Hydrobiologia 231 : 99-105, 1992 .© 1992 Kluwer Academic Publishers . Printed in Belgium .

Limits for the detection of daily otolith increments in whitefish(Coregonus lavaretus L.) larvae

Axel Klink & Reiner EckmannLimnological Institute, University of Constance, P .O. Box 5560, D-7750 Konstanz, Germany

Received 16 May 1991 ; accepted 27 June 1991

Key words: Coregonus, otolith, daily increments, resolution limit, SEM

Abstract

The formation of daily otolith increments in European lake white-fish was evaluated in the laboratoryduring 40 days at 4, 6, and 8 ° C under limited and ad libitum food supply. Daily increments were eas-ily identified in larvae reared at 8 'C . At 6 ° C and, more, at 4 'C, an unstructured perinuclear zone wasformed, and daily increments could only be recognized in the light microscope starting from 10 to 35days after hatching . In some larvae from the 4 ° C groups, no increments at all were found . Scanningelectron microscopy (SEM) could not improve increment resolution . Only those increments could beviewed by SEM which were visible in the light microscope as well . We conclude that whitefish larvaewhich experience low (4-6 'C) temperatures during their first weeks of life, hence those which hatch inthe lake, cannot be aged by the currently employed preparation techniques .

Introduction

The analysis of daily otolith increments in fishlarvae can provide valuable informations abouttheir early life history (Brothers et al., 1976; Cam-pana & Neilson, 1985) . In whitefish (Coregonussp.), this method is used e .g. for ageing larvae(Eckmann & Rey, 1987 ; Rice et al., 1985) and forback-calculation of growth during the first weeksof life (Eckmann & Pusch, 1989 ; Eckmann &Pusch, 1991 ; Rice et al ., 1987). During the firstdays after hatching, however, daily incrementscan often not be detected by light microscopy(LM). This is a minor problem in C . hoyi in whichincrement deposition begins at first feeding, i .e.about 3 d after hatching (Rice et al., 1987). But inwhitefish from Lake Constance, this lag phase,which results in the formation of an unstructured

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perinuclear zone, can extend over several weeksafter hatching, although larvae have alreadystarted to feed on zooplankton . Daily incrementsbecome recognizable only when water tempera-ture starts to increase (Rey & Eckmann, 1989) .

These problems might arise from the resolutionlimits of LM, as demonstrated for larval herringby Campana et al. (1987). Scanning electron mi-croscopy (SEM) could improve daily incrementdetection in Theragra chalcogramma (Bailey &Stehr, 1988; Nishimura & Yamada, 1984), reveal-ing increments of less than 200 nm width . Thepresent study attempts to disclose the extremetemperature and food conditions under whichdaily otolith increments in European lake white-fish larvae are just recognizable by LM . Further-more, we tested whether daily increments can bebetter resolved by SEM .

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Materials and methods

Fertilized eggs were obtained by stripping freshlycaught Coregonus lavaretus L. from Lake Con-stance. They were incubated at 0 .9 to 6.8 'C .After hatching on 14 Febr . 1990, six 16 literaquaria were stocked with 100 larvae each . Waterwas exchanged at a rate of 1-2 h -1 . Tempera-tures were gradually adjusted to 4, 6, and 8 'C .There were two aquaria for each temperaturelevel: in the first aquarium, fish were fed ad libitum(adlib) by adding zooplankton every two hours inorder to ensure a high food concentrationthroughout the day; in the second aquarium, fishwere given a limited ration (limit) by feedingzooplankton only once per day and siphoning offmost of the remaining plankton two hours later .Zooplankton was sampled every two or threedays from Lake Constance, passed through a1 mm sieve and kept at 5 ° C under gentle aira-tion. It was mainly composed of copepodites andadults of Cyclops sp. (83-98%) and Eudiaptomussp. (1-17%) .

During the 40 days experiment, temperaturesfluctuated only little :

Artificial illumination provided a 14:10h day-night rhythm. Ten fishes were sampled and deepfrozen from each aquarium after 11, 19, 26, and34 days. After 40 days, all remaining fish weresampled .

Total length (LT) of freshly thawed larvae wasmeasured to the nearest 0 .5 mm and corrected forshrinkage. From each sample taken on days 26and 40, the sagittal otoliths of 10 larvae weredissected out. One otolith was embedded inEPON on a microscope slide, the other one on a

stub for SEM. The preparations for light micros-copy were ground with abrasive paper of 360 to1200 grit. The grinding procedure was controlledunder a binokular or microscope and continueduntil a cross-section was obtained. Sections werepolished with diamond paste, cleaned with deter-gent, rinsed with distilled water and air-dried .Some of the larger (40 d old) otoliths were re-moved from the slide after grinding and polishing,embedded with the polished face down andground on the other side as well . Finally, all oto-liths were protected with a coverglass . The prep-arations were examined under a light microscopeat 100 to 1600 x magnification . The microscopewas equipped with a video camera, and all mea-surements were performed on video frames usingan interactive image analysis system . Measure-ments included : otolith length and width, hatch-check length and width, dorsal radius from hatchcheck to the outer otolith margin, and, as far asthey could be reliably identified, widths of dailyincremental zones . The otoliths for SEM wereground and polished in the same way as for LM .They were etched from 2 to 5 min in 0.1 M EDTAat pH 7 .4, dried for 24 h in a dissicator and goldcoated for 1 min at 45 mA (layer thickness ap-prox. 50 nm). The preparations were viewed in aLeitz DSM 940 at 10 kV, using the detector forbackscattered electrons .

Statistical tests were performed with SAS . Dif-ferences in final total length among the six treat-ments were tested with Duncan's multiple rangetest (p = 0.05). A possible influence of the feedingregime (adlib vs . limit) on the relation betweendorsal radius and fish total length was tested bycovariance analysis .

Results

The influences of both temperature and feedingregime on coregonid larval growth are apparentfrom the mean lengths reported in Table 1. Afteronly 40 days and with small sample sizes, how-ever, the observed differences are statistically sig-nificant only in part (cf. Duncan's grouping inTable 1) .

Experiment Daily mean [°C] Registered temperature [°C]± 1 SD (n = 39)

minimum maximum

4 1 C limit 4.3+0 .2 3 .9 4 .84 'C adlib 4.2+0 .2 3 .9 4 .76 ° C limit 6.0+0 .2 5 .5 6 .76 'C adlib 6.1+0 .3 5 .5 6 .78 1 C limit 8.1+0 .2 7 .8 8 .68 'C adfib 8.0+0 .2 7 .8 8 .6

Table 1 . Mean total lengths (LT) and dorsal otolith radii (DORR) of Coregonus lavaretus larvae reared during 40 d at threetemperatures levels and under two feeding regimes (adlib : ad libitum food supply throughout the 14 h day ; limit : limited food supply,zooplankton given only once per day) . Treatment groups with the same letter do not differ significantly in mean total length ac-cording to Duncan's multiple range test (p = 0.05) .

The otolith nucleus, the central part enclosedby the hatch check, had a mean length of81 .7 ± 11 .0 µm (n = 96) and a mean width of62 .0±7 .0pm (n=92) .

Mean dorsal otolith radii at the age of 40 d aregiven in Table 1 . Taking together all data from thesix treatment groups, the relation between dorsalsagitta radius DORR in pm and fish total lengthLT in mm is best represented by a linear regres-sion :

LT = 12.0 + 0.122 * DORR

(r2 = 0.75,n = 105) .

There is an obvious tendency, that food-limitedcoregonid larvae have larger otoliths relative totheir body size . This trend, however, cannot beconfirmed by covariance analysis (p = 0 .123) after40 days of feeding .

Typical LM images for all treatment groupsshowing the sagitta dorsal sector are presented inFigs. la-If. The hatch-check is always veryprominent (arrows in Fig . 1). Increments whichare visible inside the nucleus are deposited beforehatching and are not considered in this context .All interpretations refer to the sector betweenhatch-check and dorsal margin .

Under all experimental conditions, from 8 ° Cadlib (Fig. lf) to 4 ° C limit (Fig. 1 a), at least someincrements could be detected, but only in the twogroups reared at 8 ° C they could be counted un-interruptedly from the margin back to the hatch-

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check. These daily increments are of higher con-trast under ad libitum as compared to limited foodconditions (cf. Fig. le and lf) .

Fishes reared at 6 °C (Fig . lc,d) laid down anunstructured perinuclear zone where no daily in-crements could be identified. At best, in somesectors of the perinuclear zone some ring struc-tures were visible, but a complete and reliableenumeration of daily increments between hatch-check and periphery was not possible. The peri-nuclear zone was from 4 to 12 .5 itm wide. It wasformed during the first 9 to 18 days under ad li-bitum and during the first 10 to 25 days underlimited food conditions . Outside of this zone,daily increments can be easily identified . Underlimited food conditions, these increments are nar-rower and, as in the groups reared at 8 °C, theyare of lower contrast as compared to the larvaefed ad libitum .

In fishes from the 4 ° C adlib group (Fig . lb),no daily increments could be detected during thefirst 17 to 30 days . The perinuclear zone was from5.2 to 16.6 µm wide . Only in the outer marginalzone, very narrow increments of 0 .9 µm maxi-mum width were visible . In 2 out of 10 otoliths,however, no ring structures at all could be re-solved in the light microscope . Finally, the larvaereared at 4 ° C under limited food supply repre-sent the extreme case (Fig. 1a) : in only 4 out of10 otoliths, faint daily increments could be iden-tified in the marginal region . They numbered 23,14, 10, and 5, respectively .

Experimentalconditions

LT(nun) ± 1 SD n Duncan'sgroupingfor LT

DORR (µm)± 1 SD n

4 1 C limit 13.4+ 1 .2 10 A 16.3+4 .8 84 ° C adlib 16.1+0 .6 8 B 19.5+4.8 46 1 C limit 15 .7+0 .9 7 B 28.2+5 .0 76 ° C adlib 16.8+ 1 .9 10 B 37.8+8 .3 98 ° C limit 17.2+2 .3 10 B 47 .2+ 11 .8 98 ° C adlib 21 .5+ 1 .8 10 C 68 .6+ 16 .6 9

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Fig . 1 . Light microscopical images of ground otoliths from 40 d old Coregonus lavaretus larvae reared under different experimentalconditions (adlib : ad libitum food supply throughout the 14 h day; limit: limited food supply, zooplankton given only once per day) .a) 4 ° C limit, b) 4 ° C adlib, c) 6 ° C limit, d) 6 ° C adlib, e) 8 1 C limit, f) 8 1 C adlib . Arrows indicate hatch-check. Bar: 10 µm .

In SEM observations, the best results were ob-tained by using the detector for back-scatteredelectrons . Most parameters like duration of etch-ing with EDTA and acceleration voltage have alarge range of tolerance. Nonetheless, in severalcases daily increments were more reliably identi-fied in LM preparations . Even more surprisingly,the perinuclear zone could not be better analyzedby SEM as compared to LM : increments werevisible in some sectors only, and they could notbe followed uninterruptedly from the periphery tothe hatch-check (Fig. 2a,b) .

Discussion

The experimental conditions were chosen to dis-close the limits under which daily increments infirst feeding coregonid larvae are just recogniz-able. The temperature spectrum reflected the insitu conditions for coregonid larvae during theirfirst weeks of life (Eckmann & Pusch, 1989) .Temperatures above 8 °C were omitted, becauseunder these conditions increments are alwaysclearly recognizable (Eckmann & Rey, 1987 ; Rey& Eckmann, 1989) .

Maintaining well defined food conditions wassomewhat problematical. Fishes fed ad libitum al-

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ways stayed in dense plankton concentrations . Inthe aquaria designated for limited food supply, itwas not possible to syphon off the remainingplankton completely after two hours of feeding,so a real food limitation might be questioned .However, considering mean total lengths at theend of the experiment (Table 1), the desired effecthas obviously been accomplished, although thenarrow temperature range as well as the shortexperimental time precluded from obtaining pro-nounced and highly significant differences be-tween treatment groups .

Due to refraction and interferencies in the mar-ginal region of an otolith (cf . Fig. 1), it is notori-ously difficult to identify daily increments withcertainty in this zone, no matter under which con-ditions the larvae were reared . Therefore, an errorof 1 or 2 days may arise in age determination . Amore important problem, however, is the identi-fication of daily increments in the region close tothe nucleus .

The first problems in detecting daily incrementsby LM were encountered in larvae reared at 6 'C .Mean incremental widths in the perinuclear zoneof these larvae should range from 0 .3 to 1 .1 µm .These values were estimated from the widths ofthe perinuclear zones and the number of daysduring which these zones were formed . Detection

Fig. 2 . Scanning electron microscopical images of otoliths from 40 d old Coregonus lavaretus larvae, reared at 6 ° C under ad libitum(a) and under limited (b) food supply . Arrows indicate hatch-check . Bar: 5 µm .

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of daily increments is believed to be restrictedmainly by the resolution limit of the light micro-scope. The theoretical lowermost limit of detec-tion is determined by light wave length (Michel,1964). Two points at distanced are unequivocallyseparated from each other if there is a completeminimum between the two refraction maximaoriginating from these points . This is the case ata distance of 21r between the refraction figures,hence : d = .1/A (d = distance between two points,A = wavelength in nm, A = numerical aperture ofthe system) .

Practice, however, shows that smaller distancescan be recognized, as long as there is a minimumbetween the two refraction maxima . The individ-ual acuity of the observer is very important in thiscontext, and a conventional limit can be estimatedas : d :Z~ 3/4 *2/A.

For light emitting objects as well as for inco-herent or partially incoherent light sources, thedistance between the two main maxima can de-crease further down to iv, hence : d = 2/2A . Thus,there is no sharp resolution limit but rather acritical range from d = )./A to d = 2/2A . These val-ues of course can only be reached under optimumconditions and by using monochromatic light .

For the research microscope used in this studywith a numerical aperture ofA = 1 .3 and blue light(not monochromatic), the theoretical resolutionlimit should be in the range from 200 to 400 nm .The narrowest increments observed in this studywere more than 400 nm wide . It might, therefore,be argued that the resolution limit of the micro-scope system (plus observer's acuity) did notallow to detect more increments . This might havebeen the case in otoliths from larvae reared at4 'C, where the only visible increments near theotolith periphery exceeded 400 nm width, andwhere the mean widths of daily increments withinthe perinuclear zone were estimated to range from300 to 500 nm . In larvae reared at 6 'C, however,resolution problems alone cannot explain why anincrement-free perinuclear zone was observed . Inseveral otoliths, mean increment widths withinthe perinuclear zone should be around 1 .1 µm,and this is clearly far beyond the resolution limit .The same argument applies to the otoliths of lar-

vae reared at 8 'C: the first daily increments foundin larvae kept under limited food conditions areof very low contrast, thus barely recognizable, butthey are nearly 1 µm broad (Fig . le) .

In summary, the resolution limit of LM mightrestrict the detection of daily otolith increments inCoregonus lavaretus larvae, but this seems not tobe the most important factor which limits incre-ment visibility . From the timing of clearly visibleincrement formation it appears as if good opticalcontrast between incremental and discontinuouszone of a daily increment is only achieved whenlarvae ingest reasonable amounts of exogenousfood. At 8 'C, food ingestion starts only 2 to 3days after hatching and soon afterwards larvaehave well filled intestines (visible increments areformed immediately after hatching) . At 4 ° C onthe contrary, food ingestion starts much later andintestine fullness is generally very low (visible in-crements are formed starting from 10 to 35 d afterhatching).

Therefore, another reason for the failure in de-tecting daily increments might be a different oto-lith fine structure or biochemical composition(Gauldie & Nelson, 1988) during the periods ofendogenous and exogenous feeding .

The results of our SEM observations supportthis view . Daily increments are only resolved bySEM if discontinuous and incremental zones aredifferently etched . In the present study, only thoseincrements that showed good optical contrast inthe light microscope could also be viewed bySEM.

Detection of daily otolith increments was con-siderably improved in several species of fish bySEM examinations. In walleye pollock (Theragrachalcogramma) for example, increments with lessthan 200 nm width were identified (Bailey &Stehr, 1988), and good agreement between incre-ment counts and true age was attained (Nish-imura & Yamada, 1984) . Nevertheless, in manycases increments were resolved in certain parts ofthe otoliths only, while in other parts they re-mained unresolved (Bailey & Stehr, 1988) . Inother species like herring (Clupea harengus), de-tection of daily increments was not improved bySEM as compared to LM (Campana et al., 1987),

a result which very much resembles the observa-tions on Coregonus lavaretus larvae in the presentstudy .Comparing SEM and LM, it is recommended

to use the latter technique for the study of larvalwhitefish otoliths for the time being . The resolu-tion, and hence information, obtained is the samefor both methods and in addition there are sev-eral advantages in light microscopy : (i) the grind-ing process for SEM preparations is much moretime consuming because the cross section mustpass exactly through the otolith nucleus, while forLM preparations this is not absolutely necessary ;(ii) the percentage of successfully prepared oto-liths is consequently higher in LM studies ; (iii)viewing and measuring otoliths is much faster inthe light microscope . In summary, there is notechnique available at the moment which allowsto detect daily otolith increments in whitefish lar-vae which live at low (4-6 ° C) temperatures .These are the temperatures which the larvae en-counter after hatching in Lake Constance in lateFebruary, so their true age and their growth his-tory during the first days or weeks of life cannotbe evaluated so far .

Acknowledgements

The studywas supportedby Deutsche Forschungs-gemeinschaft within the special collaborative pro-gram (SFB 248) `Cycling of matter in Lake Con-stance' .

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