal digestion differed between whole navy beans (∼60%) and milled bean flour and bean starch (85-90%) after they were cooked under similar conditions. Starch hydrolysis (%) increased significantly when the cotyledon cells in the cooked whole navy beans were disrupted using high pressure treatment (French press). The storage of freshly cooked whole beans resulted in a lower (40-45%) starch hydrolysis whereas re-heating increased the same to 70-80% during in vitro small intestinal digestion. The SEM pictures of cooked navy bean digesta after different intervals of in vitro gastric and small intestinal digestion showed that the cotyledon cell structure is maintained well throughout the digestion period. However cotyledon cells appear shrunken and developed wrinkles during in vitro digestion. Particle size analysis of cooked bean paste taken before and after the in vitro gastro-intestinal digestion showed similar particle size distributions.