Ultrastructural Observations on the Gill Filaments of Kuruma Prawn, Penaeus japonicus Bate Exposed to Air

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1 Ultrastructural Observations on Gill Filaments Kuruma Prawn, Penaeus japonicus Bate Exposed to Air Maher SAMET United Graduate school Kagoshima University, Faculty Fisheries, Shimoarata, , Kagoshima 890, Japan Abstract The fine structure gill filaments Kuruma prawn, Penaeus japonicus, exposed to air were studied with transmission electron microcopy, were compared to normal gill filaments. After 12h 14 Ž air exposure, electron microscopic examinations gill filaments did not show any changes except in configuration mitochondria. Contrasting with normal mitochondria, which show well developed cristae an electron dense matrix, mitochondria in air exposed prawns had collapsed rearranged cristae. The normal mitochondria were called energized modified, non energized. During aerial exposure, oxygen supply to metabolically active mitochondria decreased in collapsed gills, electron transfer ATP formation could not be harnessed efficiently, thus resulting in modified mitochondria. Many studies have dealt with relationships between mortality various environmental conditions under which prawns are transported. Although no observations have been made on histological changes occurring in prawn organs during live transportation, gill is assumed to be main site respiration even in air. In cold air, prawn withst long periods without any apparent damage. Despite anatomical works on general description white shrimp P. setiferus1), branchial organ Kuruma prawn2), histology fine sturcture gills various penaeid species (Pink shrimp P. duorarum3) Brown shrimp P. aztecus4,5) few studies have been done on fine structure gills kuruma prawn P. japonicas. The purpose this study was examine ultrastuctural changes in gill filaments Kuruma prawn when exposed to air, which is practically an important condition in live transportation. Kuruma prawns are presently transported live in chilled sawdust without water. During this study, electron microscopy observations were made on branchial filament tissues normal air exposed Kuruma prawn. Materials Methods Animals Kuruma prawns P. japonicas were obtained from Aira Prawn Farming Iwasaki Industry Co., Kagoshima. They had been reared in field ponds at Ž from early summer to autumn. Prawns were acclimated in a indoor tank equipped with a central pipe, s gravel filter at bottom, fed with prawn pellets (2.5-3% body weight per day) for one month. The water temperature was controlled at 24 Ž Received : September 1, 1995 Key words: Penaeus japonicas, Fine structure gill, Air exposure

2 74 M. Samet (1996) with a cooling equipment (Rei-Sea cooler RZ-140) with a precision }0.4 Ž. Transmission electron microscopy (TEM) shown in Figs Each cytoplasmic process contains a basal nucleus, rough endoplasmic reticulum (er), golgi apparatus (G) numerous round to elongate mitochondria (m) (Fig.1). Ribosomal particles appeared to be grouped freely throughout cytoplasm. The nucleus epilial (N) lies in septum. Its cytoplasm ranges from light to deuce (Fig.1), conspicuous, ranges in shape from round to multilobed (Figs.1 2b). The most abundant organelles found in cytoplasm were mitochondria rough endoplasmic reticulum (er) (Fig.1). Abundant mitochondria in cytoplasm epilial s in filament suggest need to produce large quantities energy probably related to regulatory functions epilial s ir extended cytoplasmic sheets. The commonly encountered type were normal in appearance, having well developed cristae a uniformly electron dense matrix (Fig.2a). Five individual prawns (10.95 }0.97g) at intermolt stage were exposed to 14 Ž air (75-85% relative humidity) in an incubator for 12h. After this, gill samples were excised fixed in ice-cooled 5 % glutaraldehyde buffered with 0.1 M ƒá-collidine (ph 7.7) for 2h. Samples were repeatedly rinsed with 0.2 M ƒá-collidine left overnight in a refrigerator. The next day, y were post fixed in 1% osmium tetroxide buffered with same solution for 2h. Osmolarities fixative buffer were adjusted to approximate that to prawn gills by addition sucrose (6.5 %). After dehydration through an ethanol series, samples were embedded in Epon 812. Thin sections tissues, cut by an ultramicrotome (ULTROTOME III 8200) using glass knives, were double stained with uranyl acetate lead citrate6). They were observed under a Hitachi H-700 H electron microscope compared with normal gills individuals not exposed to air. Normal tissues were obtained from 5 similarly-sized intermolt individuals (11.08 }0.68g). Study ultrastructure normal gills was completed prior to examination gills air-exposed prawns. Results Ultrastructural observation normal gill filaments The gills normal Kuruma prawn were dendrobranchiate type, consisting a central axis that bore primary gill filaments which in turn branch into finer gill filaments. The Kuruma prawn has 18 pairs gills in branchial chamber, that is, six wall-gills, five anterior joint-gills, six posterior joint-gills a foot-gill2). The focus present study was distal filaments gills, which are smallest functional anatomical units gill system visible to unaided eye. In histological sections, normal filaments consisted a cytoplasmic sheet derived from s that are mainly medial in position. These s ir extended cytoplasms made up gill epilium are Ultrastructural observation gill filaments prawn exposed to air After air exposure, all gill tissue ular organelles retained ir normal ultrastucture with exception mitochondria regardless ir location. Most priles revealed ir size to be some what larger than those normal gill tissue. Primary degenerative changes found in mitochondria were indicated by irregularity in shape. They were packed with tubular cristae more largely spaced than those usually found in normal mitochondria had a matrix low electron density (Figs.2 b 2c). In some mitochondria, cristae were poorly developed ten found only near periphery matrix (indicated by arrows in Fig.2b). Discussion In air exposed prawns, most s gill filament did not loose ir structural integrity organization. Only fine structural changes in mitochondria with a complete rearrangement cristae were observed. These changes were attributed as effects air exposure. The cristae inner membrane mitochondria were considered to exist in two configurational states: 1) energized (found in

3 Effects Fig. 1. Longitudinal air Penaeus lial drion; G, golgi section exposed (b) japonicus. N, in er, rough (a) filament nucleus septum; apparatus. ~7,000. normal gill endoplasmic air exposure m, epi- Fig.2. on gills fine structure Epilial mitochon- electron epilial dense tae; also matrix cated when well (a) collapsed were indicated magnification asterisk found by (b) to in air near showing Normal developed exposed with cristae extensions nucleus matrix). ~36,000; as by cytoplasmic (note reticulum; High mitochondria 75 b. ~36,000. Mitochondria (asterisk notes rearranged periphery arrows). ~12,000; collapsed gill cristae cris (c) indi-

4 76 M. Samet (1996) normal gills, Fig. 2a), 2) non energized (found in air-exposed gills, Fig.2c). Mitochondria are known as site metabolic activity, constantly providing framework for electron- transfer processes that produce ATP. Electron transfer hydrolysis ATP are two enzymic means generating energized state7). On non-energized state, membrane cristae were observed to be collapsed probably due to a lack oxygen lower respiration rates. Larger sized prawns show a significant fall in ATP concentrations a rise in AMP concentrations in abdominal muscle when stored in air at 17 Ž for 24 hours8). On energized state (as in normal under water respiration) cristae retain ir tubular characteristic. Oxygen uptake measurements from prawns in air showed that prawns are initially able to consume adequate amounts oxygen to maintain energy metabolism (i. e. ATP turnover) this ability decreases markedly with lapse time8). Taylor Weateley9) say that hypoventilation occurs when crustaceans leave water (ir gill bailer beats more rapidly) despite this oxygen level in ir blood decreases. After long term air exposure, oxygen supply to metabolically active mitochondria decrease oxidation reactions in cristae could not be completed. Insufficient molecular O2 in heamolymph due to air exposure decreases rate oxidation reactions phosphorylation. The resulting lack ATP could not maintain mitochondria in energized state cristae collapse. Hence, degree gill collapse would depend upon extent gill filament dysfunction which in turn depends upon extent changes in mitochondria s. It is suggested that configurational changes are relevant to work performances coupled with ATP synsis. Acknowledgments The author would like to thank Pr. S. Higashi Faculty Science, Dr. K. Yasui Faculty Dentistry, Kagoshima University, for ir valuable advice providing facilities for electron microscopy work, to Mr. T. Kakoi for his technical assistance. This study was supported by Ministry Education, Science culture, Japan. References 1) Young, J. H. (1959) : Morphology white shrimp Penaeus setiferus (Linnaeus,1758). Fish. bull., 145, 59, ) Nakamura, K. K. Hiyamizu (1981) : Anatomy branchial organ prawn Penaeus japonicas Bate. Mem. Fac. Fish., Kagoshima Univ., 30, ) Couch, J. A. (1977) : Ultrastrural study lesions in gills marine shrimp exposed to cadmium. J. Invert. Path., 29, ) Foster, C. A. H. D. Howse (1978) : A morphological study on gills brown shrimp, Penaeus aztecus. Tissue Cell, 10 (1), ) Talbot, P., W. H. Clark, A. L. Lawrence (1972) : Light electron microscopic studies on osmoregulatory tissue in developing brown shrimp, Penaeus aztecus. Tissue Cell, 4 (2), ) Reynolds, E. S. (1963) : The use lead citrate at high ph as an electron opaque strain in electron microscopy. J. Cell Biol., 17, ) Green, D. E., J. Asai, R. A. Harris, J. T. Penniston (1968) : Conformational basis energy transformations in membrane systems. Arch. Biochem. Biophys., 125, ) Paterson, B. D. (1993) : The rise in inosine monophosphate 1-lactate concentration in muscle live penaeid prawns (Penaeus japonicas, Penaeus monodon) stressed by storage out water. Comp. Biochem. Physiol., 1068, ) Taylor, E. W. M. G. Wheatly (1980) : Ventillation, heart rate respiratory gas exchange in crayfish Autropotamobius pallipes (Lereboullet) submerged in normoxic water following 3h exposure in air at 15 Ž. J. Comp. Physiol., 138,

5 Effects air exposure on gills fine structure

Aberrant Mitochondria with Longitudinal Cristae Observed in the Normal Rat Hepatic Parenchymal Cell. Takuma Saito and Kazuo Ozawa

Okajimas Fol. anat. jap., 44 : 357-363, 1968 Aberrant Mitochondria with Longitudinal Cristae Observed in the Normal Rat Hepatic Parenchymal Cell By Takuma Saito and Kazuo Ozawa Department of Anatomy, Kansai