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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.
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Institut rur Meereskunde an der Universität Kiel, Düstembrooker Weg 20, 24105 Kiel,
Germany
. ABSTRACTI:
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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.
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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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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
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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
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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-
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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
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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).
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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.
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