Intermitochondrial junctions in the heart of the frog,Rana esculenta

Summary In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4±0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3±0.29 nm. There are 65.3±2 periodic structures per m of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11±0.3 nm thick and are separated from each other by 16.5±0.46 nm, their density being 65±2.28 per m of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.     

Cellular junctions in perifollicular contractile tissue of the rat ovary during the preovulatory period

Summary Rat ovarian perifollicular contractile tissue was examined at specified intervals prior to ovulation to determine the type, relative number, and length of cellular junctions. Rat ovaries were taken for electron-microscopic observation at 1500 h on the afternoon of proestrus (proestrus 0-h group), at 2000 h (proestrus 5 h group), at 0100 h (proestrus 10-h group) and at 1600 h on the afternoon of diestrus I. Close junctions, intermediate junctions, and gap junctions were counted and measured. The number of gap junctions 1,000 m of membrane and the number of intermediate junctions 1,000 m of membrane was significantly higher in the proestrus 10 h group as compared to the other groups. There was no difference in the number of close junctions during the periods studied. Also the length of all junctions was similar in all groups. These morphological findings are discussed in the context of a contractile role for perifollicular tissue in the ovulatory process.     

Sulfur amino acid metabolism in the developing rhesus monkey brain: Interrelationship of taurine and glutamate

The relationship of taurine to glutamate, and to other amino acids, has been examined in the occipital lobe of the developing rhesus monkey. During development taurine decreases in concentration (4.96 mol/g in fetus to 1.52 mol/g in adult) while glutamate increases (7.92 mol/g in fetus to 11.26 mol/g in adult). When the concentration of taurine is plotted against that of glutamate in fetal, neonatal and adult animals there is a significant correlation in the fetal (p<0.01) and adult (p<0.01) but not in the neonatal occipital lobe samples. This correlation in both fetal and adult brain is specific for these two amino acids. Subcellular fractionation studies further indicate that this relationship may be of special importance in nerve endings.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon

Summary The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-m long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 m transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 m and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.     

Upregulation of the expression of tight and adherens junction-associated proteins during maturation of neonatal pancreatic islets in vitro

Cell–cell contacts mediated by intercellular junctions are crucial for proper insulin secretion in the endocrine pancreas. The biochemical composition of the intercellular junctions in this organ and the role of junctional proteins in endocrine pancreatic dysfunctions are still unclear. In this study, we investigated the expression and cellular location of junctional and cytoskeletal proteins in cultured neonatal rat pancreatic islets. Neonatal B-cells had an impaired insulin secretion compared to adult cells. Cultured neonatal islets showed a time-dependent increase in the glucose-induced secretory response. The maturation of B-cells in vitro was accompanied by upregulation of the expression of some junctional proteins in islet cells. Neonatal islets cultured for only 24 h showed a low expression and a diffuse cytoplasmic location of the tight junctional proteins occludin and ZO-1 and of the adherens junctional proteins - and -catenins, as demonstrated by immunoblotting and immunocytochemistry. Culturing islets for up to 8 days significantly increased the cell expression of these junctional proteins but not of the cytoskeletal proteins vinculin and -actinin. A translocation of ZO-1 and catenins to the cell–cell contact region, as well as a higher association of F-actin with the intercellular junction, were also observed in neonatal islets following prolonged culturing. ZO-1 and -catenin were immunolocated in the endocrine pancreas of adult rats indicating that these junctional proteins are also expressed in this organ in situ. In conclusion, endocrine pancreatic cells express several junctional proteins that are upregulated following differentiation of the endocrine pancreas in vitro.     

Diacylglycerol downregulates junctional membrane permeability. TMB-8 blocks this effect

Summary We tested the question whether junctional cell-to-cell communication is regulated by the diacylglycerol branch of the phosphoinositide transmembrane signal pathway. Cultured epithelial rat liver cells were treated with the synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol, while their junctional permeability was probed with the microinjected 443-dalton fluorescent tracer Lucifer Yellow. The treatment reduced junctional permeability (without affecting Lucifer permeability of nonjunctional cell membrane). The effect was dose dependent, with a threshold of about 25 g diacylglycerol/ml in sparse cultures and about 50 g/ml in confluent cultures. The reduction of junctional permeability began within 3 min of diacylglycerol application, peaked within 20 min, and reversed spontaneously within 90 min. The phorbol ester TPA mimicked the diacylglycerol effect, but the (spontaneous) reversal was slower. We propose that cell-to-cell communication is under dual physiological control: an upregulatory one, as exerted by the cyclic AMP signal route (Loewenstein, W.R., 1985,Biochem. Soc. Symp. London,50: 43–58), and a downregulatory one, by the diacylglycerol signal route.TMB-8 (54–70 m)—a blocker of intracellular Ca²⁺ mobilization-impeded the diacylglycerol action on junctional permeability. It prevented the effect of low diacylglycerol doses completely and it markedly reduced the effect of high doses. (It also counteracted the effect of TPA.) Ca²⁺ thus emerges as a possible candidate for a role in the junctional downregulation by the diacylglycerol signal route. We tentatively advance two models. In one, leaning closely on the Calcium Hypothesis of cell-to-cell channel regulation (Loewenstein, W.R., 1966,Ann. N.Y. Acad. Sci. 137:441–472), Ca²⁺ mediates the action of the route on the channel. In the other, Ca²⁺ acts farther removed from the channel, on protein kinase C.Calmidazolium (5–10 m)—an inhibitor of calmodulin-activated proteins—did not prevent the diacylglycerol-induced reduction of junctional permeability. Nor did sodium orthovanadate (25 or 50 m)—an inhibitor of tyrosyl phosphatase-prevent the reversal of diacylglycerol-induced (or TPA-induced) reduction of junctional permeability.     

Karyometry of nuclei in liver cells

Summary In untreated adult male albino rats nuclear volume and the percentage of binucleate cells were determined in the first layer of hepatocytes adjacent to hepatic venous branches of varying diameters (<40 m, 40 m–80 m, 80 m–120m, 120 m–160 m, >160 m), and in the third and fourth layer of hepatocytes in the remainder of the perivenous parenchyma. In the first layer of hepatocytes adjacent to the vascular structures means of nuclear volume are significantly lower and percentage of binucleate cells significantly higher than in the cells of the remainder of the perivenous parenchyma. Within each area measured distribution curves of nuclear volume classes were homogeneous but showed heterogeneity in comparison with each other. The morphometric data presented in this study strongly support the opinion of the heterogeneity of liver cells in the perivenous zone, as previously postulated on the basis of histochemical investigations.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/2-1)     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

A study of the vascular arrangement in the rat adrenal gland using non-radioactive microspheres

Summary The intra-glandular vascular arrangement in the adrenal has been studied using non-radioactive microspheres injected by three different routes: in-vivo injection into the left ventricle under pentobarbital anesthesia, postmortem orthograde, and postmortem retrograde injection. The doses of microspheres were 10⁵ (average size 24.7 m), 10⁶ (15.8 m) and 10⁷ (9.9 m). The entrapment rate of microspheres by the medulla as compared with the whole gland was measured in the serially sectioned tissue (section thickness 60 m).The entrapment rates of 25-m microspheres differed between the orthograde and retrograde injections, while the entrapment rates for 15-m microspheres were essentially similar irrespective of the route of injection.Our results support the conclusion from previous microangiographic studies that the adrenal cortex and medulla are supplied by different arteries but have a common venous outflow, and that direct communication between cortical and medullary sinusoids is not likely. The medullary blood flow per gram tissue weight is estimated to be larger than cortical blood flow.     

Maturation of optics and resolution in adult dung beetle superposition eyes

Summary The maturation of the morphology and refractive-index gradients of the crystalline cones and cornea of the superposition compound eye of the nocturnal dung beetle Onitis aygulus was traced as a function of age following adult ecdysis. Intracellular recordings from retinula cells were also made to trace the maturation of angular sensitivity, thereby determining the resolution of the eye at each age. Maturation proceeded quickly during the first 3 days following ecdysis, and then more slowly, with full maturation attained by the end of the second week. The main experimental results obtained during the first 2 weeks after ecdysis were: (1) the mean length of the crystalline cones increases from 67 m to 79 m; (2) the mean thickness of the cornea increases from 36 m to 50 m; (3) the mean refractive index along the axis of the crystalline cones increases from 1.459 to 1.511 in the distal cone region, from 1.434 to 1.501 in the waist region and from 1.425 to 1.486 in the proximal region; (4) the shape of the refractive-index gradient becomes more parabolic; (5) the mean rhabdom cross-sectional area increases from 190 m² to 230 m²; (6) the angular-sensitivity function narrows, with the acceptance angle decreasing from 22 ° to 4 °. An optical ray tracing model predicts an image quality limited only by the immaturity of the optics, a prediction confirmed by the electrophysiological results. The results are compared to optical maturation in day-active moths and skipper butterflies (which mature very quickly) and discussed in relation to ecological efficiency.     

Evaluation of the glyoxal-bis-(2-hydroxyanil)-method for staining of calcium in model gelatin films and pancreatic islets

Summary The nature of tissue calcium, detectable with glyoxal-bis-(2-hydroxyanil), (GBHA), was investigated using gelatin films as model. The results indicate that in the films the procedure detects only the calcium fraction which was ionized in the original gelatin solution. The GBHA staining intensity (absorbance) appeared to be linear with the amount of ionized calcium in the range from 0 to 2 g/cm². The method allows detection of amounts of ionized calcium as low as 0.15 g/cm² or 0.0015 pg/².For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at –80°C in acetone containing 1% oxalic acid. Adjacent sections were stained with either GBHA or aldehyde-fuchsin (AF). Exocrine tissue hardly stained with GBHA whereas islet tissue stained intensely. For GBHA as well as for AF a variation in staining intensity (visual evaluation) between islets was observed. Islet GBHA- and AF-staining intensities did not correlate. The AF-staining intensity but not the GBHA-staining intensity decreased with increasing islet diameter. Also in pancreatic islet tissue the GBHA method appears to be very sensitive and reproducible and small differences in islet GBHA-staining intensity can be detected. The results indicate that between islets differences in ionized calcium content exist. These differences do not correlate with the degree of B-cell granulation.     

The electromotor system of the electric catfish (Malapterurus electricus): A fine-structural analysis

Summary The electromotor system of the electric catfish (Malapterurus electricus) consists of two large ganglion cells situated in the spinal cord, two single axons containing electric nerves and two large electric organs with several million electroplaque cells. The small, irregularly stacked electroplaque cells possess at their center a crater-like indentation from which a stalk like protrusion arises. Many synaptic contacts derived from a single axon collateral are carried on lobe-like protrusions at the terminal knob of this stalk. The electric nerve consists of a large myelinated axon (diameter: 25 m) surrounded by many layers of connective tissue cells. The two ganglion cells (200 m in diameter) are rich in elements of the rough endoplasmic reticulum, Golgi apparatus and lysosomal structures. The cytoplasm of the soma changes its appearance towards the voluminous axon hillock (50 m in diameter) which these organelles do not enter. The cell soma is perforated in a tunnel-like manner by blood capillaries, axons and processes of glial cells. The cell soma and dendrites are covered with two types of synapse. One type forms mixed chemical and electrical (gap junctions) contacts with intermediate attachment plaques. The other type is only chemical in nature. This system may be useful in the study of an identified vertebrate giant neuron.     

Pre-epithelial mucus layer in the colon of conventional and germ-free rats

Summary The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10m-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40±24 m at 120 days of age and 44±22 at 350 days of age in conventional rats, and 25±17m (120 days) and 22±10 (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free: 185±73m, conventional: 350±115m). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.     

Seasonal change of seston size distribution and phytoplankton composition in bivalve pearl oyster Pinctada fucata martensii culture farm

Seasonal changes of seston were monitored in the pearl cultivation area of Uchiumi Bay, Ehime Prefecture, Japan where the pearl oyster Pinctada fucata martensii is cultivated. The concentrations of chlorophyll a and particulate carbon, nitrogen and phosphorus were measured and the community structure of the phytoplankton investigated between June 1997 and February 1999. The seston was divided into pico- (<2 m), nano- (2–20 m) and large (>20 m) size fractions. The dominant size fraction during the study period, <2 m (38.1–51.4%), is consumed by oyster larvae but is too small for the adult pearl oyster. During the summers of 1997 and 1998 Nitzschia spp. became dominant but is indigestible for the oyster which was thus subjected to food limitation. We suggest monitoring the composition of seston and phytoplankton >2 m for evaluation of the food environment of the pearl oyster.     

Macromolecules can pass through occluding junctions of rat ileal epithelium during cholinergic stimulation

Summary Crypt, but not villus, goblet cells in the ileum accelerate their secretion of mucus within 5 min following cholinergic stimulation. This study was done to determine whether the macromolecular permeability and structure of occluding junctions in the ileum are altered during accelerated secretion. Rats were injected intravenously with horseradish peroxidase followed by carbachol (250 g/kg, subcutaneous) and the intestinal mucosa was fixed 3–12 min later. In control mucosa (saline-injected), peroxidase filled lateral intercellular spaces up to the occluding junctions of both crypt and villus epithelium, but did not enter occluding junctions or pass into the lumen. In 3 of 8 carbachol-stimulated rats, peroxidase was present within occluding junctions in crypt epithelium and in the crypt lumen, although all intermembrane junctional fusion sites appeared intact. Villus epithelial occluding junctions, in contrast, continued to exclude peroxidase. In freeze-fracture replicas of crypt cells prepared after carbachol stimulation, we detected no structural changes in strand networks of occluding junctions that could account for increased paracellular permeability.     

Islets of preadipocytes highly committed to differentiation in cultures of adherent rat adipocytes

Summary Cultures of adherent mature adipocytes, obtained from collagenase-digests of adipose tissue of the rat, invaribly contain rapidly proliferating, fibroblastlike cells despite the washing and centrifugation procedures empolyed during isolation of the fat cells. Such spindle-like cells originate from low-density structures, which we term islets, that are present, together with the mature adipocytes, in the floating layer of the digest of adipose tissue. Islets are found in preparations from adult (3–4 months old) as well as aging (17–24 months old) rats. By light-and electron microscopy, the islets appear as clusters of closely associated cells containing a variable amount of lipid-like material. Cells of endothelial or pericytic origin are also present in the islets. Within a few hours of culture, the islets give rise to those spindle-like cells that have been seen to proliferate in the cultures. By 36–48 hours, such cells begin to accumulate lipid droplets and, by 150 hours, assume the morphology of small mature adipocytes (diameter 20–35 m) with a large central lipid droplet. The pattern of differentiation of these cells recalls that of preadipocytes derived from the stromal-vascular fraction of adipose tissue digests. Nonetheless, the extent and rapidity of their adipose conversion, as well as the culture conditions necessary for differentiation, are different and suggest that these cells are a substantially uniform subpopulation of adipocyte-precursor cells highly committed to differentiation.     

Effects of phenoxybenzamine on insulin secretion from isolated rat islets of Langerhans

In isolated rat islets the ₂-adrenergic antagonist phenoxybenzamine was found to be only partially effective at relieving the inhibition of glucose-induced insulin secretion mediated by noradrenaline. Further experiment revealed a direct inhibitory effects of phenoxybenzamine itself on the secretory response to glucose. At concentrations above 1 M the antagonist inhibited insulin secretion in a dose-dependent manner, with greater than 50% inhibition at 50 M. The inhibition of secretion developed rapidly in perifused islets, and was not altered when islets were also incubated with idazoxan or benextramine, suggesting that it did not reflect binding of phenoxybenzamine to the ₂-receptor. Paradoxically phenoxybenzamine significantly increased the basal secretion rate in the presence of 4 mM glucose. The results demonstrate that phenoxybenzamine can exert direct effects on insulin secretion which are unrelated to its -antagonist properties.     

Improved islet survival and in vitro function using solubilized small intestinal submucosa

In vitro proliferation of isolated pancreaticislets has become an area of great interest given the scarcity of clinicalisletdonors and the islet mass requirements for clinical islet transplantation.Smallintestinal submucosa (SIS), a naturally occurring extracellular matrix, hasbeeninvestigated to promote wound healing, tissue remodeling and cell growth. Thisstudy evaluated recovery and function of isolated canine pancreatic isletsfollowing in vitro tissue culture. Pancreatic islets wereisolated from mongrel dogs using standard surgical procurement followed byintraductal collagenase distension, mechanical dissociation and EuroFicollpurification. Groups of purified islets were cultured in a humidifiedatmosphereof 95% air and 5% CO₂ for 48 hours in standard islet cultureconditions of CMRL 1066 tissue culture media (Gibco) which had beensupplementedwith 25M HEPES, penicillin/streptomycin and either 10% heat inactivatedfetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc.,West Lafayette, IN). The mean recovery of islets following the culture periodwas determined by sizing duplicate counts of a known volume and viability wasassessed by static incubation with low glucose (2.8 mM), highglucose (20 mM) and high glucose solution supplemented with 50m IBMX solution. Remaining islets were embeddedhistologically.From a consecutive series of six culture experiments, a significantly higher (p< 0.05) recovery of islets co-cultured with SIS was observed when comparedtocontrols. Mean islet recovery was 84.5 ± 2.9% (mean ± SEM) fromthe SIS cultured group compared with 64.7 ± 4.5% from the control groupcultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibiteda significantly higher (p <, 0.05) insulin response to the high glucosestimulus than islets cultured in the standard FCS cultured solution. Thecalculated stimulation index was 12.3 ± 3.4 for the SIS-treated groupcompared with 5.6 ± 1.8 for the standard cultured group (p < 0.05).The overall mean numbers of islets recovered following invitro culture was also higher in the SIS-treated group. Theproportion of islets with a mean diameter >150 m increasedfrom 24% to 31% in the SIS-treated group, whereas the same proportion decreasedto 18% from 22% in the control (FCS-treated) group. Histological evaluation offixed tissue samples collected following the culture period identified insulinand glucagon-secreting cells in the SIS and FCS treated groups, however ahigherfrequency of insulin positive cells were detected consistently in the SIStreated group. A proliferation marker (PCNA) identified positive cells withinboth groups as well. This study suggests that co-culture of freshly isolatedcanine islets in medium supplemented with solubilized SIS can improve thepost-culture recovery and in vitro islet function. Futureinvestigations will focus on the cellular interactions of SIS, bothinvitro and in vivo.     

Freeze-fracture and morphometric analysis of occluding junctions in rectal glands of elasmobranch fish

Summary The structure of occluding junctions in secretory and ductal epithelium of salt-secreting rectal glands from two species of elasmobranch fish, the spiny dogfishSqualus acanthias and the stingrayDasyatis sabina, was examined by thin-section and freeze-fracture electron microscopy. In both species, occluding junctions between secretory cells are shallow in their apical to basal extent and are characterized by closely juxtaposed parallel strands. Average strand number in the dogfish was 3.5±0.2 with a mean depth of 56±5 nm; in the stingray a mean of 2.0±0.2 strands encompassed an average depth of 18±3 nm. In contrast, the linear extent of these junctions was remarkably large due to the intermeshing of the narrow apices of the secretory cells to form the tubular lumen. Morphometric analysis gave values of 66.8±2.5 and 74.9±4.6 m/cm² for the length of junction per unit of luminal surface area in the dogfish and stingray, respectively. This junctional morphology is similar to that generally described for leaky epithelia. In comparison, the stratified ductal epithelium which carries the NaCl-rich secretion to the intestine is characterized by extensive occluding junctions which extend 0.6–0.8 m in depth and consist of a mean of 12 strands arranged in an anastomosing network, an architectural pattern typical of tight epithelia. The length density of these junctions in the dogfish rectal gland was 7.6±0.1 m/cm².The junctional architecture of the rectal gland secretory epithelium (few strands, large junctional length densities) is similar to that described for several other hypertonic secretory epithelia [20, 34] and is compatible with the recent model for salt secretion in rectal glands [39] and in other Cl^– secretory epithelia which posits a conductive paracellular pathway for transepithelial Na⁺ secretion from intercellular space to the lumen to form the NaCl-rich secretory product.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.