Not to be cited without prior reference to the authors leES C.M. 1995 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
I:
. ABSTRACT
/ - '
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
•

3 microstructurc analysis arid RNAIDNA ratio detemlimition on the
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,
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
riuorescent transfered to mark on the increment formed that day. After that the larvae were: carefullY
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
polarisationttlter aUached 10 a binokUlar and were fiXed on a glass slide marldng left and right side.Tbe
iarva witbout the otolith was pul into
Epperidorf via!
kept on ice. After 5 larvae were disSected (necessary tune: 30miri.) the RNAIDNA analysis
penorined.
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 of a 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 of days after the marking was peiformed. Results are giveri in Fig. 1b and show that the rings
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
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.
•
•

5
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 ä value orOAllm (Fig. 3a). Fig. 3b gives a eomparlson ofthe size ofthe inerements of all äriatysCd fed and starved llirvae showing mean values and standard deviations.
The
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
4 to day
5 and then sllghtiy increaSe<l to reach a vaIue 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
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
2; tms
Fig.
5.
From day 4 to day 7 the RNAlnNA ratio decreased from a vaJue of 7 to below
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
(Fig.
5). Fig. 6 shows the relation between mean
RNAmNA ratios arid
.
' be visabl.e on day i
1
relative otolith
, gfoWih of all aDalySed fed and starved larvae. The relative otolith groWth was calculated by setting the Width of the increment
to a vaJue' of i and ca1culating the reiative growth of the foilowrng increments in relation to that vaJue. EverY d3ta point represents the niean of
4-5 larvae. Tbe relative groWth ·of the otolith is
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
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
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. AIizMinmarkirig 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
this study would IUive been much more signmcant if sarnples from older
had been aVäilabie. Future snidies must increase the Sämplirig protocOi
to
age of
21 daYs.
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 a genernI 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
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 of the larirae have been found (eanino et aI.
1991, Frank & McRuer 1989 t
Sctzler-Hamiltori et al. 1987).
A

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

12
14
13
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0 c:
12
11
10
9
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'-
<D
.c
8
E
7
::3
Z
6
5
4
4 y =1.008x -0.09
f=0.994
n =150 a)
5 6 7 8 9 10 11 12 13 14
0) c
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E c:
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8
7 oe:(
Lo-
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(J.)
.c
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Z
E
~
0 c:
-
2
3
1
4
0
5
6
0 y =0.009 x + 0.0017
f= 0.999
n = 109 b)
1 2 3 4 5 6 7 8 9 10
Fig. I: Relation between number of increments and known age of laboratory reared cod larvae (Fig. la). Validation ofthe daily otolith deposition by marking the otolith with Alizarin (Fig. Ib).
•
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8 9
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6
7
5
4
Y=0.333x +5.55
rZ =0.099
n =61
0
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13
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0
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00 a)
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19
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18
17
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1:
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0
16 -
15
14
13
12
11
10
9'
20
4 5 6 7 8 9 .10
Radius cf the otolith cere left [~m]
11 12 y=0.633x +5.26
r2=0.299
n=61 v v v f} vv v v v v
V
V v b)
9 10 11 12 13 14 15 16 17 18 19 20
0
-
c
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E
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-
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10
9
8'
4
3
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1
0
7
6
5
Radius left otolith [~m] y =1.037x-0.16
r2=0.863
n=61 c c) 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 on
4-12 day old cod larvae.
,
Values are means calculated from 4 measurements on the otolith in fouT different directions. Linear regression model was fitted to the data.

14
1.4
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.....
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c
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E
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(.) c
1.2
1.0
0.8
0.6
0.4
0.2
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e-~
-~
[J a)
2 4· 6 8
10 12
1.4
1.2
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E 0.8
o c
0.6
0.4
-+--...-_--r--_-...-_-...-_--r--_-or-~
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
(Fig. 3b). Error bars give the standard deviation.
cod larvae
•

..
15
1.0 . . . , . . . - - - - - - - - - - - - - - - - - - - ,
• Fed
Starved
0.8
.-..
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::l.
....... 0.6
•
~
0.4
o
0.2
a)
0.0
-..I-----..--__ r - - - - , - - - - - r - - - - - r - - - - - !
.......
1.6
.-..
~1.4
~
1.2
ca
~
1.0
0.8
0.6
0.4
2 4 6 8 10
Ageindaysafterha~h~g
12 14
2.2 - - - - - - - - - - - - - - - - - - - - - - .
2.0
• Fed
starved
1.8
0.2
0.0
b)
- - - , . . - - - - - - . r - - - - !
2 4 6 8 10 12 14
Age in days after hatching
Fig. 4: DNA- content (Fig. 4a) and RNA- content (Fig. 4b) of cod larvae in relation to age and feeding situation. Starving larvae (n= 45) were deprived of food starting on day 4 and day 7 and compared to fed larvae (n= 86). Error bars give the standard deviation.

- - - -
- - - - - - - -- - - -
:'
.
r
16
9 z
0
:=( z a::
5
4
6
3
8
7
2
1
0
0
2 4 14 6 8 10
12
Fig. 5: RNNDNA ratios of cod 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 bars give the standard deviation.
4.5
12
0
Z a::
Q)
-
CI)
:J ro
> c: rn
Q)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
8 10 m--~
7 8 "
10
9 11
l!l
•
0.5
1.0
1.5
2.0
2.5
3.0
Fig.
6: Relation between relative otolith growth offed and starved cod larvae and RNAIDNA ratios.
For calculation of relative growth the width of ring number 4 was set to one.
Tbe numbers give the age in days.