leES C.M. 1995

Not to be cited without prior reference to the authors

P:13

Theme Session on causes of observed Variations in fish growth (Theme session P)

DOES THE OTOLITH STRUCTURE REFLECT THE NUTRITIONAL CONDITION

OF A FISH LARVA?

- A COMPARISON OF OTOLITH STRUCTURE ANO BIOCHEMICAL INDEX

(RNAIDNA RATIO) DETERMINED ON COO LARVAE -

Cl'emmesen, C. and Ooan, T.

'. . .

Institut rur Meereskunde an der Universität Kiel, Düstembrooker Weg 20, 24105 Kiel,

Germany

. ABSTRACTI:

/-'

Cod'larvae from laboratory rearing experiments aged from 1 to 12 days after hatching fed and

deprived of food were analysed. Tbe number of increments on the otolith and the width of

these increment~ were detennined t0:Bether with the RNNDNA ratios on the same larva.

Alizarin marking of the otoliths was performed to validate the formation of daily increments.

e Cod larvae reared at 6°C formed the first ring right after hatching and deposited increments on

a daily basis. A comparison ofthe measurements between the right and the left lapillus showed

that the measurements between the right and the left otolith can differ, if the radius is taken.

Specially the otolith-core showed high individual variability. Whereas the sum of the

increments didn't differ between both otoliths. Until day 10 after hatching, while the larvae

were still feeding on their yolk, the extemal food situation didn't effect the increment width of

the otoliths or the RNA/DNA ratios. In larvae older than 10 days the widths of the daily

increments was dependend on the nutritional situation and RNAIDNA ratios decreased in

starving larvae in comparison to feeding larvae. RNAIDNA ratios and increment widths were

correJated.

2

INTRODUCTION

Fluctuations in the size of fish populations may occur as a consequcnce of changes in the

annual influx ofyoung or recruiting fish. Recruitment variations are compoundcd by the effects

of human exploitation and oftcn attributed to thc effects of environmental variations on the

survival of egg and larval stages. The success or failure of annual recruitment can have a

number of different abiotie and biotic causes. High mortality rate during early stages is

considered one ofthe major factors causing stock fluctuations. The lack offood or amismatch

in larval fish and food organisms distribution are a principal cause for poor year dass strength

(Hewitt et al. 1985, Hjort 1914, Hunter 1976, Lasker 1978).

Tbc rclationship between RNA and DNA is an index of the cell's metabolie intensity and has

been used to measure recent growth in fish (Bulow 1987, Bucldey 1984, Bucldey & Lough

1987, Hovcnkamp 1990, Hovenkamp & Wilte 1991) and has proven a useful indicator ofthe

mitritional condition as shown in severallarvciI fish studies (Bucldey 1980, 1984, Bucldey &

Lough 1987; Clemmesen 1987, 1994, Fukuda et al. 1986; Martin et al. 1985, Raae et al. 1988,

Robinson & Ware 1988).

All condition indices determine the nutritional condition at the time of catch reflecting the

situation ofthe last hours, days or wecks before catching depending on the methods used. It is

sometimes difficult to predict if the larvae are on the way of improving or deteriorating their

condition. RNAIDNA ratios are commonly used to back- and forecast the growth and survival

potential of larvae in order to help predicting recruitment. The validity of the RNAIDNA ratio

can be irnproved by incorporating otolith increment structure s~dies (daily increment studies)

on same subsarnples to have the growth history ofthe larvae as weil as the condition at catch.

Otolith structures reflecting daily patterns were described by Pannella (1971, 1974) and

experimentally proven by Stuhsaker & Uchyama (1976). Brothers et aI. (1976) used otolith

increment structure to determine the age oflarvae and juvenile fish.

The environment experienced by a larva will influence it's otolith structure as could be shown

in severallaboratory experiments (panella 1980, Radtke & Dean 1982, Neilson & Geen 1982,

Dale 1984, Bergbahn & Karakiri 1990, Zhang & Runham 1992). Studies on Norwegian spring

spawning herring and North Sea autuUlll spawning herring have shown the potential of using

otolith microstrueture in recruitment research (Fossum & Moksness 1993).

Daily ring formation was to be validated in this study on laboratory reared cod larvae by using

Alizarin marking (BIom et al. 1994). The aim of this study was to combine otolith

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3

microstructurc analysis arid RNAIDNA ratio detemlimition on the same individual larva to

compare thc cffeet offood deprivation on the Width ofthe daily increment imd the RNAIDNA

ratio and evaluate the potential use ofthis combinatiori for recruitment research.

MATERIALS AND METHODS

eod huvae were reared in the Havsflskeiaooratoriet Lysekil, Sweden from 03.05.94 ­

27.05.94. Adult rod caiight in November 1993 in the Bornholnt. BaSin were tranSported to

Lysekii, adaptCd tci the higher satiruty (30 ppm) arid kept in 10000 liter tanks at 6-7°C as brood

Stock: for the eXperiments. eod spawned naturally, the fertilized eggs were tranSfered irito 100 I

tankS using a 300Jlrn planktori riet. The eggs float ori the surface and are in~ved by a gentle

waier flow (0.5 Vrnin.). Temperaiure in the incubation timk was 6-7°C, I~ae hatched after 13

days and were transferecl to 100 I rearing hUlks. LaiVae were fed startirig at day 4 after

hatching with the ratifer Brachiollus plicatilis reared on the flagellate ISOclzrysis galbana.

Temperature throughout the experiment varied from 6°C to 7.5°C and Salinity waS between 33

änd 34ppm. TankS were illuminated by natur8.l day light giving a 16bour day! 8 hour night

cyc1e. LarVae were fed thfee times a day at 8:00, 12:00 and 16:00 at ci derisitY of 0.65

Brachiollus!ml. Tbe daily food nitiori aceuinulated io 2 BrachiOlius/inI.

For marking ofthe otolithS 100 larvae were transfered to a 5 liter containing Alizarin (50rng,1,

Tsulcomoto 1988,; BIom ct ai. i994) on day 4 after hatching arid kepi there for 16h to set ci

riuorescent mark on the increment formed that day. After that the larvae were: carefullY

transfered to clean sea water. SampIes ofthe larvae were taken daily stärling on day 4 at 11:00

after the larVae bad been fed at 8:00. Five larVae per sampie ~ere iaken, transfered rnto

Eppendorf vials and storoo in liquid nitrogen until the end of the' experirm~nt. After thai the

sanipIes were pul into a -70°C fTeezer and left there until analysis.

Prior to the determination ofRNA- and DNA- contents the standard length ofthe larvae was

measured arid the otolith dissected. Sagittae and lapÜli were identmoo using apolarisation­

ttlter aUached 10 a binokUlar and were fiXed on a glass slide marldng left and right side.Tbe

iarva witbout the otolith was pul into an Epperidorf via! kept on ice. After 5 larvae were

disSected (necessary tune: 30miri.) the RNAIDNA analysis was penorined. The anatytical

procedure was perforrned aceording to the method by Clenirnesen (1993) using a specmc

nucieic acid fluorescent dye - Ethidiuin BroriUd (EB)- for DNA arid RNA. In order to measure

4

the DNA-content ofa simple, RNA was enzymatically digested with RNase arid the remain.ißg

DNA was determined with EB.

Measurements on the otoliths were performed using a digitalized computer aided video system

with a CCD camera (panasonic WVCL 700) connected to a fluorescence mikroskop (Zeiss

Axioplan) using 1000-times magnification. ~f the three otoliths the lapilli were chosen, since

they are the largest at time ofhatching up to age 25 in cod larvae (Bergstad 1984). Both lapilli

were used for the analysis and the radii; number of increments and width were measured four

times in four different directions on the otolith.

Results

To determine the accuracy ofthe age determination using the number ofincremerits the results

of the readings on the lapilli were compared with the kriown age of the laboratory reared cod •

larvae. Results are shown in Fig. la. 147 out of 150 analysed otoliths showed the dcposite ora

daily increment starting on the day of hatch. Only 2% of the analysed lapilli showed one ring

less than cxpected.

16hrs treatment with Alizarin resulted in an ~range coloured increment deposited on thc day of

marking. It could latcr be identified undcr the fluorescence- mikroskop. Tbe number of

increinents formed after the Alizärin-marked increment were counted and set into eomparlson

with the known number ofdays after the marking was peiformed. Results are giveri in Fig. 1b

and show that the rings after the marking are deposited on a daily basis and give further

validation ofthe formation ofthe increments in the cod otolith starting on the day ofhatching.

A comparison of the measurements performed on the len and on the right otoliths showed that

the radius or diameter ofthe otolit~ isn't only dependent on the growth ofthe daily increments •

but very much affected by the size of the otolith's core. Tbc size distribution of the otolith

cores between the left and the right side showed that the core size varied between 4.5Jlm and

13Jlffi, but didn't showa statistica1ly different distribution between len and light side (t-test, p<

0.05). The comparison ofthe otolith core ofthe left and the right otolith measured on the same

individuallarva revea1ed that there was a diffences in the size of the core when len and right

otolith were analysed (Fig.2a).

A similar picture is seen when the radius of the left and the right otolith on the same larva are

compared. The radius measurement between both sides was different (Fig.2b).

In comparison the sum of the incrernents deposited on the left and the right otolith did not

differ (Fig.2c). Tbe variability between the size ofthe len and right otolith is mainly caused by

the difference in the size ofthe COTe.

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To evaluate the effect of food availabiÜty or food withdrawal on the width of the daily

incremerits two inclividuaI laivae fed or .starved starting on day 4, were compared. Up to day 6

the larvae seem to deposite heterogenous increments dependi~g on their individual yolk

content. At day 7 the siZe ofthe increments seems to be detennined by the food situation (Fig.

3a). With increasmg age (ring number) the width ofthe increments increased in fed larva up to

a Width of 1.31lm whereas the starved larvae showed a constant decrease in the width of the

incremerit arid reached ävalue orOAllm (Fig. 3a). Fig. 3b gives a eomparlson ofthe size ofthe

inerements ofall äriatysCd fed and starved llirvae showing mean values and standard deviations.

The vanabilitY in the sire ofthe dally incremerits is high iri both groups but 'a comparison ofthe

mean vaIues shows a trend of inereäsing increment width in fed larvae Startirig on day 7 and

decreäSing width oithe iricrerrients in the 8tarved group.

DNA-coriierit of fed larvae was stable between day 4 arid 5. After that larvae starte<! to grow,

DNA-content increased and reached a value of 0.6J1g/larvae (Fig. 4a). It is striking that the

DNA.;'ooritetit of the starved larvae is bigher betwecn day 6 and day 10. Larvae from the

StarVirig group mus! have been larger at the start of the eXperiment. There is no significant. '

difierence in the DNA-ci>nierit between the staived and red larvae between day 10 arid 13 (Fig.. ,

4a). A ccimparison oe the RNA::'content of fed arid starved cod Jarvae is given in Fig. 4b. Tbe

RNA-eon6eiitratiori decreasoo from day day 4to day 5and then sllghtiy increaSe<l to reach avaIue of äpproxiniately 1-1.21lgllarva in the feeding group.Except for the vaJue at day 9 the

RNA-eontent ofthe Starved larvae was lower thaß in the fed larvae. Although a st~ong overia~

ofthe vaIues was seen a trend to higher RNA-content in the fed group compared to the starved

group (Fig. 4b) was shown. Tbe RNAmNA ratios during the course oe the experiment are

given mFig. 5. From day 4 to day 7 the RNAlnNA ratio decreased from a vaJue of 7 to below

2; tms seems to oe duc to yolk-absorption causing a decrease in condition. Between day 7 arid .

day 12 the RNAJriNA ratio in the starved gfoup decreased further, on the contrary the fed

group shöwoo a sllght fuciease. The effect of food aVm1ability starts to be visabl.e on day i 1

(Fig. 5). Fig. 6 shows the relation between mean RNAmNA ratios arid mean relative otolith. ' ,

gfoWih of all aDalySed fed and starved larvae. The relative otolith groWth was calculated by

setting the Width of the increment 4to avaJue' of i and ca1culating the reiative growth of the

foilowrng increments in relation to that vaJue. EverY d3ta point represents the niean of 4-5larvae. Tbe relative groWth ·of the otolith is red~Ceci compared to the fed group. The

RNAIDNA ratios of fed larvae doubled betweCn day 6 and day 12, whereas the vaJue in the

stai-ved group was reduce to hatrofthe ratio. It c3ri be seen that the feeding Jarvae häve rugher

6

RNNDNA ratios and a higher growth of the daily inerements showing that both the

RNNDNA ratio and the inerement growth are eoupled.

DISCUSSION

Tbe study showed that eod Iarvae fonn then fIrst increment on the day of hatching. This couId

be shown by the age determination based on increment numbers in comparison to known

laboratory age as weil as by the Alizarin- marking results whieh validated the deposition of

°daily increments. These results are in agreement with Dale (1984) who determined the·

incrernent formation in cod larvae using Electron- Transmission- Mikroscopy. By using a 12

hour day/ 12hour night cyele laboratory reared cod larvae produced an increment every 24

~ .Neilsen & Geen (1982) showed difTerences in the size of the otoliths taken from the len or

right labyrinth in salmonids (OllcOrCh)71Chus tslumytscha). Therefore both len and right

otoliths were measured and compared in this study. Tbe results clearly show that care has to be

taken when measuring the total size (radius), since differences between left and right otoliths

occurred. It could be shown that these difTerenees were not due to the growth of the daily

increments, but dependend on the size of the otolith core at the time of hateh. The individual

egg-development might efTect the size of the core as welI as environmental faetors. Influences

on the formation ofthe otolith eore should be further analySecl.

It is known that environmental faetors Iike constant darkness (Dale 1984)or too high

ternperatures (Mosegaard et al. 1988) can efTect the inerement structure. Based on the

assumption that somatic growth is reflected in the growth of the otolith a weil growing larva

should deposit a wider daily inerement than a starving larvae with slower growth. Maillet &

CheckIey (1990) and Zhang & Runharn (1992) determined changes in the otolith strueture in

starving laboratory reared Atlantic menhaden and Oreochronis ni/oti/us.

The individual observations on the width ofthe otoliths in this study showed that the growth of

the increments was effected by the availability ofextemaI food sources and showed differences

in the growth ofthe increments between feeding and starving Iarvae starting on day seven.

DNA can be used as an indicator of cell number, reflecting the growth of the larva. During

yolk absorbtion the DNA- content was stable but incieased with age starting on day 7. Tbe

DNA-content wasn't affected by the feeding situation. RNA- content started to inerease at day

"a-

.7 ' '

7 in the fecding group getting more pronounced on day 11, whereas the RNA- content in the

starved group deereases: The Same picture was shown for the RNAIDNA ratio. Surriing on

day 11 the RNAIDNA ratio aitd the RNA~coriierii ~eflect the different nutritional situations.

Results shown here are in agreement with Stlldies on cod larvae by BuckJey (1979) arid results

on herring Iarvae by Clemrnesen (1987, 1994). The minimum RNNDNA ratio of 1 Suggested

by CleImriesen (1994) neccessaiy for the Jarva to survive is also found in this study. AIizMin­

markirig didn't effect the RNA- and DNA measurements, since the concentrations of nucJeic

acids detemuned on marked and unmarked cod larvae didn't differ (Doan pers. comin.).

Unforttlnaiely Sarilpling 'ofthe Iarvae couId not be performed after day 12, it is postuIated thai

the trends shoWn in this study would IUive been much more signmcant if sarnples from older

larvae had been aVäilabie. Future snidies must increase the Sämplirig protocOi up to an age of

21 daYs.

An evaiuation of nutritional condition can not be performed in larvae before first feeding

neither by analyzing the increrrient structure nor by measuring biochemical eomposition. DUe

to tbe process of internal yolk absorption arid learning to capture food items the conditiori of

fed iutd stiu"Ved larvae in the first days after hatching is not significaittly different. Therefore

that life stage shows agenernI problem in condition analysis independent ofthe technique used(See Clemrnesen 1994).

First observations shoWing the relationship between groWth rates over the last' five days (as

determined from the width of otolith daily fucrements) and proteiD groWth rates based on

nucJeici acid determiriations are available for North Sea plaice ~ovenkamp 1990) but have not.

been performed Oll the same iarva. To our knowlegde this Study is on ofthe first to deterinine

otolith rillcrostrueture arid RNAIDNA ra.tio analysis on the same Jarva. During the yolkSac '

phase no clear differences between feeding and starving larvae could be found. After yolk

absOrption the effect of an external food supply could be measured. The trend starts to be

visable on day 7 arid incrcases on day 11. High RNNDNA ratios are coupled with gi'eater

increment width in fed laivae. Lower RNNriNA ratios and sm3.ller increment widths are fbund

in starved larvae.

BiocheInicai indicators (RNAmNA ratios) have also been iJsed to demoilStrate Stärvaiion

morialitY in the field and correIations between food avaiIabiIity änd condition ofthe larirae have

been found (eanino et aI. 1991, Frank & McRuer 1989t Sctzler-Hamiltori et al. 1987). A

8

positive relationship ofRNAIDNA ratio and prcy abundance has been shown for stripped bass

larvae (Martin et al. 1985) and larvae of the Atlantic cod and. haddock (Buckley & Lough

1987). By coupling RNAfDNA ratio detenninations and otolith increment structure analysis on

the same larva the decision whether the larva is improving or deteriorating it's condition should

be possible. Tbe validity of these studies for th<: recruitment problem could be further

improved.

Whether the findings in this study determined on laboratory rcared larvae can also be found in

field caught larvae has to be further evaluated. Results by (Clemmesen in prep.) on field caught

anchovy larvae (Ellgraulis allchoita) revea1ed that the sum of the last increments was

correlated with the RNAIDNA ratio, mcaning that the effect of lack of food in the wild should

be possible to detennine by the combination ofbiochemical and otolith studies.

ACKNOWLEDGEMENT

We thank the staff of the fishery department of the University of Marine Sciences in Kiel and

the staffat the Institute ofMarine Research in Lysekil for their support. Special thanks to 1ana

Pickova for rearing of the cod larvae and sharing her expertise. \Ve thank Karin Burkert for

helping in the laboratory. We are indebted to Prof. Dr. D. Schnack for his support. Partial

funding was provided by the Institute in Kiel, the Deutsche Forschungsgemeinschaft (DFG, NE

99/23-1) and the European Union (EU-Am. 294 1226).

REFERENCES

Bergbahn, R and Karakiri, M., 1990: Experimental induction of biological tags in otoliths ofO-group plaice Pleurollectes platessa by starvation, temperature and UV-B radiation. Mar.Ecol. Prog. Ser., Vol. 67,227-233.

Bergstad, O.A, 1984: A relationship between the number of growth increments on theotolithes an age of larval and juvenile cod, Gadus morhua L. In: Tbe propagation of cod,Gadus morhua L. Flodevigen Rapportser., 1,251-272.

BIom, G., Nordeide, 1., Svasand, T. and Borge, A, 1994: Application of two fluorescentehemicals, alizarin complexone and alizarin red S., to mark otoliths of atlantic cod, Gadusmorlma L. Aquaculture and Fisheries Management 25, Supplement 1,229-243.

Brothers, RB., Mathews, C.P., and Lasker, R, 1976: Daily growth increments in otoliths fromlarval and adult fishes. Fish. Bull. U.S. 74, 1-8.

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",' " ... .,.'.,...,'

.9

........ ,.'...:....

BuckIey, L.l., 1979: Relationship beiween RNAIDNA ratio, prey density. arid gfowth rate inAtlantic Cod (GadliS morhua) larvae. 1. Fish. Res. Bd. Can. 36: 1497- 1502.

1_. .' •• " .

BuekIey, L. J.; 1980: Changes in. the ribomicleie acid, deoxyriboriueleie aeid and. proteincontent during ontogenesis in winter flounder, Pscudoplellroi,ectcs americalills , and the effeetofstarvation. Fish BulI. U.S., 77: 703-708. .

Bueidey, L. I.; i984: RNAIDNA ratio: an index oflarval fish growih in the sea. Mai". BioI., 80:291-298.

BuckIey, L. 1.; Lough, RG., 1987: Recent growth, ehemieaI eomposition arid prey field ofhaddock (MeliIllogramnllis acglcfillus ) and cod (Gadus morhua) larvae and post larvae OllGeorges Bank, May 1983. Can J.Fish. Aquat. Sei., 44: 14-25.

Buiow, F.l., 1987: RNA:DNA ratios as imUcators.ofgroWth in fish: a review.In SUnUneifelt,RC. and Hall,G.E.(eds). Age arid growth offish. Iowa State University Press, AInes. : 45-64.

'"

• eanirio, M.F.; Bailey, KM.; Ineze, L.S., 199i: Temporal and geographie differences in feedingapd nutritional condition of waIJeye potJoek larvae 111crag,.a chalcogramina in SheJikof Strait,GulfofAJaska. Mac. EcoI. Prog. Sero 79: 27-35. .~

Cleinritesen, C. 1987. Laboratory studies on RNAIDNA ratios of starved and fed .. hciririg(Clupea harcllgus) arid tUrbot (Scophthalmus maximus) Iarvae. J. Cans. Int. Explor. Mer 43:122-128.

Clemniesen, C., 1988: A RNA and DNA fluoreseenee technique to evaluate the nutritionalcoridition ofindividual marine fish larvae. Meeresforseh. 32: 134-143.

Clemmeseri, C., 1993: Improvements in the fluorimetrle determination ofthe RNA and DNAContent ofindividuaJ marine fish larvae. Mar. EcoI. Prog. Sero 100: 177-183.

Clernrnesen, C., 1994: The effect of food avaÜibility, age aild size on the RNAJriNA ratio ofiridividually nleasui-oo herring larvae: laboraiory calibmtion. Mar. BioI. 118: 337-382.

DaIe, T., 1984: Embyögericsis and Growth ofOtoiiths in the eod (Gadus morhua L.) m: Tbcpropagation ofcod; Gadus morlma L. FWdevigen Rapportser. I, 231-151.

Fossum, P. and MokSness, E.; 1993: A study ofspring- aild autümri-spaW11ed hernng (CiupeaIulrCllguS L.) larvae in the Norwegian coastal current during spring 1990. Fish. Oceanogr. 2:2,73-81.

Frank, :K.T.; McRuer, l.K, 1989: Nutritionai status of field-colleaed haddock(Melailog,.ammus aeglefillus) larvae from southwestem Nova Scotia : an asSessmentbased onmorphometric and vertieal distribution data. Can. l. Fish. Aquat. Sei. 46 (sUppt I): 125-133.

Fukuda, M; Nakario, H.; Yamamoto, K, 1986:. Biochemical cluÜtges in Pacific herrlng dUringearly deveIopmental stages. Bul1. Fac. Fish. Hokkaido University 37(1):30-37.

Hewitt, R.P.; TheiIaeker, G.H.; Lo, N.C.H., 1985: Causes ofmortaIity in youngjaek mackerel.Mar. EcoI.Prog.Ser. 26: 1-10. .

10

IIjort, J., 1914: Fluctuations in the great fisheries ofNorthem Europe viewed in the light ofbiological research. Rapp. P. -v. Reun. Cons. Penn. int. Explor. Mer 160: 1-228.

Boude, E.D., 1989: Comparative growth, mortality and energetics of marine fish larvae:temperature and imptied latitudinal effects. Fish. Bull. V.S. 87(3): 471-495.

Hovenkamp, F., 1990: Growth diferences in larval plaiee (Pleuronectes platessa L.) in theSouthem Bight of the North Sea as indicated by otolith increments and RNNDNA ratios.Mar.Ecol. Prog. Sero 70: 105-116.

Hovenkamp, F.; Witte, J.I.]., 1991: Growth, otolith growth and RNNDNA ratios of larvalplaice Pleuronectes platessa in the North Sea 1987 to 1989. Mar. Ecol Prog. Sero 58: 201­215.

Hunter, J.R, 1976: Report of a colloquium on larval 6sh mortality studies in their relation tofisheries research. Jari. 1975. NOAA tech. Rep. V. S. Dep. Commerce NMFS Cire. 395: 1-5.

Lasker, R, 1978: Tbe relation between oceanographic eonditions and larval anehovy food inthe Califomia eurrent: identification offactors contributing to recruitment failure. Rapp.Proc.­v..Reun. Cons.lnt. Explor. Mer. 173: 212-230.

Laurence, G.C., 1978: Comparative growth, respiration and delaycd feeding abilities of lavateod (Gadus mor/ma) and haddock (Melanogrammus aeglejilms) as influenced by temperatureOduring laboratory studies. :Mar. BioJ. (BerJ.) 50, 1-7.

Maillet, G.L. and Checkley, D.M., 1990: Effeets of starvation on the frequeney of fonnationand width of growth increments in sagittlte of laboratory reared Atlantic menhaden BrevortiaI)rallllus larvae. Fish. Bull. U.S. 88: 155-165.

Martin, F. D; \Vright, D.A.; :Means, lC.; Setzler Hamilton, E.F., 1985: Importance of foodsupply to nutritional state of larval striped bass in the Potomac river estuary. Trans. Am. Fish.Soe. 114: 137-145.

•

'.Mosegaard, H., Svedang, H. and Tabennan, K., 1988: Uncoupling of somatie otolith growth •rates in aretie eharr (Sal},'elilms alpillus) as an effect of differences in temperature response.Can. J. Fish. Aquat. Sei. 45: 1514-1524.

Neilson, J.D. and Geen, G.H., 1982: Otoliths of chinook saImon (OllcorYIlc/ms tshawytscha):Daily growth increments and faetors influencing their production. Can. J. Fish. Aquat. Sei. 39,1340-1347.

Panella, G., 1971: Fish otoliths: Daily growth layers and periodieat patterns. Seienee 173,1124-1127.

Panella, G., 1974: Otolith growth pattern: an aid in age detennination in temperate and tropica1fishes. In: T.B. Bagenat (cd.). Tbe agiog offish. Vnwin Bros. Ldt. Surrey, England. 28-39.

PaneUa, G., 1980: Growth patterns in fish sagittae. In: Skeletal Growth ofAquatie Organisms:Biologieat Records ofEnvironmental Change. Plenum Press, New York, 519-560.

11·

Raae, A.l, Opstad, 1., Kvenseth, P. & Walther, B.T., 1988: RNA, DNA and protein duringearly development in feeding and starved eod (Gadus morhua L.) larvae. Aquaeulture 73,247-259.

Radtke, RL. and Waiwood, K.G., 1980: Otoloith formation and body shrinkage due tofixation in larval cod. (Gadus morhua L.). Can. Tech. Rep. Fish. Aquat. Sei. 929, 1-10.

Radtke, RL. and Dean, 1M., 1982: Increment formation in the otoliths of embryos, larvae andjuveniles ofthe mummichog, Fundu/us heteroc/itus. Fish. Bull., U.S. 80,201-215.

Robinson, S.M.C. ; Ware, D.M., 1988: Ontogenetic development of growth rates in larvalPacific herring, C/upea harengus pa//asi, measured with RNA-DNA ratios in the Strait of

. Georgia, British Columbia. Can J. Fish . Aquat. Sei. 45: 1422-1429.

Setzler-Harnilton, E.M.; Wright, F.; Martin, F.D.; M"tllsaps, C.V.; Whitlow, S., 1987: Analysisof nutritional condition and its use in predieting striped bass recruitment : field studies. Am.Fish. Soc. Symp. 2: 115-128.

• Stuhsacker, P. and Uchiyama, 1976: Age and growth ofthe nehu Stophonls pupureus (pices:Engraulidae), trom the Hawaiian Islands as indicated by daily growth inerements of sagittae.Fish. BuH. U.S. 74, 9-17.

Tsukomoto, K., 1988: Otolith tagging of ayu embryo with fluorescent substances. NipponSuisan Gakkaishi 54(8), 1289-1295.

Zhang, Z. and Runham, N.\V., 1992: Effects of food radiation and temperature level on thegrowth of(Oreochrollis ni/oticlis L.) and their otolithes. J. Fish. Bio!. 40,341-349.

•

12

14

13 y =1.008x -0.09f=0.994

12 n =150cn...-c:

11<DE~ 100c:

'+- 90'- 8<D.cE 7::3Z

6

5 a) •4

4 5 6 7 8 9 10 11 12 13 14

Age in days after hatching

0) 10c~'-

y =0.009 x + 0.0017ro 9E f= 0.999c: 8 n = 109·ero.~ 7oe:(Lo-

6Q)iS= •caCf) 5+-'c:Q) 4E~ 30c:- 20L-(J.)

1 b).cE::J 0Z

0 1 2 3 4 5 6 7 8 9 10

Days after Alizarin - marking

Fig. I: Relation between number ofincrements and known age oflaboratory reared codlarvae (Fig. la). Validation ofthe daily otolith deposition by marking the otolithwith Alizarin (Fig. Ib).

1,\,-, .• ,,'. ~l

........ 13E 12::L.

Y=0.333x +5.550

'"--"- 11.s:::. rZ =0.0990>'C 10 n =61 0~

8 9.s:::.~ 8 00-0

7 00Cl>

.s:::. 0- 6 0-0CI)

50

a)::JUm 4a::

4 5 6 7 8 9 .10 11 12

Radius cf the otolith cere left [~m]2019 y=0.633x +5.26 v........

E 18 r2=0.299• ::L. v--.. 17 n=61 v.s:::. V~ 160

'. - 150

1: 14 v0>'L: 13CI)

::s 12 v f}U Vm 11 vv va:: v b)10 v

9'

9 10 11 12 13 14 15 16 17 18 19 20

........ Radius left otolith [~m]E 10 c::L.......

9.s:::. y =1.037x-0.16~ 8'0 r2=0.863-0 7 n=61-.s:::.0> 6'CCI) 5-cCl> 4E~ 30.~ 2- c)0 1E::J 0W'

o 1 2 3 4 5 6 7 8 9 10

Sum of increments left otolith [~m]

Fig. 2: Correlations between the radii ofthe core (Fig. 2a), the radii ofthe total lapillus (Fig. 2b)and the sum ofthe increments (Fig. 2c) ofthe left and the right lapilli based on measurements on4-12 day old cod larvae. ,Values are means calculated from 4 measurements on the otolith in fouT differentdirections. Linear regression model was fitted to the data.

0.6

0.4 -+--...-_--r--_-...-_-...-_--r--_-or-~

.....c:Q)E 0.8~oc

.r:.~ 1.0'::

14

1.4

1.2 ~ fed\........ \E::L 1.0 b.~

.r:. "-....."'C e-~':: 0.8.....

-~cQ)

"-~E~ 0.6 starved(.) 'o--ac

0.4 "- [J

a) •0.2

O· 2 4· 6 8 10 12

Increment number

1.4

Fed1.2

o 1 2 3 4 5 6 7 8 9 10 11 12

Increment number

Fig. 3: daily otolith growth (increment width) oftwo individual cod larvae (Fig. 3a).Mean increment width of 59 fed cod larvae compared to 27 starved cod larvae(Fig. 3b). Error bars give the standard deviation.

..15

1.0 ...,...-------------------,

a)0.0 -..I-----..--__r----,-----r-----r-----!

.-..C>::l........ 0.6

~ca

~ 0.4o

0.2

• Fed

o Starved0.8

•2 4 6 8 10 12

Ageindaysafterha~h~g

14

• FedliI starved

2.2 ----------------------.

2.0

1.8

1.6.-..~1.4.......~ 1.2ca~ 1.0

~ 0.8

0.6

0.4

0.2 b)0.0 -..I------..---.,.....--__---,..------.r----!

2 4 6 8 10 12

Age in days after hatching

14

Fig. 4: DNA- content (Fig. 4a) and RNA- content (Fig. 4b) ofcod larvae in relation to ageand feeding situation. Starving larvae (n= 45) were deprived of food starting on day 4 and day 7and compared to fed larvae (n= 86). Error bars give the standard deviation.

--- -- --- -- -- ----- ----

:' .r

169

8

• Fed7 0 Starved

6

«z 50:=(

4za::

3

2

1

0 •2 4 6 8 10 12 14Age in days after hatching

Fig. 5: RNNDNA ratios ofcod larvae in relation to age and feeding situation. StaIVing larvae (n=45)were deprived of food starting on day 4 and day 7 and compared to fed larvae (n= 86). Error barsgive the standard deviation.

12

•• fedl!l starved

8 10

m--~7 8" 10

ID--G1l9 11

1.5 2.0 2.5 3.0

Relativ otolith.. growth (mean values)

4.5

4.0

-CI)3.5Q)

:Jro> 3.0c:rnQ)

E 2.5.......,«z 2.00~Z 1.5a::

1.0

0.5

1.0

Fig. 6: Relation between relative otolith growth offed and starved cod larvae and RNAIDNA ratios.For calculation of relative growth the width ofring number 4 was set to one.Tbe numbers give the age in days.