Xenobiocic-induced imbalance between production and ulilisation of cylosolic lipid provides biochemical evidence of structural and functional alaerations in animal tissues . The present study investigates metal induced accumulalion of unsaturated neutral lipid inthe hepatopancreas of the penaei

Manisseri, Mary K

Menon, N R

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The FO\Inh Indian Fisheries Forum Proceedings 24·211. Novcmlw-'r. I996. Kochi. p. 143· 145. Puhlisl"lolJ in 1999. Xenobiotic Induced Functional Alterations in Hepatopancreas of the Penaeid Shrimp Metapenaeus dobsoni (Miers) MARY K. MANISSERI, and N.R. MENON' Central Marine Fisheries Research Institute, Kochi 682014. 'School of Marine Sciences, Cochin University of Science & Technology, Kochi 682016. Abstract Xenobiocic-induced imbalance between production and ulilisation of cylosolic lipid provides biochemical evidence of slruclUrnl and functional alaerations in animal tis.~cs . lbe presem sludy invtsligalcs metal induced accumulalion ofunsaruraled neulrnllipid in !he hepalOp3.rtereaS of the penaeil shrimp M~t~~na~us dobs~n~ (~;e~) exposed to culture medium conlllining 0.05 and 0.15 ppm copper and 0.005 and 0.015 ppm mereu~. Accumula~~~ of neul,:,,"1 IIP.ld In arumal" exposed 10 0.05 ppm copper stCm5 to be higher lhan that in animals exposed to 0.15 ppm. ~uggestIng the poS.~lblhty or a blpilaslc effect of themetal. Shrimp exposed 10 mercury showed reasonably high coocenlration of neUlrnllipid In the hepatopancreas. the mobilization being more at higher meta1 concentration. A significant observation made during the present study was on the formation of greatly enlarged heterogenous Iy.so.umlcs as a result of exposure to heavy metals. Introduction Cellular accumulation of neutral lipid as a result of exposure of organisms to xenobiotics and the use of this phenomenon as a sensitive biochemical index to assess the cellular reactions to stress from pollution have been reported by Moore (1985), Capuzzo and Leavitt (1988), Lowe (1988), and others. Among the studies on the toxic effects of copper in crustaceans are those of Chen (1985) on Pellaeus monodon. Liao and Hsieh (1988, 1990) on P. japollicus and Macrobrachium rosenberg;;, Koivisto el al., (1992) on cladocerans, and Weis and Weis (1992) on Balallus ebumeus. Mercury, which has no established biological functionk also is one of the highly toxic heavy metals, affecting biological systems even at very low concentrations. Harmful effects of this heavy metal have been reported in Penaeus japonicus (Chuang el ali ' 1991), Penaeus shrimp and crab (Palmer el aJ. , 1992), P.aztecus (Palmer and Presley, 1993), Callianassa tyrrhena (Thaker and Haritos, 1993) and Pleoticus muelleri and Artemesia longinaris (Marcovecchio, 1994). It has been noticed that mercury toxicity causes structural and functional alterations at the molecular level, interferes with biochemical functions and cellular respiration and affects growth and development in crustaceans. It is in this context that a study has been undertaken on the effect of copper and mercury on the effect of copper and mercury on accumulation of neutral lipid in a commercially important penaeid shrimp, Melapellaeus dobsoni distributed abundantly in tropical waters. Material and Methods Juvenile Metapenaeus dobsolli measuring 25-35 mm in total length (from the tip of rostrum to the tip of tel son) were collected from an aquaculture farm at Vypeen (76' 10' Long E , and 10' 0' Lat N); transported to the laboratory in oxygen filled polythene bags and acclimatised to a salinity of 20 ± 2 ppt. 143 The test animals were fed ad libitum on fresh clam meat. Copper sulphate and mercuric chloride were used as sources of heavy metals. Test animals were exposed in batches often to sublethal doses of 0.05 and 0.15 ppm copper and 0.005 and 0.015 ppm mercury in a semi flow-through system for 15 days at room temperature (28 ± 2OC). Experiments were run using 3 replicates for each treatment and control. Test medium was well-aerated. Left over feed and faecal matter were siphoned out and 213 rd of the test solution in each treatment was replenished every 24h. Three animals (belonging to the intermoult stage) were selected from each replicate of the different treatment on closure of the experiment for histochemical studies. Hepatopancreas was dissected out immediately after sacrificing each animal and fixed ad a whole tissue in hexane using liquid nitrogen. Sections, 10 microns in thickness, were taken using a Bright Cryostat, ;maintaining a chamber temperature of -20 ± I ' C; post-fixed in calcium formal at 4'C for 15 min; rinsed in distilled water and placed in 60% triethylphosphate (in distilled water) for 3min. Staining was done in a 1% solution of Oil Red 0 in 60% triethylphosphate, for 15 min. at 20'C (Moore, 1988). The sections were then rinsed in 60% triethylphosphate for 30 seconds, rinsed in distilled water and mOilnted in an aqueous mounting medium, glycerin jelly. Unsaturated neutral lipid content of the epithelial ceils as indicated by a red colouration was determined by microscopical assessment of the sections. Results and Discussion The intensity of colour and the area of colouration indicate the quantity of neutral lipid available in the hepatopancreatic tissue from various treatments. Hepatopancreas necessarily has a storage, though small, of neutral lipid as evidenced by the colouration in the control animals (Fig. 1.1). Difference in the intensity of neutral lipid concentration in the hepatopancras of animals exposed to 0.05 and 0.15 ppm copper gives the evidence that lipid accumulation is more when the medium contains 0.05 ppm copper (Fig. 1.2 and 1.3). Since there is no mechanism by which the bulkness of the organelles containing lipids could be assessed based on colour, it is not possible to ascertain whether the reduced number and size of pigmented areas in the hepatopancreas of animals exposed to 0.15 ppm copper does really indicate, variation in the quantities of lipid distributed in the cytoplasm and Iysosomes or whether the metal shows biphasic effect on lipid accumulation in the hepatopancreas of Metapenaeus dobsoni. Fatty degeneration and increased cellular lipid content associated with heavy metal stress includes neutral lipid within the enlarged secondary Iysosomes as well as lipid droplets distributed in cytoplasm. Evidently, the quantitative distribution of neutral lipid in the secondary Iysosomes and cytoplasm of the hepatopancreas of animals exposed to 0.05 ppm copper seems to differ considerably from that seen in the animals exposed to 0.15 ppm copper. In the case of shrimp exposed to mercury, the variation in the neutral lipid concentration was cospicuous. Animals exposed to 0.015 ppm mobilized more lipid to the Fig. I.!. Hepatopancreas of M. dobson; maintained under controlled conditom: showing neutrallipid.X50 Fig.I.2. Heavily accumulated neulra1 lipid in the hepatopancreas of M. dobson; exposed to 0.05 ppm copper. XIOO Fig. 1.3. Hepatopancreas of M. dobson; exposed to 0 .15 ppm copper with lesser accumulation of neutral lipid. X 100 Fig. IA. Hepruopancreas of M. dobson; showing increased accumulation of neutral lipid when exposed to 0.015 ppm mercury. X IOO Fig. 1.5. Lesser accumulation of neutral lipid in the hepaIopancrcas of M. dob.wn; exposed to 0.005 ppm mercury. X 100 Ftg. I .6. Hepatopnncreas secondary Iysosomes in the hepatOpanCrea.1; of M. dobson; exposed to 0.05 ppm copper. X200 144 hepatopancreas (Fig. 1.4). However, from the intensity of colour it looks that animals exposed to 0.005 ppm also have reasonably high concentration of lipid in the hepatopancreas (Fig. 1.5) In this case also accumulation of n~utral lipid was much ;more than what was observed in the control group. A significant observation made during the present study was the formation of a different type of greatly enlarged secondary lysosome in metal abused animals. These lysosomes containing granular matrix. were found in very large numbers in the epithelial cells of the shrimp exposed to 0.05 ppm copper (Fig. 1.6). The heterogenous Iysosomes, resulting probably from the fusion of smaller Iysosomes, were surrounded by layers of discrete lipid droplets. The number of heterogenous secondary Iysosomes observed in animals exposed to 0.15 ppm copper and 0.015 ppm mercury was comparatively less (Fig. 1.3, 1.4 and 1.5). The hepatopancreas of the control animals was totally devoid of the heterogenous secondary Iysosomes. Xenobiotic induced sublethal cellular pathology rellects perturbations of function and structure at the molecular level and the primary events are generally associated with particular types of subcellular organelle such as Iysosomes, endoplasmic reticulum and mitochondria (Moore, 1985). According to Capuzzo and Leavitt (1988) changes in the lipid content and the lipid: protein ratio of digestive gland of Carcinus maenas were rellected along an ascending pollution gradient. However, not much literature is available on this phenomenon in crustaceans in general and shrimps in particular. In crustaceans. hepatopancreas plays a major role in the absorption and storage of lipid material. Mobilisation, accumulation and metabolism of lipid reserves influence growth, moulting, reproduction and adaptive responses to stress. Moore (1988) reporting fatty degeneration and associated lysosomal accumulation of unasaturated neutral lipid in the digestive cells of Mytilus edulis and Littorina lit/area from polluted waters, suggested this phenonmenon as a useful indicator of pathological alterations. Such enlarged and lipid enriched lysosomes frequenly showed reduced membrane stability, resulting in fragi lity and thereby augmenting transference of part of the increased cytoplasmic lipid to the Iysosomes by autophagy. Storvh and Kleinfeld (1985) also provided evidence for increased autophgy associated with reduced lysosomal membrane stabiltiy. Increased quantity of neutral lipid notived in the hepatopancreatic cells of shrimp in the present study a lso could probably be assoc iated with accumulation in the en larged secondary Iysosomes and distribution as droplets in the cytoplasm, as a consequence of exposure to sublethal doses of heavy metals. An increase noticed in the neutral lipid content of the hep.topancreatic cells of Metapenaeus dobson;, at a highcr concentration of mercury in the test medium indicates that the enhanced lipid content is toxic dose-dependent. This is in agreement with the inference drawn by earlier workers. However. the maximum lipid accumulati o n o h",crvcd In individuals exposed to lower concentration of copper needs explanation. Some xenobiotics have been known to exerl biphasic effects on organisms. Moore (1988) in a study on Mytilus edulis exposed to a diesel oil and copper mixture observed greatest lysosomal membrane stabililY in the low-exposure individuals. Here the xenobiotic is expected to have induced a stabilising effect on the lysosomal membrane. A similar biphasic effect of copper could probably be the reason for the larger quantity of neutral lipid found in the M. dobsol/i exposed to lower concentration of the metal, in the present study also. Most of the structural alterations observed in the invertebrate digestive epithlium brought about by exposure to pollutants are known to be due to increased lipid accumulations associated with enlarged secondary Iysosomes. Pathologically enlarged heterogenous secondary Iysosomes, developed as a consequence of pollutant exposure, were first reported by Lowe (1988) in a study on Mytilus edu/is exposed to different levels of diesel oil and copper mixture. According to the author their numbers and sizes seem to be directly proportional to the levels of xenobiotic the animal is exposed to. Occurrence of heterogenous secondary Iysosomes in large numbers in the hepatopancreatic epithelial cells of Me/apel/aeus dobsol/i exposed to 0.05 ppm copper as noticed in the present study is probably an indication of cellular pathology. Their absence in the control animals supports Lowe's (1988) statement that these Iysosomes are developed as a result of exposure to pollutants. However, a reduction in the number, in animals exposed to a higher level of copper (0.15 ppm) might indicate a certain level of biphasic effect exerted by the metal. Further, the lesser number of heterogenous secondary lysosomes, as noticed in animals exposed to sublethal levels of mercury would indicate a less pronounced induction of such lysosomes as a result of mercury toxicity. The fact that the heterogenous secondary Iysosomes are surrounded by layers of lipid droplets could result in the distributioh of lipid in the cytoplasm as well. The present study clearly shows that the lipid content in heavy metal stressed animals is at any point of time, more than that in the control, suggesting histochemicsl estimations of neutral lipid as useful tool to assess chronic stress from sublethal doses of xenobiotics. References CapuzlO, J.M. and Leavill, D.F., 1988. Lipid composition of the digestive glands of Mytilus edulis and carr:iflusnJnena in responsse to pollutant gradients. Mar. Ecol.Prog. Ser., Prog. Ser., 46: 139-145. Chen, H.e., 1985. Water quality criteria fro farming the grass shrimp, Pellaeus monodoll. In: Y. Taki, J.H. Primavera and J.A. L10brera (Editors), Proc. First International Conference on the culture of penaeid prawns/shrimps. Iloilo city, Philippines, 4-7 Dec. 1984. 9Abst): 165. 145 Chuang, Nin Nin and Yang, Bei Chia, 1991. Purification and properties of a beta-mannosidase from shrimp (Pellaeus japollicus)hcpatopancreas . Compo Biochem. Physiol. , 98B: 627-63. Koivisto, S., Kotola, M. and Walls, M., 1992. Comparison of five cladoceran species in short and long-term copper exposure. Hydrobiologia. 248: 125-136. Liao, I.e. and Hsieh, e.S., 1988. Toxicity of heavy metals to Penaeus japonicus~ I. Toxicities of copper, cadmium and zinc to Penaeusjaponicus. 1. Fish. Soc. Taiwan, 15:69-78. Liao, I.e. and Hsieh, e.S., 1990. Toxicity of three heavy metals to Macrobrachium rosenbergii. In: R. Hirano and I. 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Palmer, S.1 ., Presley, B.1. and Taylor, R.1. , 1992. Mercury bioaccumulation in oysters, Crassostrea virgillica, blue crabs, Callinectes sapidus and Penaeus shrimp in a contaminated estuary. J. Shellfish Res., II :204. Storch, J. and Kleinfelf, A.M., 1985. The lipid structure of biological membranes. Trends Biochem. Sci. , 10:418-420. Thanker, A.A. and Haritos, A.A. , 1993. Mercury bioaccumulation and effects on soluble peptides, proteins and enzymes ·in the hepatopancreas of the shrimp Callianassa tyrrhena. Final reports on Research Projects Activity- G. Athens-Greece UNEP, 48:89-104. Weis, J.S. and Weis, P., 1992. Construction materials in estuaries: Reduction in the epibiotic community on chromated copper arsenate (CCA) treated wood. Mar. Ecol. Prog. Ser., 83:45-53. 

Xenobiotic induced functional alterations in Hepatopancreas of the Penaeid shrimp Metapenaeus dobsoni (Miers)

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Xenobiotic induced functional alterations in Hepatopancreas of the Penaeid shrimp Metapenaeus dobsoni (Miers)

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