Biochemical changes taking place in ovary, hepatopancreas, muscle and haemolymph of P. indicus

during ovarian maturation have been studied and quantified. The trends in the fluctuation of metabolites

indicate that in ovary th~re is considerable accumulation of protein, lipid, cholester9l and carotenoids

but loss of moisture and nucleic acids. Translocation of these metabolites takes place from the chief synthetic

and storage organ, hepatopancreas, via haemolymph. During the initial stages of yolk synthesis,

ovary is autosynthetic as indicated by high RNA values and RNNprotein ratio. Subsequently the mode

of yolk formation shifts to heterosynthesis mainly through the uptake of lipid from hepatopancreas.

RNNDNA ratio has been found to be useful as an index of ovarian growth

Diwan, A D

Mohamed, K S

30-34Biochemical changes taking place in ovary, hepatopancreas, muscle and haemolymph of P. indicus during ovarian maturation have been studied and quantified. The trends in the fluctuation of metabolites indicate that in ovary there is considerable accumulation of protein, lipid, cholesterol and carotenoids but loss of moisture and nucleic acids. Translocation of these metabolites takes place from the chief synthetic and storage organ, hepatopancreas, via haemolymph. During the initial stages of yolk synthesis, ovary is autosynthetic as indicated by high RNA values and RNA/protein ratio. Subsequently the mode of yolk formation shifts to heterosynthesis mainly through the uptake of lipid from hepatopancreas. RNA/DNA ratio has been found to be useful as an index of ovarian growth

Mohamed, K Sunilkumar

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Biochemical changes in different tissues during yolk synthesis in marine prawn Penaeus indicus  H.Milne Edwards

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-Indian Journal of Marine Sciences Vol. 21 , March 1992, pp. 30-34 Biochemical changes in different tissues during yolk synthesis in marine prawn Penaeus indicus H. Milne Edwards K Sunilkumar Mohamed· & A D Diwan Central Marine Fisheries Research Institute, P.B. No. 2704, Koehl (Cochin) 682 031, India Received 27 March 1991; revised 19 Sep,emher 1991 Biochemical changes taking place in ovary, hepatopancreas, muscle and haemolymph of P. indicus during ovarian maturation have been studied and quantified. The trends in the fluctuation of metabolites indicate that in ovary th~re is considerable accumulation of protein, lipid, cholester9l and carotenoids but loss of moisture and nucleic acids. Translocation of these metabolites takes place from the chief syn-thetic and storage organ, hepatopancreas, via haemolymph. During the initial stages of yolk synthesis, ovary is autosynthetic as indicated by high RNA values and RNNprotein ratio. Subsequently the mode of yolk formation shifts to heterosynthesis mainly through the uptake of lipid from hepatopancreas. RNNDNA ratio has been found to be useful as an index of ovarian growth. Oogenesis in crustaceans includes synthesis of nut-ritive yolk in the ooplasm to meet the basic require-ments of embryortic development, independent of the maternal orgartism' . Studies on yolk production in crustaceans have focussed on the potential tissue sources of the yolk components like hepatopan-creas, muscle and epidermal adipose tissues2 - 7• In most crustaceans, yolk materials produced in the extra-ovarian sites are transported to the ovary through the haemolymph (heterosynthesis). A re-cent ultrastructural and histochemical study of the ovary in Penaeus indicus has indicated that the bio-synthesis of yolk in this penaeid prawn is probably by auto-heterosynthetic means". However the quali-tative and quantitative information on the mobiliza-tion and build up of orgartic reserves in relation to reproduction in culturable species of penaeid prawns is lacking. In view of the significance of such information in the scientific management of brood-stock in prawn hatcheries, the present investigation on female P. indicus has been carried out·to quantify variation in protein, carbohydrate, lipid, cholester-ol, carotenoid, DNA, RNA and moisture in various tissues like ovary, hepatopancreas, muscle and haemolymph during different maturity stages. Materials and Methods Live females of P. indicus (120-170 rnm total length) were collected from the sea off Cochin by short duration otter trawling. The prawns were then ·Present address: C M.F.R.I., P.B. No. 244, Bolar, Mangalore 575001 , Kamataka, India transported live to the laboratory and kept in plastic pools containing seawater (sal. 28-32 x 10 - 3) and were later segregated according to a 5 point matur-iry scale'. Samples of haemolymph (HL), ovary (OV), he-patopancreas (HP) and muscle (MS) from prawns of all maturity stages were analysed for total protein'o, lipid" , carbohydrate 12, cholesterol 13 and carote-noid 14 contents. HL samples were collected by di-rect cardiac puncture using a hypodermic syringe rinsed in an anticoagulent (10% trisodium citrate). OV, HP and MS tissues were analysed for moisture, RNA" and DNA'6 contents in addition to the above. Carotenoids were not estimated in MS tissues be-cause of undetectable levels. Dried (70°C) and pow-dered tissue samples were used for the estimation of protein, lipid, carbohydrate and cholesterol, while for the estimation of moisture, carotenoids, DNA and RNA fresh tissues were used. Six replicates were carried out for each estimation. Analysis of variance (ANOVA) was performed to test significant differences in biochentical values of a particular metabolite during different maturity stages. Results and Discussion The results show cyclic variation and accumula-tion of organic reserves in HL, OV and HP of P. in-dicus during yolk synthesis. Proteins-Significant (P< 0.01 ) hike in the pro-tein levels observed (Fig. lA) during active phases of vitellogenesis in HL as well as OV indicates prob-able plasmal transport of proteins from an external • MOHAMED & DIWAN: BIOCHEMICAL CHANGES IN PENAEUS INDICUS 31 source to the ovary. In the American lobster Homo-rus americanus, total serum protein was in peak le-vels during the mature stage due to the mobilization of proteins to the ovary I" Although the protein con-tent in HP was poor, there was a progressive reduc-tion in protein levels in this organ during vitellogen-esis indicating that considerable mobilization of proteins takes place from HP. Other studies··19 on yolk protein synthesis in penaeids have shown that ovary is the prime source of yolk proteins. However, in a recent study on P. vannamei, Quackenbush6 re-ported that HP produces a subunit of egg yolk pro-tein which is transported to OV through HL. He al-so suggested that egg yolk protein could serve as a lipid shuttle to the developing ovary. Since the quan-tum of proteins transferred from HP to OV in P. in-dicus is small and as almost all of the lipid yolk is derived from HP, these proteins may also function as lipid carriers. Moreover, an earlier study" has shown that pinocytotic vesicles along the oolemma! z W ... o "' .. 20 _ov .--.... HL I ___ • MS v----'9 HP L ___ +-----l", 1 1 r ... " ... ;<. \.. ...... -J ,' \ , , / , , ' , , , , / , , , , 'f 120 10 ""-, i i-ii: ~--_r----~----~r_----r-----~--~~ l I!! .. " ~2 .. "' .. u / / / / -1 ' A'/ \ t--·,~-· t------+-----+------1 r / \I III IV V Maturity stages ~ o :II ~ 2 % Fig. I-Trends in variation of protein (A ) and carbohydrate (8 ) in HL. av, MS and HP during different maturity stages [Vertical lines indicate standard deviation {SD)I wall of P. indicus aids in active intake of organic ma-terial from outside source into oocytes. Although MS protein did show significant (P< 0.01 ) variation., MS RNNprotein ratio (see Fig. 5A) was uniform throughout indicating that protein synthesis did not take place in MS in relation to yolk synthesis. Earlier studies l7, have also shown that maturity stages did not alter MS biochemical com-position of Indian penaeid prawns. Carbohydrates-Differences in carbohydrate content (Fig. IB) in HL, OV and HP were signifi-cant at 1 % level, while in ¥S, it did not show any significance. In HL, carbohydrate content increased four-fold during stage N but decreased drastically in spent prawns. Similarly in Parapenaeopsis hard-wickii'°, it was reported that an incessant hike in HL glucose levels occurs alongwith ovarian develop-ment. The trend of HL and hepatic carbohydrate content observed presently indicates translocation of these substances to ovary for utilization in the synthesis of yolk and perhaps also as a fuel during 20 10 T i t ------+-----f------f-------1 i ;---~--~~--_,-----r----~--+~ o • l B • " .. ... .. .. ~ o % U • --- -----+-----+-----1\ II IV V MD~rity stages :II .. .. % 0 .4 0 .2 Fig. 2- Trends in variation of lipid (A ) and chok'stcrol (R) in HL. av. MS and HP during differenl m.lIUril Y slagcs [Vertical lines indica!..: Sf) ! 32 INDIAN J MAR SCI, VOL. 21, MARCH 1992 the viteUogenic process. There is evidence to show that in P. notalis glycogen is mobilized from HP to the gonads during ovarian development21. Upidy-Variation in lipid levels (Fig. 2A) in HL, OV and HP were significant (P< 0.01) indicating mobilization of lipids from HP to OV via HL. In slage V, spent recovery, HL lipid content remained at comparatively high levels due to possible resorp-tion of relict oocytes, which involves retransport of lipid material from the ovary back to the storage site. This may also be due to the rapid rematuration capability of this species22• Relatively high lipid le-vels present in HP of spent prawns also support this view. Hepatopancreas has been identified as the princi-pal storage site for lipids in cruslaceans2J, and in penaeid prawns, origin of ovarian lipids is reported to be the hepatopancreas24• Apparently large amount of this stored lipid is mobilizt!d to OV from HP in P. indicus during sJages II, III and IV through HL. The process of synthesis, accumulation and mobilization of lipid from HP is seemingly continu-ous as indicated by the peak value (52%) in sJage III and the marginal decrease thereafter in sJages IV and V. Further, resorption of relict oocytes and re-maturation can also account for high lipid content in HP of spent prawns. MS tissues were poor in lipids and no definite trend could be observed. Cholesterol-HL and OV showed (Fig. 2B) con-siderable accumulation of cholesterol during vitel-logenesis. Concurrently there was a decrease in cho-lesterollevels after stage III in HP indicating its con-tinuous accumulation and transport to OV via HL. Muscle tissues did not show any significant var-iations while variations in HL, OV and HP were significant (P< O.ot). Cholesterol has been demon-strated to be the dominant sterol in the ovary of penaeidsi 5. Cruslaceans lack the ability to synthes-Carotenoidy-The deepening colouration of OV during maturation was due to the increase of carote-noid pigments from slage I to IV. The increasing trend of carotenoid content (Fig. 3B) in HL is ap-parently owing to its transfer from HP to OV. HP was the richest source of carotenoids and peak va-lue was observed in slage III after which the values slabilized between 45 and 47 I'g.mg- 1. The hepato-pancreas in crustaceans plays a major role in the ab-sorption of carotenoids from food and has been ob-served to show fluctuation during viteUogenesis27 . Ceccaldi and Martin28 also showed that in Carcinus maenas carotenoid pigments are transferred from HP into HL during viteUogenesis. However, the pre-cise biological function of carotenoids in crusla-ceans is stiU vague2 •. Muscle tissues did not have de-tecJable quantities of carotenoids and differences in HL, OV and HP were significant at 1 % level. Ribonucleic acid-RNA content (Fjg. 4A) in OV decreased significantly (P< 0.01) from an initial peak of 51I'g.mg- 1 in Slagel to 16 I'g.mg- 1 in slage IV and was restored to the initial concentration in ao .. '" " ... !!60 o " 40 10 I~ rN " , - 10 '"-I .-- MS ~ HP ------- ------+-----+---1 " / 40 ize cholesterol although they are necessary to supp- 50 ress the permeability of phospholipid membranes' ·' _ ./·r1··· '1 and therefore it is presumed that cholesterol present . F'O in HP is derived solely from the diet. Moisture-Moisture (Fig. 3A) was the principal component of ripe ovary although it showed a dec-lining trend in both OV and HP and maximum va-lues were seen in spent prawns. Percenlage var-iations in OV and HP were significant (P< O.ot) while in MS it was less and not significant (P> 0.01). In Metapenaeus affinis and Portunus pe/agicus, an inverse relationship was observed between water content and gonad development"'o. It is possible that continued deposition of organic materials in ovarian and hepatic tissues results in loss of water content. 010 ii. .. ... o '" -<. u II 111 Moturrty .Ioge. , IV V Fig. 3-Trends in variation of IIlOistun (A) and carolenoids (B) in HL. OV. MS and HP during differenl maturity Slages IVerticallines indicale SOl • MOHAMED & DlWAN: BIOCHEMICAL CHANGES IN PENAEUS INDICUS 33 stage V. RNA content of MS also showed significant variation (P<0.05). In lIP it showed significant (P< 0 .01) increase from stage I to N and sharply decreased ii1 spent animals. RNNprotein ratio in OV decreased to very low levels in the ripe stage (Fig. 5A). High values were observed in stage I and V coinciding with peak protein synthesis. In lIP, this ratio peaked in stage ill due to high metabolic rates taking place in the organ. In MS, the ratio did not vary with maturation. Despite their importance to crustacean biology, nucleic acids have been investigated only to a limit-ed extent'!, particularly in relation to reproduction. During the early vitellogenic phase of oocytes, RNA level and RNN protein ratiq in the ovary of P. indf. cuy was consistently high due to the autosynthetic manner of protein synthesis and the same was re-duced when heterosynthesis was predominant. The RNA content in tissues is known to be high corre-sponding to the period of maximum protein synthe-sis32. In ocypodid crabs, oocyte resorption resulted in reduction of RNA levels in the ovary and oogoni-~ x CI > o 6 -7"<ID o E ~ ~ -:;: z '" -20 o. "7 0.3 o E .. -3 .. z o 0 .1 II 111 IV v Motll'ity s10ges • , , , '" '" , I> o aI proliferation coincided with elevated RNA, DNA and protein contents". ' Deoxyribonucleic acid-Mean DNA content (Fig. 48) in OV showed a declining trend which was sign-ificant (P< 0.01) with ovarian maturation and low-est value was noticed in stage N and highest in stage V. This is probably due to rapid increase in cyto-plasmic volume rather than an actual decrease in content. However, in spent prawns, ovaries undergo quick recovery with mitotic multiplication of oogo-nial cells and this explains the remarkable increase in DNA content during this stage. In MS, the varia-tion was marginal (P< 0.05) and in lIP, there was a gradual build up from stage I to V (P< 0.(1). Hepat-ic DNA levels were consistently high due to the syn-thetic activity taking place in them. In OV, RNNDNA ratio showed a steady in-crease, peaking in stage N and declining in stage V (Fig. 58). In lIP, the ratio peaked in stage ill and decreased in later stages. RNNDNA ratio of MS did not show much fluctuation. RNNDNA ratios have been frequently used as growth index in • 10 '" :0 Goo ~ "TO ;; '" ;loa o , .. z '" 10 _ ov ~ HP .. --- .. , .------------...... ----- -----.. ... ______ ~--__ -..... -------4'-------II II IV v Maturity .t*" 400 ... Fig. 4-Trends in variation of RNA (A) and DNA (B) in OV, MS Fig. 5-Variation in RNNpro tein ratio (A ) and RNNDNA ra-and HP [Vertical lines indicate SD} tio (B) in OV, MS and HP during different maturity stages. 34 INDIAN J MAR SCI, VOL. 21 , MARCH 1992 Table I-Percentage biochemical composition of mature yolk in P. indicus Metabolite Percentage Moisture 65.41 wetwt Protein 39.30 drywt Upid 29.14 drywt Carbohydrate 2.80 drywt Ololestero) 4.14 drywt Carotenoid 0.59 wetwt DNA 0.01 wetwt RNA 1.63 wetwt fishes32 and prawns34 and the hike in the ratio in the OV indicates its rapid growth by accumulation of yolk. In crustaceans, composition of yolk varies from species to species I. The percentage biochemical , composition of mature yolk is given in Table 1. Wa-ter formed the principal component of yolk and proteins and lipids constituted the major organic re-serves. Other metabolites were present in small quantities only. This study also indicated that a sign-ificant portion of proteins and carbohydrates and al-most all of lipids, cholesterol and carotenoids found in the yolk were derived from HP via HL. Yolk (pro-tein) synthesis takes place in the ovary only during the early stages of vitellogenesis. Therefore, biosyn-thesis of yolk is seemingly carried out through dual processes, auto-synthesis and heterosynthesis, and the latter appears to be the predominant one. Acknowledgement Thanks are due to Drs E G Silas and P S B R James for facilities and encouragement. The authors are grateful to leAR, New Delhi for financial sup-pon during the study. References 1 Adiyodi R G & Suhramoniam T, in Reproductive biology of invertebrates. Vol. I. edited by K G Adiyodi & R G Adiyodi (Wiley. Chichester ) 1983. 443. 2 Paulus J E & Laufer H, 1m J Invertebrate Reprod INv, 11 (1987) 29. 3 Tom M. Goren M & Ovadia M. Inl J Ihvertebrate Reprod Dey. 12 (1987) I. 4 Yano I & Chinzei Y. Comp Biochem Physio, 86 B (1981) 213. 5 Quackenbush L S & Keeley L L, Bioi Bul, 175 (1988) 321. 6 Quackenbush L S, Comp Biochem Physio~ 94 B (1989) 253. 7 I'enafIorida V & MiUamena 0 M, Israeli J AqlUJcuItu,.." 42 (1990) 84. 8 Mohamed K S, Studies on the reproductive endocrinology of the penaeid prawn Ponaeus indicus H. Milne Edwards, Ph.D. thesis, Cochin University ofScieoce and Technology, 1989. 9 Rao P V,FAO Fish Rep, 57 (1968) 285. 10 Gornall A G, BradawiU C J & David M M, J Bioi Chem. 177 (1949) 751. II Folch J, Lees N & Sloane-Stanley G H, J Bioi Chern, 220 (1957) 497 . . 12 Dubois M K. Gilles K A, Hamilton J K, Rebers P A & Smith F, Anal Chern, 28 (1956 ) 350. 13 Hestrin S,JBiol Chern, 180 (1949) 249. 14 OlsonJ A. Nut Rep 1nJ, 19 (1979)807. IS Dagg M J & UnlepageJ L, Mar Bioi, 17 (1972) 162. 16 Cerriotti G, JBiol Chern, 198 (1952)297. 17 Achu\bankuny C T & Parulekar A H, Mahasttgar-Bull Natn Inst Oceanogr, 17 (1984) 239. 18 Barlow J & Riggeway G T, J Fish Res Boord C"""tia, 26 (1969)2101. 19 Rankin S M, Bradfield J Y & Keeley L L, Inl J /"",nebrate Reprod lJey, IS (1989) 27. 20 Nagabhushanam R & Kulkarni G K, Hydrobio, 70 (1980) 29. 21 Trujillo R L & Luna 1 F, Rev Invest Mar, 2 (1981) 141. 22 Mohamed K H, Muthu M S & Laxminarayana A, Rematura-lion of fNlUJeid p1TJMInS after first spaMlFting in controlled S)'S"" tems, Paper presented at First lndo--Pacific symposium on Invertebrate Reproduction. AI1IlIII8III>od, 1981. Z3 Chang E S & O'Connor J 0 , in The Biology of crustacea, Vol. 5, edited by L H Mantel, (Academic Press, New York) 1983,263. 24 Castille F L & Lawrence A L, J CrustJJcean Bio, 9 (1989) 202. 25 Middledltch B S, Missler S R, Hines H B, Mevay J P, Brown A. Ward 0 G & Lawrence A L, J Chromatogr, 195 (1980) 359. 26 Morris R J, Annitage M E. Ballantine J A & Lavis A, J Exp Mar Bioi Eco, 53 (1981 ) 105. 27 Castillo R. Nip-e- Sadargues G & Lenel R, in CarOienoid chemistry and biochemistry, edited by G Brinon & T W Goodwin, (Pergamon Press, Oxford) 1982, 211 . 28 Ceccaldi H J & Manin J L M. C R Soc Bio, 163 (1969) 2638. 29 Goodwin T W, The biochemistry of the carotenoid.<, Vol. 2, Animals, (Chapman & Hall, New York) 1984, pp. 115. 30 Pillay K K & Nair N B. Mar Bio, 18 (1970) 167. 31 Claybrook 0 L. in The biology of crustacea, Vol. 5, edited by L H Mantel, (Academic Press, New York) 1983, 163. 32 Buckley LJ. Mar Bio, 80 (1984) 291. 33 Nadarajalingam K & Subramoniam T, Mar Ecol Prog Ser. 36 (1987) 43. 34 Thomas G & Diwan A D. Proc [ndum Acad Sci (Anim SCI). 99(1990) 125. 

Biochemical changes in different tissues during yolk synthesis in marine prawn Penaeus indicus H. Milne Edwards

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Biochemical changes in different tissues during yolk synthesis in marine prawn Penaeus indicus H. Milne Edwards

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