Application of RNNDNA ratio and tryptic enzyme activity on laboratory-reared and wild-caught herring larvae - Short communication -

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1 8 Application of RNNDNA ratio and tryptic enzyme activity on laboratory-reared and wild-caght herring larvae - Short commnication - Catriona Clemmesen and Bernd Ueberschiir Institte for Marine Sciences, University of Kiel, DUsternbrookcr Weg 2, D-W-23 Kiel, Federal Repblic of Germany Abstract RNA/DNA ratio and tryptic enzyme activity were shown to be appropriate indicators to detennine starving herring larvae or herring larvae in bad ntritional condition in laboratory rearing experiments. Based on the laboratory reslts, the methods were applied to evalate the ntritional condition of larvae from a field sample and to detennine the proportion of starving larvae. Keywords: herring, larvae, ntritional condition, starvathm, RNA!DNA ratio, tryptic enzyme activijy Introdction For stdies on the recritment process of fish larvae and in aqacltre, indicators are needed for the detennination of the n!ritional condition. RNAJDNA ratios and tryptic enzyme activity measrements were fond to be appropriate indicators to describe the ntrilional condi!ion (Bckley 1979, Clemrnesen 1987, Hjelmeland et al. 1984, Martin et al. 1985, Pedersen et al. 1987, Pedersen and Hjelmeland 1,988, Ueberschar 1985). Highly sensitive florescence techniqes were developed to measre both,indicators in individal marine fish larvae (Clemrnesen 1988, Ueberschar 1988). Before these rne!hods can be applied to fish larvae with nknown ntritional condition, they have to be calibrated with fish larvae reared nder defined conditions in the laboratory. The paper presents an application of RNA/DNA ratio and tryptic enzyme activity detenninations on laboratory-reared fed and starved herring larvae and gives an example for the evalation of the ntritional condition of herring larvae from a field sample based on the laboratory reslts. VCH Verlagsgesellschaft mbh, D-694 Weinheim, 1993

2 228 Catriona Clemmesen and Bernd Ueberschiir Materials and methods Larval fish. Herring (Clpea harengs) laivae aged 9-65 days for the RNAJDNA ratio measrements and aged 1-45 days for the tryptic enzyme activity determinations were reared nder laboratory condition as described by Ueberschar (1985) and Clemmesen (1987). Laivae were fed with Brachions plicatilis (5/ml) andartemia naplii (1/mL) or deprived of food for inteivals of 6-9 days (RNAJDNA ratios) or 2-6 days (tryptic enzyme activity measrements). Herring laivae were sampled in the English Chat1el.in Janary 1986 by sing a MOCNESS- system (.3mm mesh size, Wiebe et al. 1976). Fish laivae were individally collected from the sample, shock froz.en in liqid nitrogen and stored at -74 C. Measrement of RNA/DNA ratio and tryptic enzyme activily.rna and DNA contents of the laivae were detem1ined by sing the florescence techniqe by Clemmesen (1988) with some modit:ications (Clemmesen 199). The tryptic enzyme activity measrements were perfom1ed according to the florescence techniqe described by Ueberschar (1988). 7 j:: ll::'. z... z ll::'. FED LARVAE : f STARVING LARVAE r LARVAL LENGTH (mm SL) Fig. 1. RNAJDNA ratios of laboratory-reared fed and 6-9 days starved herring larvae. Data points represent 5 to 15 larvae measred individally. Lines were fitted by linear regression analysis with: y = x, r =.62 for fed larvae, and y = x, r =.25 for starving larvae. In addition, the 95% confidence level of the regression is presented.

3 RNAJDNA ratios and tryptic activities in fed and starved herring larvae 229 Reslts A comparison of the RNNDNA ratios of laboratory-reared fed and starving herring larvae is shown in Fig. 1. Tryptic enzyme activity measrements of laboratory-reared larvae are given in Fig. 2. The RNNDNA ratios as well as the tryptic enzyme activities were significantly lower in the starving larvae than in fed larvae. RNNDNA ratios and tryptic enzyme activities of both fed and starving larvae showed a linear increase with larval length, bt the slopes of the regression lines fitted to the data were different between fed and starving larvae. Based on the reslts of RNNDNA ratios and tryptic enzyme.activities determined on starving larvae from the laboratory, an estimation of the ntritional condition of fieldcaght herring larvae was perfom1ed. For both indicators, twice the standard deviation (2s) of the regression for laboratory-reared starving larvae was defined as the critical level for determination of larvae as starving, inclding 95% of the vales fond in the 1.6% <{ 1.2% a:::: <{...! 1.% ' % <{.6%... - a:::: 1.4% O FED LARVAE e STARVING LARVAE.4%.2%.% LARVAL LENGTH (mm TL) Fig. 2. Tryptic enzyme activity of laboratory reared and 2-6 days slarved herring larvae. Data points are means of 5 lo 15 larvae measred individally. Lines were fitted by linear regression analysis with: y = x, r =.62 for fed larvae, and y = x, r =.61 for starving larvae. In addition, the 95% confidence level of the regression is presented.

4 23 Catriona Clemmesen and Bernd Ueberschiir staiving grop. Based on their RNAJDNA ratios, 12.5% of the laivae from the field sample were staiving (Fig. 3) compared to 16.% as determined by their tryptic enzyme activity vales (Fig. 4). Discssion The present stdy shows that both indicators, the RNAJDNA ratio and the tryptic enzyme activity, are appropriate to distingish between feeding and staiving fish laivae. The laboratory rearing reslts clearly demonstrate significantly lower vales in starving laivae for both indicators. Since the RNAJDNA ratios and tryptic enzyme activity vales in starving larvae showed a lower coefficient of variance compared to fed larvae, they were sed for the evalation of the ntritional condition of field larvae. 6 O STARVING LARVAE LAB ti FIELD-CAUGHT LARVAE I- a:: <l'. z 5 4 l : ::mr ; Q....g o ti oo o@ o o to 2s LARVAL LENGHT (mm SL) Fig. 3. Evalation of the ntritional condition of herring larvae sampled from the English Channel based on RNA/DNA ratios of laboratory reared starving herring larvae. Data points represent 5 to 15 larvae measred individally in relation to their length. The solid line shows the linear regression fitted to the data of laboratory reared starving larvae (see Fig. 1). The dotted line represents twice the standard deviation (2s) of the regression and gives the critical levels below which larvae were determined as starving.

5 RNA/DNA ratios and tryptic activities in fe d and starved herring larvae 231 8% O::'. _J "'-. f f a.. - ::'. 6% 4% 2% O STARVING LARVAE LAB b. FIELD-CAUGHT LARVAE % LARVAL LENGTH (mm TL) 2 25 Fig. 4. Evalation of the ntritional condition of herring larvae sampled from the English Channel based on tryptic enzyme activity measrements of laboratory reared starving herring larvae. Data points represent 5 to 15 larvae measred individally in relation to their length. The solid line shows the linear regression fitted to the data of laboratory reared starving larvae (see Fig. 2). The dotted line represents twice the standard deviation (2s) of the regression and gives the critical levels below which larvae were determined as starving. The actal vales of the RNNDNA ratios and tryptic enzyme activity measrements might differ between species, therefore, at present, it seems advisable to se laboratory reslts on starving larvae only to detem1ine the ntritional condition of larvae from field samples of the same species. The stdy shows that both indicators are able to detect larvae, which were exposed to starvation intervals in the range of 4-9 days. When estimating the proportion of starving larvae from a field sample, both methcxls correspond well. In the short-term range, the tryptic enzyme activity reacts faster to food deprivation than the RNNDNA ratio, as shown by Ueberschar and Clemmesen (1992). The tryptic enzyme activity was already si gn ificantly lower in starving larvae after 2 days of food deprivation, if compared to fed larvae, whereas the RNNDNA ratio needed a starvation period of 3-4 days to reslt in si gn ificantly lower vales compared to fed larvae. In this stdy as well, starvation intervals from 2-6 days already lead to si gn ificantly lower tryptic enzyme activity vales in starving larvae, whereas the RNNDNA ratio is more conservative and is more

6 232 Catriona Clemmesen and Bernd Ueberschiir sefl for evalation of longer starvation intervals in the range of 6-9 days. In ftre experiments, the effect of temperatre on the RNA/DNA ratio and tryptic enzyme activity in relation to different starvation periods will have to be evalated. Higher temperatres might lead to a faster decrease of both indicators. The vales for RNA/DNA ratios and tryptic enzyme activity given here might, therefore, be reached in shorter time intervals. The actal vales for both indicators from larvae sffering from long-term starvation inflences shold not be affected by temperatre. The reslts of this stdy demonstrate the seflness of RNA/DNA ratios and tryptic enzyme activity for the detection of starving larvae in the field. It is assmed that these methods are valable for assessment of rearing conditions in aqacltral sitations inclding optimm food density, qality of the food, grow1h rates and feeding activity. Acknowledgement Part of this stdy was financed by the Federal Minister for Science and Technology (BMFT) nder contract No. MFE 541/9. References Bckley Ll (1979) Relationship between RNA/DNA ratio, prey density and growth rate in Atlantic Cod (Gads morha) larvae. J Fish Res Bd Can 36: Clemmesen C (1987) Laboratory stdies on RNA/DNA ratios of starved and fed herring (Clpea harengs) and trbot (Scophthalms ma.xims) larvae. J Cons Int Explor Mer. 43: Clemmesen C (1988) A RNA and DNA florescence techniqe to evalate the ntritional condition of individal marine fish larvae. Mccrcsforschng 32: Clemmesen C (199) Improvements in the florimetric determination of the RNA- and DNAcontent in individal marine fish larvae. ICES CM 199/L: 98 Biol Ocean Comm, 14 pp. Hjelmeland K, Hse I, Jorgensen T, Molvik G, RhJ (1984) Trypsin and trypsinogen as indices of growth and srvival potential of cod (Gads morha L.) larvae. In: Dahl E, Danielssen DS, Moksness E, Solemdal P (eds) The propagation of cod, Gads morha, Part 1. Institte of Marine Research, Flodevigen Biological Station, pp Martin FD, Wright DA, Means JC, Setzler-Hamilton EF (1985) Importance of food spply to ntritional state of larval striped bass in the Potomac river estary. Trans Amer Fish Soc 114: Pedersen BH, Nilssen EM, Hjelme!and K (1987) Variations in the content of trypsin and trypsinogen in larval herring (Clpea harengs) digesting copepod naplii. Mar Biol 94: Pedersen BH, Hjelmeland K (1988) Fate of trypsin and assimilation ef!iciency in larval herring (Clpea harengs) digesting copcpod naplii. Mar Biol 97: Ueberschiir B (1985) Unterschngen tiber die Verdangsenzyme von Fischlarven. MSc thesis, University of Kiel, 111 pp. Ueberschiir B (1988) Determination of the ntritional condition of individal marine fish larvae by analysing their proteolytic enzyme activities with a highly sensitive florescence techniqe. Meeresforsch. 32: Ueberschiir B, Clemmesen C (1992) A comparison of the ntritional condition of herring larvae as determined by two biochemical: methods - tryptic enzyme activity and RNA!DNA ratio measrements. ICES Jornal of Marine Science, in press. Wiebe PH, Brt KH, Boyd SH, Morton AW (1976) A mltiple opening/closing net and environment sensing system for sampling zooplankton. J Mar Res 34: