Fisheries Research 68 (2004) 57–65

Seasonal variations of RNA/DNA ratios and growth ratesof the Alboran Sea sardine larvae (Sardina pilchardus)

Teodoro Ramırez∗, Dolores Cortés, Alberto Garcıa, Angel CarpenaInstituto Español de Oceanograf´ıa, Centro Oceanográfico de Málaga, Puerto Pesquero s/n,

Apdo. 285, 29640 Fuengirola, Malaga, Spain

Received 28 March 2003; received in revised form 2 February 2004; accepted 22 February 2004

Abstract

RNA/DNA ratios and larval growth of winter and spring-spawned sardine larvae (Sardina pilchardus), from the southerncoast of Spain were studied in 1997 and 1998. Seasonal variations of larval growth and RNA/DNA ratios were analysedin relation to environmental variables. Oceanographic features in the area of study are strongly influenced by the flow ofnutrient-poor Atlantic water, which enters the Alboran Sea through the Gibraltar Strait. In 1997, the influence of Atlanticwater was stronger in spring than in winter, leading to lower salinity in spring 1997. The predominance of Atlantic waters couldcause lower chlorophyll-a concentration in spring 1997 than expected for this season. In addition, lower microzooplanktonbiomass was observed in spring 1997 than in winter 1997. In concordance with microzooplankton biomass, the RNA/DNAratio of sardine larvae was higher in winter 1997 than in spring 1997. No seasonal differences in somatic or otolith growthrates were observed in 1997, which could be due to a time lag in the response of larval growth to changing environmentalconditions. Unlike the previous year, during 1998, no seasonal changes were observed in the amount of incoming Atlanticwaters, as evidenced by salinity values. In 1998, temperature and microzooplankton biomass were higher in spring than inwinter, causing higher somatic and otolith growth rates in spring 1998. The higher temperatures observed in spring 1998compared to winter 1998 could be responsible of a decline in the RNA/DNA ratio of spring-spawned sardine larvae incomparison to winter-spawned larvae. Larval growth and RNA/DNA ratios may show a different response to changes in theenvironment, particularly temperature. It its known that temperature has a positive effect on growth rates but it has a negativeeffect on the RNA/DNA ratio, then it is recommended to bear in mind these effects, particularly when joint analysis of larvalgrowth and RNA/DNA ratios are carried out.© 2004 Elsevier B.V. All rights reserved.

Keywords:Nutritional condition; RNA/DNA ratio; Larval growth; Otolith; Sardine larvae

1. Introduction

Sardine (Sardina pilchardus) is one of most abun-dant species of the ichthyoplankton in the northwest-ern Alboran Sea (Garcıa et al., 1988; Rodrıguez,

∗ Corresponding author. Tel.:+34-952-47-19-07;fax: +34-952-46-38-08.E-mail address:teodoro.ramirez@ma.ieo.es (T. Ramırez).

1990). The spawning period of sardine in the AlboranSea is from October to May, with a peak in winter(January–February) (Rodrıguez, 1990). In the north-ern sector of the Alboran Sea, the main sardine spawn-ing grounds are located off the coast of Malaga. Thisarea has peculiar oceanographic features that make itappropriate as sardine nursery grounds, and relativelyhigh abundance of sardine eggs and larvae have beenfound there (Garcıa et al., 1988; Rodrıguez, 1990).

0165-7836/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.fishres.2004.02.008

58 T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65

In addition, several studies have showed that fish eggand larval distributions in the Alboran Sea are closelylinked to its hydrography (Lafuente et al., 1998).

The influence of Atlantic waters on the northern sec-tor of the Alboran Sea is reflected in the oceanographicfeatures of this area. The upper layers of the watercolumn are occupied by modified Atlantic water, withsalinities usually ranging from 36.2 to 36.6 psu. Belowthis layer more dense and saline Mediterranean wa-ters are found. The isohaline of 37.5 psu is consideredas the interface between Atlantic and Mediterraneanwaters (Parrilla and Kinder, 1987). In the northwest-ern Alboran Sea different upwelling mechanisms leadto periodic local increases of productivity along theSpanish coast (Sarhan et al., 2000), and high concen-trations of nutrients and chlorophyll-a have been asso-ciated to these upwelling phenomena (Delgado, 1990;Champalbert, 1996; Sarhan et al., 2000).

As with other species of clupeoids, recruitment ofsardine in the North Alboran Sea shows a high inter-annual variability (Giráldez and Abad, 1991). Mortal-ity during the early life stages in small pelagic speciesis considered to be the main cause of interannualvariability in recruitment (Bailey and Houde, 1989).Mortality of fish larvae is mainly due to starvationand predation (May, 1974; Bailey and Houde, 1989).Both processes are related, since starving larvae haveslower growth rates (Buckley, 1984) making themmore vulnerable to predators than fast-growing larvae(Folkvord and Hunter, 1986; Purcell et al., 1987).The joint assessment of growth and nutritional con-dition in field-caught larvae could provide valuableinformation on survival probability, allowing a betterunderstanding of the main factors affecting year-classstrength (Ferron and Leggett, 1994; Clemmesen andDoan, 1996).

Nutritional condition of fish larvae can be estimatedby the RNA/DNA ratio (Buckley, 1984; Ferron andLeggett, 1994; Bergeron, 1997; Garcıa et al., 1998;Buckley et al., 1999). In addition, the RNA/DNAratio has been used as an indicator of recent growthin fish larvae (Buckley, 1984; Hovenkamp and Witte,1991; Westerman and Holt, 1994). These studies haveshown that well-fed and fast-growing larvae havehigher RNA/DNA ratios than slow growing ones.

Larval growth can be inferred from otolith mi-crostructure (Campana and Jones, 1992). Incrementdeposition in otoliths seems to be controlled by an

endogenous rhythm that can be modified by environ-mental factors (Campana and Neilson, 1985). It hasbeen shown that increment widths of otoliths respondto changes in feeding conditions and temperature(Govoni et al., 1985; Heath, 1992; Folkvord et al.,2000). Recent otolith growth, estimated from thewidth of the last three to six increments, has been usedas an index of recent somatic growth in fish larvae(Suthers, 1996; Clemmesen and Doan, 1996; Folkvordet al., 2000), and it has been related to the RNA/DNAratio (Clemmesen and Doan, 1996; Suthers, 1996).

The main aim of this work is to analyse theseasonal variations in growth rates and RNA/DNAratios of winter and spring-spawned Alboran Sea sar-dine during two consecutive years (1997–1998), andtheir relations with the seasonal changes in the sur-rounding environment, namely temperature and foodavailability.

2. Materials and methods

Sardine larvae were collected in February and April1997 and in February and May 1998. Samplings werecarried out during 1–2 days in a small area (approxi-mately 6 km2) located off the coast off Malaga (Fig. 1).Larvae were collected by tows with a bongo 90 netequipped with 1 mm mesh, at a speed of two knots. Inorder to minimise variability in RNA/DNA ratios dueto diel fluctuations in RNA content (Chıcharo et al.,1998), all tows were carried out at night. During sam-plings, temperature and salinity in the water columnwere registered by a CTD Seabird-25. Besides, wa-ter samples at three different depths were taken with5 l Niskin bottles for chlorophyll-a analyses. Meso-zooplankton and microzooplankton samples were col-lected by tows with bongo 40 net (200�m mesh) andbongo 10 net (53�m mesh), respectively.

After the tows, the larvae captured were quicklyseparated from the plankton samples and identifiedunder a binocular microscope. Afterwards, they werekept in cryogenic vials containing filtered seawater(Whatmann GF/F filters) and frozen in liquid nitrogen.At the laboratory larvae were preserved at−80◦C.Mesozooplankton samples were preserved in 10%formaldehyde buffered at pH 8 with borax. Microzoo-plankton samples were kept in Petri dishes and frozenat −20◦C for biomass estimation. For chlorophyll-a

T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65 59

Fig. 1. Map of the studied region showing the sampling area.

analysis 2 l of seawater were filtered through What-mann GF/F filters, and the filters were frozen in dark.Chlorophyll-a was extracted with acetone 90% for 24 hat 4◦C in the dark, and subsequently analysed by spec-trophotometry (SCOR-UNESCO, 1966). Samples ofmesozooplankton were analysed under a binocular mi-croscope, samples were sorted and sardine eggs werecounted. Likewise, the main groups of zooplanktonwere identified. Microzooplankton biomass was de-termined after drying the samples to a constant weightat 60◦C.

Standard length of sardine larvae was measuredby an image analysis system (Garcıa et al., 1998).Sagittae were extracted and mounted with nail lac-quer onto slides for age estimation. Daily incrementswere counted as described inRamırez et al. (2001).After otolith extraction, larvae were homogenised at0◦C in 690�l of Tris-buffer (0.05 M Tris, 0.1 M NaCl,0.01 M EDTA, pH 8.0) containing 10�l SDS (0.7%)by means of ultrasonic pulses (2× 10 s). The ho-mogenate was centrifuged at 6000 rpm for 8 min, at4◦C. Two aliquots of the supernatant (100�l each)were taken for the fluorimetric determination of nu-cleic acids. DNA was determined after treatment withRNAase. One aliquot of the homogenate was trans-ferred to a vial and 10�l of RNAase (0.2 mg ml) wereadded, and the mixture was incubated at 37◦C for30 min. A second aliquot of 100�l of supernatantwas transferred to another vial for total nucleic acids(RNA + DNA) determination. The fluorescent dyeethidium bromide was used for quantification of nu-

cleic acids. Calibration curves were made with DNAfrom calf thymus. RNA content was calculated ac-cording toLe Pecq and Paoletti (1966).

Individual daily growth rate in length and dailyotolith growth were estimated according toRamırezet al. (2001). Individual recent otolith growth was es-timated averaging the width of the last six increments.Prior to statistical analysis all data were transformedto natural logarithms (ln). Differences in average stan-dard length between seasons and years were testedby one way ANOVA, using natural logarithm trans-formed data. Differences in growth rates betweenseasons were tested by ANCOVA, using ln age ascovariant. In addition, seasonal differences in bio-chemical parameters were tested by ANCOVA usingln SL as covariant. Correlation analysis was used tostudy the relationships between RNA/DNA ratios andlarval growth. Statistical analysis was carried out withthe software package Statistica 5.1 for Windows. Allstatistical tests were carried out at significance levelof α = 0.05.

3. Results

3.1. Environmental data

In winter 1997, the average temperature in the up-per 20 m was 14.7◦C, while in spring 1997 it was16.3◦C. Seawater temperature was 15.1◦C in winter1998, while in spring 1998 it was 17.6◦C. Salinities

60 T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65

also showed differences between seasons and years,particularly in 1997. In 1997, the average salinitiesregistered in winter were higher than those observed inspring, 37.65 and 36.87 psu, respectively. These salin-ities indicate that there was a stronger influence of At-lantic waters on the studied area in the spring of 1997than in the winter of 1997. In 1998, salinity was onlyslightly higher in spring than in winter (Table 1), indi-cating that the influence of Atlantic waters was similarin both seasons.

Due to the increase of primary production duringspring, chlorophyll-a concentrations were generallyhigher in spring than in winter, in both years (Table 1).In winter 1997, chlorophyll-a concentrations werelow, as expected for this time of the year (Camiñaset al., 1998). However, in spring 1997 chlorophyll-aconcentration was lower (0.66 mg m−3 in average)than expected for this season (Camiñas et al., 1998).This relatively low concentration of chlorophyll-ain spring 1997 could be due to the intrusion of At-lantic waters, which have lower salinity and nutrientconcentrations than the Mediterranean waters (Minaset al., 1991; Camiñas et al., 1998). In winter 1998,chlorophyll-a concentration was slightly higher thanexpected for this time of the year, while in springchlorophyll-a concentrations correspond to the usualvalues found in the area of study during this season(Table 1).

Microzooplankton biomass in the present studywas considered as a relative index of potential foodavailability for sardine larvae (Chıcharo et al., 1998;Garcıa et al., 1998). In 1997, microzooplanktonbiomass was higher in winter than in spring, 13.3 and3.7 mg m−3, respectively, indicating lower food avail-ability in spring 1997. In contrast, microzooplanktonbiomass was lower in winter 1998 than in spring

Table 1Mean values and standard deviations (S.D.) of salinity (psu), tem-perature (◦C), and chlorophyll-a (mg m−3) in winter and spring(1997–1998)

Season/year Salinity Temperature Chlorophyll-a

Mean S.D. Mean S.D. Mean S.D.

Winter/1997 37.65 ±0.14 14.7 ±0.3 0.40 ±0.10Spring/1997 36.87 ±0.04 16.3 ±0.4 0.66 ±0.25Winter/1998 37.51 ±0.15 15.1 ±0.3 0.93 ±0.14Spring/1998 37.61 ±0.19 17.6 ±0.9 1.54 ±0.22

1998, 15.5 and 29.8 mg m−3, respectively, indicatingthat potential prey density was higher in spring. Thesevalues are within the range found in other areas fromthe western Mediterranean (Garcıa et al., 1998). Thelow microzooplankton biomass found in spring 1997could be due to the lower productivity found duringthis season, possibly caused by the influence of At-lantic waters. Likewise, zooplankton abundance waslower in the spring of 1997 (174 individuals m−3)than in the winter of 1997 (827 individuals m−3). Ingeneral, copepods were the most abundant group ofthe zooplankton. In the winter of 1997, copepods ac-counted for 82.6% of total individuals, 57% in spring.In the winter of the 1998, zooplankton abundance wasmuch lower (1222 individuals m−3) than in spring(3742 individuals m−3). In winter 1998, copepods ac-counted for 65.8% of the total abundance, contrastedwith 88.2% in spring.

3.2. Size distribution and hatching dates

The sardine larvae caught in winter 1997 wereslightly smaller than those collected in spring 1997(Table 2) (ANOVA, F = 10.87, P < 0.005). In con-trast, larvae collected in winter 1998 were on averagelarger than larvae caught in spring 1998 (ANOVA,F = 7.48, P < 0.01) (Table 2). The size frequencydistribution of sardine larvae was notably differentbetween years (Table 2). In general, larvae collectedin 1998 were much larger than those collected in1997 (ANOVA, F = 26823.74, P < 0.0001). Thenumber of larvae within the same size range in spring1997–1998 and winter 1997–1998 was low and insuf-ficient to study the interannual variations. Therefore,only seasonal variations (winter–spring) within eachyear are considered in this study.

Table 2Mean values, ranges and standard deviations (S.D.) of standardlength (mm) and RNA/DNA ratios of sardine larvae in winter andspring (1997–1998)

Season/year Standard length RNA/DNA n

Mean Range S.D. Mean Range S.D.

Winter/1997 11.9 7.4–16.7 ±1.7 2.8 1.5–4.5±0.5 123Spring/1997 12.7 7.5–19.3±2.0 2.4 1.8–3.5±0.5 116Winter/1998 20.7 11.0–28.9±3.8 3.4 1.8–6.2±0.7 161Spring/1998 19.4 9.9–27.3±3.2 3.0 1.6–5.1±0.7 105

T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65 61

Hatching dates, obtained from daily incrementscount, indicated that sardine larvae collected in winter1997 hatched between 22 January and 5 February,while larvae collected in winter 1998 hatched from15 December to 8 February. In spring 1997, hatchingdates of larvae go from 1 April to 19 April, while inspring 1998 larvae hatched from 9 March to 30 April.These results suggested that the spawning in winterand spring occurred earlier in 1998 than in 1997.

3.3. Larval growth and RNA/DNA ratios

3.3.1. Winter–spring 1997The growth pattern of sardine larvae in winter and

spring 1997 was described by the growth models:SL = 4.2+ 1.496 age0.665, r2 = 0.56,P < 0.001 andSL = 4.2 + 1.525 age0.663, r2 = 0.70, P < 0.001.Otolith radius was related to standard length in win-ter and spring through the power equations: OR=0.353 SL1.658, r2 = 0.76,P < 0.001 and OR= 0.113SL2.120, r2 = 0.81, P < 0.001, respectively.

In 1997, there was no difference in size at age be-tween seasons (ANCOVA,F = 1.04, P > 0.05).In concordance, somatic larval growth was similar inwinter and spring (ANCOVA,F = 1.34, P > 0.05),declining with larval age (r = −0.46, P < 0.001).

In addition, no seasonal difference was found inotolith radius at age between winter and spring (AN-COVA, F = 1.46, P > 0.05). Likewise, average in-crement widths at age were similar for both seasons(ANCOVA, F = 1.30, P > 0.05). Moreover, no dif-ferences in recent otolith growth, as estimated from theaverage width of the six last increments, were detectedbetween seasons (ANCOVA,F = 0.47, P > 0.05).

The RNA/DNA ratio showed a high variability, bothin winter and spring (1997) spawned sardine larvae(Table 2) (Fig. 2). The RNA/DNA ratio did not showany relation with somatic growth in length in winter orspring (1997) (P > 0.05), but it showed a significantcorrelation with recent otolith growth (Table 3).

RNA content of sardine larvae was significantlyhigher in winter than in spring 1997 (ANCOVA,F =644.72, P < 0.0001). In contrast, spring-spawnedlarvae had higher DNA content than winter-spawnedlarvae (ANCOVA, F = 314.35, P > 0.0001). TheRNA/DNA ratio of sardine larvae was higher in win-ter 1997 than in spring 1997 (ANCOVA,F = 71.93,P < 0.0001) (Table 2) (Fig. 2).

5

4

3

2

1

0

RN

A/D

NA

0 5 10 15 20 25

Standard length (mm)

Winter 1997Spring 1997

Fig. 2. Relationship between RNA/DNA ratio and standard lengthof sardine larvae in winter and spring 1997.

3.3.2. Winter–spring 1998Standard length at age data for winter and

spring-spawned larvae were fitted to the growth mod-els (SL= 4.2+ 1.061 age0.761, r2 = 0.86,P < 0.001and SL= 4.2+1.491·age0.689, r2 = 0.71,P < 0.001,respectively). Otolith radius was related to standardlength, both in winter and spring, through the equa-tions: OR = 0.091 SL2.289, r2 = 0.89, P < 0.001and OR = 0.308 SL1.880, r2 = 0.73, P < 0.001,respectively.

ANCOVA showed that standard length, at any givenage, was higher in spring than in winter (ANCOVA,F = 42.32, P < 0.0001) (Fig. 3). During 1998,daily growth rates were higher for spring-caught lar-vae than for winter-caught larvae (ANCOVA,F =87.58,P < 0.0001) (Fig. 4), although in both seasonsthe growth rates decreasing with increasing larval age(r = −0.46, P < 0.001 andr = −0.30, P < 0.001,

Table 3Correlation coefficients (r) of relationships between RNA/DNAand growth rates

Season/year RNA/DNA n

DLI ROG

Winter/1997 0.01 ns 0.29∗∗ 123Spring/1997 0.01 ns 0.33∗∗∗ 116Winter/1998 0.33∗∗∗ −0.21∗∗ 161Spring/1998 0.45∗∗∗ −0.15 ns 105

DLI: daily length increment (mm per day); ROG: recent otolithgrowth (�m); ns: not significant.

∗∗ P < 0.01.∗∗∗ P < 0.001.

62 T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65

35

30

25

20

15

10

5

0

Sta

nd

ard

len

gth

(m

m)

0 20 40 60 80

Increment number

Winter 1998Spring 1998

Fig. 3. Standard length at age of sardine larvae in winter andspring 1998.

respectively). On average, otolith radius at age washigher in spring than in winter-spawned larvae (AN-COVA, F = 113.02, P < 0.0001). Moreover, the in-crement widths at age were wider in spring 1998 thanin winter 1998 (ANCOVA,F = 77.73, P < 0.001)(Fig. 5). In agreement with these results, the six lastincrements were on average wider in larvae caughtin spring compared to those collected in winter (AN-COVA, F = 67.39, P < 0.0001), indicating that re-cent otolith growth in 1998 was faster in spring thanin winter.

In winter 1998, the RNA/DNA ratio was positivelyrelated with somatic growth, while it was negativelyrelated with recent otolith growth (Table 3). In spring1998, the ratio was correlated to somatic growth, al-though the correlation coefficient was low (Table 3),but the RNA/DNA ratio was not related with recent

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

mm

/day

0 20 40 60 80

Increment number

Winter 98Spring 98

Fig. 4. Relationship between daily growth rates (mm per day) anddaily increments of sardine larvae in winter and spring 1998.

0

1

2

3

4

5

6

0 10 20 30 40 50 60 70Increment number

Ave

rag

ein

crem

ent

wid

th(

m)

Winter 1998

Spring 1998

m

Fig. 5. Average otolith increment widths at age of sardine larvaein winter and spring 1998.

Fig. 6. Relationship between RNA/DNA ratio and standard lengthof sardine larvae, in winter and spring 1998.

otolith growth. Sardine larvae caught in the springof 1998 had higher RNA (ANCOVA,F = 35.03,P < 0.0001) and DNA (ANCOVA, F = 76.75,P < 0.0001) than larvae caught in winter 1998. How-ever, the RNA/DNA ratio was significantly higher inwinter-spawned larvae than in spring-spawned larvae(ANCOVA, F = 34.15, P < 0.0001) (Fig. 6).

4. Discussion

Temperature and food availability are the mainfactors affecting growth and RNA/DNA ratios of fishlarvae at the sea (Buckley, 1984; Heath, 1992; Ferronand Leggett, 1994). Therefore, changes in these envi-ronmental factors should be reflected in sardine larvalgrowth and RNA/DNA ratios. The lower salinity in

T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65 63

spring 1997 compared to winter 1997 indicates that inspring there was a marked influence of Atlantic wa-ters in the area under study. In the northern sector ofthe Alboran Sea environmental conditions are greatlydetermined by the influence of Atlantic waters, whichenter the Alboran Sea through the Gibraltar Strait. At-lantic waters occupy the upper layers of the AlboranSea (Lafuente et al., 1998), and they are characterisedby relatively low salinities and low nutrient concen-trations (Minas et al., 1991). In addition, low primaryproduction in the Alboran Sea has been associated toAtlantic water masses (Minas et al., 1991). Thus, thelow concentration of chlorophyll-a and zooplanktonbiomass observed in spring 1997 was probably dueto the intrusion of nutrient-poor Atlantic water in theregion.

The RNA/DNA ratio has been used as a reliableindex of larval condition (Buckley, 1984; Ferronand Leggett, 1994; Bergeron, 1997). In 1997, theRNA/DNA ratio was higher in winter-spawned larvaethan in spring-spawned larvae, suggesting that nutri-tional condition was better in winter than in spring.This result is in agreement with the higher micro-zooplankton biomass detected in winter compared tospring, indicating higher food availability in winter.

Several authors have found a negative relationshipbetween RNA/DNA ratio and temperature (Buckley,1982; Goolish et al., 1984; Ferguson and Danzmann,1990). These studies showed that fish acclimated tocold waters had higher RNA content and RNA/DNAratio than warm acclimated ones.Goolish et al. (1984)reported that the increase in the RNA/DNA ratio atlower temperatures is due to a compensatory mech-anism for lower RNA activity, which produces anincrease in the RNA concentration. It has been sug-gested that a temperature difference of around 2◦Cis necessary to produce a significant effect on theRNA/DNA ratio (Buckley et al., 1999). In the presentstudy, the temperature difference between winter1997 and spring 1997 was 1.6◦C (on average), whichis slightly lower than the temperature suggested byBuckley et al. (1999). Therefore, in 1997, the effect oftemperature could have shown some influence on theseasonal difference in the RNA/DNA ratio. However,the higher food resources in winter 1997 were likelythe main factor affecting the RNA/DNA ratio, lead-ing to an increase of the RNA/DNA ratio of sardinelarvae in winter compared to spring.

In 1997, no differences were found in larval growthbetween winter and spring. The results showed thatsomatic growth rates and recent otolith growth rateswere similar in both seasons. Thus, the seasonal dif-ferences observed in RNA/DNA ratios in 1997 werenot reflected in larval growth rates. This was consis-tent with the weak correlation observed between theRNA/DNA ratio and recent otolith growth and withthe lack of correlation between RNA/DNA ratiosand somatic larval growth. This discrepancy betweenlarval growth and RNA/DNA ratios could be due tothe different speed with which these two parametersrespond to changes in the environment. According toFerron and Leggett (1994)changes in temperature orfood availability are reflected first on the RNA/DNA,but their effects on growth rates lag behind. Labo-ratory studies have reported that the standard lengthof starved larvae declined significantly only afterseveral days following food deprivation, indicatingthat starved larvae continue growing for some timealthough at slower rates (Theilacker and Porter, 1995;Suneetha et al., 1999; Folkvord et al., 2000). In ad-dition, recent otolith growth is suitable to detect in-creases in food and growth conditions, but it could bea less good indicator for deteriorating feeding condi-tions (Folkvord et al., 2000). In contrast, the responseof nutritional condition to deteriorating feeding condi-tions is fast (Buckley, 1984; Ferron and Leggett, 1994),and the RNA/DNA declines rapidly under starvationperiods (Clemmesen, 1994; Folkvord et al., 1996;Westerman and Holt, 1994; Suneetha et al., 1999). Allthese findings suggested that, due to the more con-servative nature of otolith growth, the effect of rapidchanges in environmental conditions may be delayedor even not detected neither in somatic growth orrecent otolith growth, while these changes would bereflected in the RNA/DNA ratio.

Relatively high concentrations of chlorophyll-a andzooplankton biomass are usually found in the area ofstudy during spring (Camiñas et al., 1998). However,in spring 1997 both chlorophyll-a concentration andzooplankton abundance were low for this time of theyear, indicating that environmental conditions wereunusual. These conditions were probably caused byan intrusion of surface Atlantic water depleted innutrients, leading to a decline in productivity. Theincursion of Atlantic water could have occurred somedays prior sampling. This hypothesis is consistent

64 T. Ram´ırez et al. / Fisheries Research 68 (2004) 57–65

with fluctuations in the direction and velocity of theAtlantic Jet, which can happen in a small time period(several days), affecting oceanographic conditionsalong the Spanish coast (Cheney and Doblar, 1982;Sarhan et al., 2000; Reul, 2000). Thus, a decline infood availability during the days previous to samplingcould cause lower RNA/DNA ratio of the sardinelarvae collected in spring 1997. In contrast, somaticgrowth and recent otolith growth could be less re-sponsive or less sensitive to deteriorating feedingconditions than the RNA/DNA ratio.

In 1998, no differences in salinity were found be-tween seasons. However, higher temperatures andmicrozooplankton biomass were found in spring 1998than in winter 1998. In agreement with these results,somatic and otolith growth rates of sardine larvaewere higher in spring 1998 than in winter 1988.However, in 1998 the RNA/DNA ratio was lower inspring-spawned larvae than in winter-spawned larvae.A lower RNA/DNA ratio in spring 1998 compared towinter 1998 does not necessarily reflect a poor nu-tritional condition of spring-spawned sardine larvae.As discussed above, the RNA/DNA ratio is affectednot only by food availability but also by temperature,therefore, the temperature effect must be considered.In spring 1998, the water column was characterisedby thermal stratification. The average seawater tem-perature in spring 1998 was 17.6◦C. In contrast, inwinter 1998 the average temperature was 15.1◦C.The temperature difference between seasons was2.5◦C, which is higher than temperature suggestedby Buckley et al. (1999)for causing a significanteffect on the RNA/DNA ratio. Therefore, the highertemperature observed in spring 1998 could lead to adecline of the RNA/DNA ratios during this seasoncompared to winter 1998 (Buckley et al., 1999). Ap-parently, the higher food availability found in springdid not compensate the effect of temperature on theRNA/DNA ratio.

Because the RNA/DNA ratio is temperature depen-dent, care must be taken when nutritional condition isassessed in field-caught larvae, since the negative re-lationship between RNA/DNA and temperature maylead to biased estimates of larvae under starvation. Inthe present study, the lower RNA/DNA ratios foundin the spring of 1998 compared to the winter of 1998were probably due to the high temperatures observedin spring.

Temperature and food availability are the primaryfactors affecting larval growth at the sea (Buckley,1984; Heath, 1992). However, in the field it is diffi-cult to assess the influence of these to variables onlarval growth, since the temperature effect on growthrates may be cancelled by the effect of prey densityand vice versa (Heath, 1992). Some authors have sug-gested that the use of nutritional condition indices suchas RNA/DNA ratio in addition to larval growth maybe valuable to assess the effect of environmental fac-tors on larval growth and survival (Clemmesen andDoan, 1996; Folkvord et al., 2000). However, somaticand otolith growth may be more conservative thanthe RNA/DNA ratio to changing environmental condi-tions (Folkvord et al., 2000) which would complicatethe interpretation of results obtained from the field.In addition, increasing temperature has usually a pos-itive effect on larval growth (Buckley, 1984), while ithas a negative effect on the RNA/DNA ratio (Buckley,1982; Goolish et al., 1984). Therefore, in those worksstudying larval growth and RNA/DNA ratios simulta-neously, the effect of temperature on these parametersshould be considered cautiously.

Acknowledgements

This work was supported by the EU-FAIR Pro-gramme (PARS—Project FAIR-CT 96-1371) and theSpanish Institute of Oceanography (IEO). We thankthe IEO Project ECOMALAGA for field work facili-ties. We also thank to Soluna Sallés by her help withzooplankton samples.

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