Busquets, Xavier

Iglesias, Magdalena

Massutí, Sofía

Morales, Beatriz

Biblioteca Digital de les Illes Balears

An attempt to determine variability of 
RNA/DNA ratios during Dicentrarchus labrax 
larval development 
Magdalena IGLESIAS, Beatriz MORALES, Sofia MASSUTÍ and 
Xavier BUSQUETS 
SHNB 
SOCIETAT D'HISTORIA 
NATURAL DE LES BALEAR S 
Iglesias, M., Morales, B., Massutí, S. and Busquets, X. 2002. An attempt to determine 
variability of RNAIDNA ratios during Dicentrarchus labrax larval development. Boll. 
SOCo Hist. Nat. Balears, 45: 15-20. ISSN 0212-260X. Palma de Mallorca. 
RNA and DNA content of seabass (Dicentrarchus labrax) larvae were determined from 
day 10 to day 60 after hatching in an aquaculture facility. Our results show a two-phase 
modulation ofthe RNAlDNA ratio throughout seabass larvae development. From day 10 
after hatching there is an increase in the RNAIDNA ratio reaching maximal values at day 
30 of age. Then, the RNAlDNA ratio decreased reaching minimal values at day 60 after 
hatching. These results are consistent with a high rate of protein synthesis during the first 
30 days of seabass development compared with the rest of the developmental period stu-
died. The results are compared with that of otolith growth carried out for the same spe-
cies larvae. 
Keywords: marine fish, larvae, DNNRNA ratio, Dicentrarchus labrax. 
APROXIMACIÓ A LA DETERMINACIÓ DE LA VARIABILITAT DE LA RELACIÓ 
DNA/RNA DURANT EL DESENVOLUPAMENT LARVAL DEL LLOBARRO 
Dicentrarchus labrax. S'ha determinat el contingut de DNA (acid desoxiribonucleic) i 
de RNA (ilcid ribonucleic) en larves de llobarro (Dicentrarchus labrax) d'edats compre-
ses entre 10 i 60 dies de vida, procedents d'una planta de cultiu. Els resultats obtinguts 
mostren dues fases diferenciades de desenvolupament d' aquestes larves, basades en la 
relació RNAlDNA avaluada. S'observa un augment de la proporció de RNNDNA des 
del dia 10 fins el dia 30 de vida, per a decreixer a continuació, trobant els valors mínims 
en les larves de 60 dies de vida. 
Paraules clau: peixos marins, larves, relació DNNRNA, Dicentrarchus labrax. 
Magdalena IGLESIAS, Centro Oceanográfico de Baleares, Muelle de Pelaires s/n, 
07070 Palma. e-mail:magdalena.iglesias@ba.ieo.es; Beatriz MORALES. CSIC-UIB-
Institut Mediterrani d'Estudis Avan(:ats, Miguel Marqués 21, 07190 Esporles. Sofia 
MASSUT! Planta de Acuicultura «Es Murterar»; Alcúdia. Xavier BUSQUETS. INSAU-
NI, Palma. 
Recepció del manuscrit: 19-abr-02; revisió acceptada: 16-mai-02. 
16 Boll. Soco Hist. Nat. Balears, 45 (2002) 
Introduction 
Determination of larval condition has 
widespread applications in the field of fish eco-
logy, as an indicator of sub-Iethal effects. A met-
hodology used to as ses s larval condition based 
on the quantification of larval nucleic acids 
(RNAlDNA) concentration allows to infer 
growth and nutritional status accurately 
(Buckley, 1984; McLaughlin et al., 1995). This 
method is based on the assumption that the con-
centration of RNA changes as a function of the 
amount of protein synthesis occurring in the cell 
whereas DNA concentration indicates the num-
ber of cells in an organism (Buckley, 1980; 
1984). Therefore, the RNAlDNA ratio is consi-
dered a measure of protein synthetic activity per 
cell (Buckley, 1984; Bulow, 1987; Robinson and 
Ware, 1988; Hovenkamp, 1990; Hovenkamp and 
Witte, 1991). In this contribution we applied this 
technique to asses larval condition during deve-
lopment of a temperate water species reared at 
controlled conditions (D. labrax, L. 1758). 
Material and methods 
a) Rearing conditions 
Larvae were obtained from naturally 
spawning adult sea bass larvae, hatched from the 
same spawning and reared at the Es Murterar 
Aquaculture facility (Mallorca, Spain). The 
experimental conditions were as follows: sea 
bass were kept indoors in a 1200-liter tank filled 
with freely circulating seawater. Initial specimen 
density was 39 larvae/l. Naturallight cycles were 
maintained. Food (Artemia spp) was supplied ad 
libitum and according to the nutritional require-
ments of the sea bass during the experimental 
periodo Water temperature (measured daily at 
noon) was kept at 19-20°C and the salinity at 39 
pSU. Specimens were hatched in February 1996 
and were killed at regular ten days interva1s. 
Larvae were measured to the nearest 0.1 mm 
under a dissecting rnicroscope, dried on blotting 
paper, and weighed to the nearest 1 Ilg. The lar-
vae were subsequently frozen in liquid nitrogen. 
b) Nucleic acid extraction 
Total RNA and DNA were extracted from 
larvae samples by repeated washing of the 
homogenised tissue with organic solvents 
(Sambrook, 1989) with modifications. Briefly, 
each larvae was homogenised in 1 mi saturated 
phenol (pH 7.5) plus 100 111 of distilled water, 
during 30 s using an Ultra-Turrax T25 homoge-
neizer (IKA, Staufen, Germany) and then incu-
bated for 5 mino at room temperature. Two hun-
dred 111 of chloroform were then added to homo-
genates, vortexed for 30 s, and then incubated at 
room temperature for 5 mino The samples were 
centrifuged at 10,000 g for 10 min at 4°C. The 
aqueous phases were separated and mixed with 
500 111 of 100% isopropyl alcohol. The mixtures 
were briefly shacked, incubated for 10 min at 
room temperature and then centrifuged at 10,000 
g for 10 min at 4°C. The nucleic acid precipitate 
(DNA plus RNA) was washed twice with 1 mi of 
75% ethanol and dissolved in DNAse-RNAse-
free water (nucleic acid solution). 
e) Enzimatic determination of the relative 
content of DNA and RNA 
This technique is based on the method des-
cribed by Clemmesen (1988). Two aliquots (ter-
med RNA and DNA aliquots) of the nucleic acid 
solution were processed in parallel. To the RNA 
aliquot (5 Ill) 5 111 of DNAse solution (Dnase 1, 
Rnase free, lOUl1l1 Boehringer Mannheim, 
Germany) and 40 111 of DNAse buffer (40 mM 
Tris-HC!. PH 7.9, 19 mM NaC!, 6 mM MgCh, 10 
mM CaCh) were added. To the DNA aliquot (10 
Ill), 2 111 of RNAse buffer (Boehringer 
Mannheim, Germany) 5 111 of RNAse (RNase, 
Dnase free, 0.5 (g/Ill; Boehringer Mannheim, 
Germany) and 33 111 of nuclease-free water were 
added. Both samples were incubated during one 
hour at 37°C. The reactions were terminated by 
adding 900 111 of nuclease-free water and placing 
the tubes at room temperature. 
d) Quantification of DNA and RNA by fluo-
rescence analysis 
DNA and RNA concentration were quanti-
fied by a method based on that of Higuchi and 
DoIlinger (1992) originally described for quanti-
M. Iglesias et al., Variability of RNA/DNA of Dicentrarchus labrax 17 
fication of amp1ified DNA sequences. Briefly 
DNA and RNA aliquots (50 ¡.tI) were mixed with 
water to a final volume of 900 ¡.tI. Then, 50 ¡.tI or 
SYBR Green II (FMC BioProducts, Rockland, 
ME, USA) solution at 1/100 dilution was added. 
Fluorescence was measured in a fluorescence 
spectro-fluorometer (model RF-1501; Shimadzu, 
Japan). For SYBR Green II fluorescence deter-
mination excitation was at 490 nm and emission 
at 520 nm. The amount of fluorescence of each 
sample was measured and the fluorescence units 
were evaluated using standard curves which con-
sisted of 5 points of DNA (herring sperm DNA, 
Sigma Chemical CO. Saint Louis, MO, USA) or 
RNA (E. coli ribosomal RNA, Sigma Chemical 
CO. Saint Louis, MO, USA) content (1-5 ¡.tg, 
resulting in a linear relationship). The standard 
curves were performed prior each set of samples 
fluorescence deterrninations. 
Results 
In the aquaculture facility sea bass larvae 
size increased from 4.5 mm totallength (TL) at 
10 days of age to 19 mm TL at 60 days following 
a linear relationship (r = 0.77) (Table 1, Fig.1A). 
In the other hand, larvae weight increased from 
0.48 mg at 10 days of age to 30.67 mg at 60 days 
reaching a plateau at 50 days (Table 1, Fig. lB). 
The DNA content increase fram 2.50 ¡.tg at 
age 10 to 42 ¡.tg at age 60, showing a big increa-
se between 40 and 50 days (Table 1; Fig. 2 A). 
The RNA content increase from 2.6 ¡.tg at age 10 
to 32.68 ¡.tg at age 50, showing a dcrease at age 
60 (21.7 ¡.tg) (Table 1; Fig. 2 B). 
The RNA/DNA ratio was 1.04 initially, 
increasing to near 2 at day 30. Then the 
RNA/DNA ratio decreased reaching minimum 
values (0.5) at day 60 (Table 1, Fig. 2 C). 
Focusing in these results we consider two age 
groups, 10 to 40 days and older. The non-para-
metric Mann-Whitney test showed significant 
differences on the RNA, DNA and RNAlDNA 
content between those groups (Table 2, Fig. 2 C). 
Discussion 
RNA/DNA ratios are considered as useful 
independent growth measure of individual fish 
larvae (Buckley, 1984). DNA of individual cells 
is fairly constant, so the DNA content of whole 
animals increases proportional to increases in 
cell number (growth). However, RNA content of 
cells is variable, and reflects active protein synt-
hesis. RNA/DNA ratios have been found to be 
accurate indicators of recent feeding of larvae, 
higher ratios indicate better feeding condition 
(Canino, 1994). 
First our results show an increase of the 
RNA/DNA ratio from 1.04 at 10 days of deve-
lopment to 1.86 ( 0.29 at day 30 of development 
(Table 1; Fig. 2 C). Clemmesen (1989) proposed 
that the RNA/DNA ratio of 1 is an indicator of a 
critical level of feeding conditions. In the acua-
Age Mean length Mean weight MeanDNA Mean RNA Mean n 
(mm) (mg) (¡.tg) (¡.tg) RNA/DNA 
10 4.50 0.48 2.50 2.6 1.04 1 
20 9.06 (0.70) 2.11 (0.22) 9.53 (6.96) 12.62 (8.27) 1.67 (1.02) 5 
30 10.80 (0.84) 5.70 (1.07) 14.18 (5.09) 25.56 (7.63) 1.86 (0.29) 5 
40 14.33 (1.15) 9.67 (5.04) 12.76 (7.96) 19.53 (15.45) 1.44 (0.30) 3 
50 17.50 (0.58) 32.93 (3.72) 38.84 (12.17) 32.68 (11.77) 0.90 (0.34) 4 
60 19.00 (LOO) 30.67 (8.27) 42.00 (17.17) 21.70 (7.68) 0.53 (0.04) 3 
Table 1. Summary of lhe nata analysed on Dicentrarchus labrax reared larvae in relation to age (days from eclo-
sion).s.d. in brackets. n : number of fish analysed. 
Taula 1. Resum de les dades analitzades de les larves cultivades de Dicentrarchus labrax en relació a l'edat (dies 
després de l'eclosió), desviació estimdard entre parentesi, n,' nombre d'exemplars analitzats. 
18 Boll. SOCo Hist. Nat. Balears, 45 (2002) 
Table 2. Results of the Mann-Whitney test on the Dicentrarchus labrax larvae. n : number of fish. s.d. : standard 
deviation. p : 
Taula 2. Resultats del test de Mann-Whitney sobre les larves de Dicentrarchus labrax. n: nombre de peixos. s.d.: 
desviació estimdard. p: probabilitat. 
Age (days) 
DNA (¡.¡.g) 10-40 
>40 
RNA (¡.¡.g) 10-40 
>40 
RNAJDNA 10-40 
>40 
culture facility seabass larvae started exogenous 
feeding on age 15-16 days (Artemia spp.). Our 
RNAJDNA ratio data suggest that seabass larvae 
in captivity are at the level of critical feeding 
25,-----------------------------, 
A 
20 
I 
rs 
-' 10 ~ 
:'S 5 
10 
40 B 
.[ 30 
f-
J: (!) 
~ 20 
w 
~ 10 
:'S 
o 
10 
20 30 40 50 
LARVAE AGE (days) 
• 
• 
20 30 40 50 
LARVAE AGE (days) 
. 
. 
60 
• 
60 
n 
14 
7 
14 
7 
14 
7 
mean s.d. p 
11.38 6.59 0.0001 
40.20 13.23 
18.00 11.29 0.07 
27.98 11.1 
1.64 0.64 0.003 
0.74 0.31 
during yolk utilisation, but improving rapidly 
once exogenous feeding started and follows a 
continuous improvement in feeding condition 
reaching its maximum at day 30 of development. 
The critical condition demonstrated by larvae 
before the exogenous feeding started may be 
explained by the fact of its high rate of RNA 
synthesis (indicating protein synthesis) as its 
shown in Figure 2 B. Protein synthesis is a high 
energy-requiring process that rapidly exhaust 
yolk energetic reserves. Once the abundant and 
more energetic exogenous feeding started, the 
high ratio of protein synthesis can be maintained 
without energy deficit and the larvae rapidly 
improve its condition and size (Table 1; Fig. 1 
A). An increase of RNA/DNA with larval size 
has been also reported previously on herrings 
(Clernmesen, 1994). 
In the other hand, our results clearly show 
that beyond day 30 of development, the 
RNAIDNA ratio diminish steadily reaching low 
values (lower than the critical ratio of 1) at day 
50 (0.90 ± 0.34) and even lower at day 60 (0.53 
± 0.04) (Table 1; Fig. 2 C). Seabass initiate meta-
morphosis precisely between 50-60 days of deve-
lopment. This, correlates with the observed slow 
down of larvae weight (Table 1; Fig. lB) and 
Fig. 1. A- Linear relationship (r = 0.77) between totallength (mm) and age (days after hatching) of sea bass (D. 
labrax) larvae from aquaculture facility. B- Relationship between total weight (mg) and age of sea bass larvae from 
aquaculture facility, reaching a plateau at day 50. 
Fig. 1. A- Relació lineal (r = 0.77) entre longitud total (mm) i edat (dies després d'incubació) de llobarro (D. 
labrax) larves d'aquicultura. B- Relació entre pes total (mg) i edat de larves de llobarro d'aquicultura, atanyent 
una pendent horitzontal el dia 50. 
M. Iglesias et al., Variability of RNA/DNA of Dicentrarchus labrax 19 
active protein synthesis (RNA/DNA ratio). 
Stage-dependent RNA/DNA ratio s have been 
reported in sole (Richard et al., 1991). Thus, our 
larvae were undertaking a critical stage corres-
ponding to the end of metamorphosis with low 
60.---------------------------~ 
50 
40 
~ 30 
~ 
o 20 
A 
10 
O~~w-~~~-u~~~~~~ 10 60 
40 
30 
~ 
« 20 
z 
ex: 
10 
2.5 
2.0 
« 
z 1.5 o 
4: 
z 
ex: 1.0 
0.5 
O 
B 
10 
e 
r--
10 
20 30 40 50 60 
p<0.005 
* 
* n 
tO 50 60 
LARVAE AGE (days) 
RNA/DNA ratio s despite the increase in larval 
size. This indicate that metamorphosis is a criti-
cal period in seabass development in terms of 
feeding conditions (as indicated by RNA/DNA 
ratios) probably by the increased metabolic 
expenses due to increased mobility and body 
changes (formation of scales, etc). 
Daily growth increments of the otolith is 
another method of assessing larval growth and 
condition (e. g. Govoni et al., 1985, Suthers et 
al., 1996). Our results of RNA/DNA ratio as 
individual seabass larvae growth measure are in 
concordance with available data on otolith 
growth (Gutiérrez and Morales-Nin, 1986), that 
demonstrated in seabass larvae reared in an 
aquaculture facility a similar biphasic paUern in 
the thickness of the daily growth increments. 
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Aproximació a la determinació de la variabilitat de la relació DNA/RNA durant el desenvolupament larval del llobarro Dicentrarchus labrax

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