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utritional condition of Argentine anchovy Engraulis anchoita larvae inonnection with nursery ground properties
arina V. Diaza,b,∗, Marcelo Pájarob, M. Pilar Olivarc, Patricia Martosb, Gustavo J. Macchia,b
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Rivadavia 1917, C1033AAV Ciudad Autónoma de Buenos Aires, Buenos Aires, ArgentinaInstituto Nacional de Investigación y Desarrollo Pesquero (INIDEP), Paseo Victoria Ocampo Nro. 1, B7602HSA Mar del Plata, ArgentinaInstitut de Ciències del Mar (CSIC), Passeig Marítim 37-49, Barcelona 08003, Spain
r t i c l e i n f o
rticle history:eceived 15 November 2010eceived in revised form 15 February 2011ccepted 28 February 2011
Nutritional condition of anchovy Engraulis anchoita larvae from Northern Argentinean sea was assessedand compared in three areas characterized by dissimilar oceanographic features (estuarine, mixed andshelf waters). Morphometrical, histological and biochemical (RNA/DNA) indexes were used and larvaewere compared through different developmental stages. Multivariate analyses performed on normal-ized morphometric variables showed that larvae from mixed waters where characterized by higher bodydepths; variables that indicate a better nutritional condition. Mean histological condition index obtainedfor larvae at intermediate developmental stages was significantly lower in estuarine waters than thoseestimated with samples from mixed or shelf waters. Anchovy larvae from the mixed water area showed ahigher slope in the log RNA versus log DNA relationship than larvae from the other areas, which indicatesthat the former were in better condition. Also, standardized RNA/DNA index (RNA/DNAs) resulted slightly
better in mixed waters than in shelf area, for larvae at intermediate developmental stages, but due tothe high variability of RNA/DNA ratio, no significant differences were found between areas. Finally, usingliterature equations relating RNA/DNAs, temperature and growth, growth rate and growth performancewere estimated. Mean growth performance did not differ between areas and was temperature indepen-dent. In conclusion, larval condition was relatively high at all stations, but our results suggested that itwas slightly better in frontal areas characterized by water masses with intermediate salinities. We also
chovy
provide evidence that an
. Introduction
The Argentine anchovy Engraulis anchoita Hubbs and Marini935 is, in terms of biomass, the greatest fish resource in the South-est Atlantic Ocean, and plays a key role as a trophic support
or several commercially exploited species such as hake (Hansen,004). It is distributed in a wide latitudinal range (from 23◦ to7◦S). At least two populations of E. anchoita occur separately atpproximately 41◦S: the Northern or Bonaerensis and the Southernr Patagonian stock (Hansen et al., 1984; Sánchez, 1995). Com-ercially, the Northern stock is the most important, with annual
alues of biomass estimated between one and six million metricons (Hansen, 2004). During spring the Northern stock is found
ostly in waters shallower than 50 m, where massive spawningccurs (Pájaro, 1998; Sánchez and Ciechomski, 1995). Anchovy eggs
∗ Corresponding author at: Instituto Nacional de Investigación y Desarrollo Pes-uero (INIDEP), Paseo Victoria Ocampo Nro. 1, B7602HSA Mar del Plata, Argentina.el.: +54 223 4862586x248; fax: +54 223 4861830.
and larvae may be detected all over the year with a main peak ofabundance in spring and a secondary one during autumn (Pájaroet al., 2009). As a consequence of the wide latitudinal spawningrange showed by Argentine anchovy, early developmental stagescould be found in areas with different oceanographic regimens,prey concentrations and likely very close to their tolerance limitsof temperature and salinity. Martos et al. (2005) studied the rela-tionship between physical variables and the abundance of adultanchovies within the main area of distribution of the northern stockduring spring. These authors define seven “hydrographical environ-ments” according to ranges of both temperature and salinity at seasurface.
The Argentine Sea is a large region comprising several areascharacterized by different oceanographic conditions. The studyarea of the present investigation includes two frontal systems, theRío de la Plata estuary and El Rincón estuary (Acha et al., 2004)
(Fig. 1). Mesoscale fronts are important features of the Argentineshelf, creating a diversity of spawning habitats for fish and differentbreeding conditions for eggs and larvae (Sánchez and Ciechomski,1995). High concentrations of ichthyoplankton, zooplankton, andfish have been detected in both estuarine frontal systems. These
controlled. A disruption of water column stratification and mixing
he different hydrographical areas studied: estuarine area (E), mixed waters areaM) and shelf waters area (S). Arrows indicate location of the four transects (1–4)sed to describe vertical temperature and salinity profiles.
ystems are characterized by a high primary productivity, drivenainly by the nutrient input from river discharges and by the high
ertical stability of the water column (Acha et al., 2004; Carretot al., 1986; Vinas et al., 2002). Nutrient enhancement, whichesults in an increased primary and secondary production, is usu-lly observed in frontal zones (Mann and Lazier, 1996). Thus, frontsrovide advantages for various types of organisms, but could be aisadvantageous zone for others due to nutritional stress (Olson,002) or predation (Acha et al., 2004; Bailey and Houde, 1989).
Northern population of E. anchoita is dominant in the ecosystemnd varies radically in annual abundance (Hansen, 2004). Planktiv-rous fishes are a key component of pelagic ecosystems controllinghe food web in various ways: top-down on plankton populations,ottom-up on predators, or both types simultaneously, also termedwasp-waist” control (Bakun, 2006; Cury et al., 2000; Hunt andcKinnell, 2006). If the herein studied ecosystem is under wasp-aist control the collapse of a dominant prey species, such as
. anchoita, could generate drastic changes at higher and lowerrophic levels. Thus, if fisheries remove substantial amounts ofnchovies, the implications for the other species in the ecosystem,ust be carefully considered.Mechanisms of population control have been an important focus
f inquiry in fisheries research. The assessment of what determinesbundance is complex since many factors acts simultaneously. It isnown that populations’ dynamics of planktonic fish are affected byhe influence of environmental conditions. Even though it is underiscussion at which developmental stage recruitment is deter-ined, larval mortality is still considered very important in this
oncern. Factors affecting survival of young fish are diverse. Manyuthors agree that main sources of mortality are predation and star-ation (Bailey and Houde, 1989). In contrast to predation, whichs difficult to measure; starvation mortality can be assessed byhe measurement of the nutritional condition (Ferron and Leggett,994). As review by Leis (2006), larvae are highly aware of theirnvironment, and thus behaviour, along with hydrography, muste considered in order to explain variations in marine fish pop-lation recruitment. Much more needs to be learned about the
ntogeny of behaviour and sensory abilities in clupeiform larvaen order to include this information in recruitment models. In addi-ion, it is probable that behaviour could be highly influenced byhysiological state.
rch 109 (2011) 330–341 331
Different criteria have been developed to assess nutritionalcondition of fish larvae based on the differences that starvationproduces in body form (Ehrlich et al., 1976; Frank and McRuer,1989; Powell and Chester, 1985; Theilacker, 1978), condition factor(Ehrlich et al., 1976), chemical cell constituents (Clemmesen et al.,1997; Håkanson, 1989) and histological integrity (Catalán et al.,2006; McFadzen et al., 1997; O’Connell, 1976; Theilacker, 1978).
Little is known on the influence of oceanographic features ofthe E. anchoita nursery areas on their larval nutritional condition.Ciechomski et al. (1986) studied the weight-length condition fac-tor along development of larvae from Northern stock, howeverrelationship with oceanographic features were not analysed. Otherauthors had studied anchovy condition in an upwelling area offSanta Marta Cape (Southern Brazil) and a tidal mixing frontal areaoff Península Valdés (Argentinean Patagonia) by means RNA/DNA(Clemmesen et al., 1997) and histological methods (Sieg, 1998).They had found better condition indices off Argentinean Patagoniathan off Brazil, but they could not relate this fact with hydro-graphical conditions or prey densities. Later, Diaz et al. (2009)demonstrated that the use of morphometrical variables and weightallow to find differences in growth rate and nutritional conditionamong anchovy larvae collected in areas with different oceano-graphic scenarios of the Argentinean Sea.
The main objective of the present work is to compare nutritionalcondition of E. anchoita larvae from the Northern stock collected inthree areas characterized by dissimilar oceanographic features, bymeans of morphometrical, histological and biochemical (RNA/DNAindex) techniques. This is a first approach to understand nutritionalcondition of the species during larval stage employing such a vastdiversity of methodologies.
2. Materials and methods
2.1. Study area
The study area included coastal and shelf sectors of the BuenosAires province between 34◦S and 41◦S up to a depth of approx-imately 140 m (Fig. 1). Previous studies suggest the presence ofthree productive systems: coastal, shelf, and shelf break or Malv-inas (Carreto et al., 1986) separated by two semi-permanent fronts:coastal and shelf break. The coastal system has depths of less than50 m and a vertically homogeneous water column throughout theyear. Chlorophyll a and nitrate concentrations are generally low.The continental shelf system, under the influence of Subantarcticwaters transported by the Malvinas Current, exhibits seasonal ver-tical stratification and two defined chlorophyll a maxima in springand fall. The Malvinas system is influenced by the shelf break frontand has high phytoplankton densities in summer and fall (Carretoet al., 1995). Coastal water masses show great variability, as theyare modified by the continental discharge of the Río de la Plata, Col-orado, and Negro rivers, and by high salinity waters moving fromthe south (Guerrero and Piola, 1997).
The Río de la Plata estuary is an extensive and shallow coastalplain estuary at 35–36◦S. It receives freshwater from the sec-ond largest South American basin, with a mean discharge of22,000 m3 s−1 (Framinan and Brown, 1996). The system is char-acterized by a strong vertical stratification: freshwater flowsseaward on the surface while denser shelf water intrudes alongthe bottom, taking the shape of a salt wedge. The dynamics of theupper water layer discharging over the continental shelf is mainlydriven by wind stress, while the bottom layer is topographically
of the salt wedge occurs after several hours of strong onshorewinds (Guerrero et al., 1997).
The coastal regime in the region called El Rincón 39◦S–41◦S ischaracterized by vertical homogeneity due to tidal forcing, and a
332 M.V. Diaz et al. / Fisheries Research 109 (2011) 330–341
Table 1General information about samplings and material employed from the different studied areas to determine nutritional condition of Engraulis anchoita by means of morpho-metrics, histology and RNA/DNA index. FCI: Fulton’s Condition Index, PCA: Principal Component Analysis, HCI: Histological Condition Index.
Area Number of stations Number of larvae analysed
FCI PCA HCI RNA/DNA
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Estuarine 19 105Mixed 78 585Shelf 36 284
oastal front separating diluted coastal water, coming from theegro and Colorado rivers (960 m3 s−1 total average discharge), and
helf waters. Salinity horizontal gradient is increased by the pres-nce in the continental shelf of high saline waters originated in Gulfan Matías (a major coastal basin isolated from the shelf by a 60 mill). The El Rincón system comprises a coastal estuarine zone andshallow sea thermal front oriented North-South at the 40–50 m
sobaths, persistent throughout the year. Bathymetry and the meanhelf circulation contribute in maintaining this front (Acha et al.,004; Lucas et al., 2005).
Water temperature (◦C) and salinity at 5 m depth were mea-ured with a CTD in each sampling station and vertical profilescross a series of four transects (indicated in Fig. 1) were depictedn order to describe the oceanography of the studied areas.
.2. Larval source
Larvae were collected from a research survey carried out inorthern Argentinean coasts during October–November 2006,ithin the spawning peak of the E. anchoita Northern stock. Sam-les were taken with a 300 �m mesh Pairovet net from two metersrom bottom to surface. Slow vertical hauls of short duration (ca.min) were conducted in order to minimise larval damage for his-
ological purposes and tissue degradation for RNA/DNA analysis.Sampled larvae for morphometric analysis were fixed in 5%
uffered formalin and those used for histological analysis wererst fixed in 10% buffered formalin (pH 7) and later transferredo ethanol 70%. Larvae that were sorted for RNA/DNA analysis weremmediately stored frozen in liquid nitrogen. In the laboratory,arvae were identified and measured. Larvae employed for bio-hemical purposes were freeze-dried and stored at −80 ◦C untilnalyses were carried out.
Taking into account Martos et al. (2005) classification oforthern Argentinean water masses, we decide to combine hydro-raphical environments in three areas depicted in Fig. 1 as:stuarine area (E), characterized by waters with low salinities<30); Mixed waters area (M) with intermediate salinities and shelfaters area (S) showing high salinity waters (>33.5).
Nutritional condition of anchovy larvae was thus comparedetween these areas by means of morphometrical, histological andiochemical techniques (Table 1).
.3. Morphometrics
Standard length (SL) and total dry weight (W) of each larvaN = 974) were obtained in the laboratory. Measurements were
ig. 2. Engraulis anchoita larva (12 mm SL) showing the morphometrical measurements tepth posterior to cleithrum (BDPC), body depth at the anus (BDA) and eye diameter (ED
68 28 24447 85 21188 82 23
made to the nearest �m using a Wild M5 stereoscope equippedwith an eyepiece graticule. SL was taken from the tip of the snout tothe end of the notochord. Measurements were made a few monthsafter samplings in order to allow larvae to reach a stable shrinkagesize. No corrections of sizes due to shrinkage or handling duringcapture were made, due to the lack on information in this regardsfor E. anchoita. Larvae were rinsed in distilled water during 48 h,dried at 60 ◦C during 24 h and weighed to the nearest �g employ-ing a Sartorius microbalance. Additionally, 5 other morphometriccharacters were measured on each larva (N = 703): head length (HL)from the tip of the snout to the cleithrum, body depth at cleithrum(BDC), body depth posterior to cleithrum (BDPC), body depth at theanus (BDA) and eye diameter (ED) measured as the media betweenthe maximal and minimum diameter (Fig. 2). All comparisons weremade within a restricted body size in order to diminish the effectsof allometric growth of body parts during the anchovy larvae devel-opment (4–12 mm SL).
Condition was estimated employing Fulton’s Condition Index(FCI); calculated employing the following equation:
FCI = W × 100
SL3
This condition index is based on the hypothesis that individualsof higher weight of a certain length are in better condition thanthose of lower weight.
A Principal Component Analysis (PCA) was also used to deter-mine if morphological differences could be detected among larvaefrom the three studied areas. Analyses were performed usingInfostat® as statistic software. In order to remove the effect of size,morphometric variables employed in the PCA were normalizedaccording to Lleonart et al. (2000) and Catalán (2003) to a referencelength of 8 mm SL:
Y∗i = Yi
[X0
Xi
]b
where Xi is the length of the individual i, X0 the reference length(8 mm SL) and b the allometric parameter which relates the variableunder study and the length. Consequently, a particular observeddata (Xi; Yi) is converted into a theoretical (X0; Y∗
i).
2.4. Histology
Larvae selected for histological analysis were dehydrated in anethanol, cleared in xilol and embedded in Paraplast® (N = 195). Sag-ital sections of 4 �m thick were mounted and stained with Harris’s
aken: standard length (SL), head length (HL), body depth at cleithrum (BDC), body).
Table 2Histological criteria used to classify different organ developmental stages for larvalEngraulis anchoita larvae, taken from Sieg (1998). DS: developmental stage.
DS I Yolk sac stage. Yolk mass clearly detectable or remnants anteriorto developing liver.
DS II No yolk detectable, first small swim bladder bulge, straightdigestive tract, ventral jaw with a single layer of chondrocytes.
DS III Swim bladder enlarge with multiple cell layers, pancreas startscranial extension, oesophagus epithelium height increased, gutepithelium still unfolded, ventral jaw possess several layers ofchondrocytes.
DS IV Swim bladder further increased, shows small cavity and firstcranial tubular branch, ventral midgut mucosa folded, dorsal rowstarts folding, hindgut still unfolded.
DS V Swim bladder with different types of cells, ventral and dorsalmidgut mucosa folded, hindgut starts folding.
DS VI Swim bladder either with a very thickened wall or greatlyextended because of first filling, oesophagus cell height further
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increased, now with several cell layers, pancreas extends craniallybeneath the glomerulum and caudally above the anterior part ofthe midgut, hindgut folding complete.
ematoxylin and eosin counterstain (H–E). Several tissues wereiagnosed but only gut, liver and pancreas were considered reliable
ndicators of anchovy larvae nutritional condition (Sieg, 1998) andhus employed to calculate a Histological Condition Index (HCI).he histological conditions were established based on criteria usedy O’Connell (1976) for Engraulis mordax and Sieg (1998) for E.nchoita. Multiple sections were examined to assess the degree oflteration throughout the specimen and each selected tissue vari-ble assigned to one of three grades. Grade 3 indicated healthy,intermediate and 1 a degraded histological condition. The HCI of
ach larva was then characterized as the mean score for all variablesn all tissues. Larvae were coded to exclude personal bias during theistological diagnostics by knowing where the individuals came
rom.Temperature influences larval metabolism, velocity of growth
nd organ development, as well as the relationship between organevelopment and length. Therefore, as there was a difference inean temperature between the three sampling areas, comparisons
hould be made within developmental stages rather than larvalength. We assigned a developmental stage (DS) to each larva hereintudied according to the classification proposed by Sieg (1998), whoefined six developmental stages for E. anchoita larvae as showed
n Table 2.
.5. RNA/DNA index
Analysis of the whole larval RNA and DNA concentrations waserformed by a modification of the protocol published by Caldaronet al. (2001) and Caldarone (2005) for fish larvae. The main modi-cation was related to the use of 1 ml of assay sample instead oficroplate. Larvae were freeze-dried and stored at −80 ◦C until
nalyses were carried out (N = 68). The protocol involves mechan-cal and chemical homogenization of each larva and subsequentuorescence-photometric measurements using ethidium bromideEB) as a specific nucleic acid fluorochrome dye. Fluorescence was
easured on a Bowman spectrofluorometer (excitation: 360 nm,mission: 590 nm). Total nucleic acid concentrations were firstetermined and then samples were incubated with ribonuclease. The fluorescence due to total RNA, mainly ribosomal, was calcu-
ated as the difference between total fluorescence (RNA and DNA)nd the fluorescence measured after ribonuclease treatment, which
s assumed to be due to DNA. Since we followed a standard pro-ocol based on the use of EB and detergent (Belchier et al., 2004;aldarone et al., 2001; Caldarone et al., 2006), which has been pre-iously used in some similar species and stages, we assume thatesidual fluorescence was insignificant and did not add DNase. Esti-
rch 109 (2011) 330–341 333
mates of the DNA and RNA content of each larva were obtainedfrom calibration curves established using standard DNA and RNAin the appropriate range of concentrations. We measured endoge-nous fluorescence (before EB addition) from the first set of samples,which resulted negligible; therefore it was not considered in cal-culations of nucleic acids concentrations. The average ratio of theslopes of DNA and RNA standard curves was 4.48 ± 0.34.
All RNA/DNA values obtained from our analyses were standard-ized (RNA/DNAs) according to the procedure described in Caldaroneet al. (2006) using 2.4 as the reference slope-ratio value. RNA/DNAs
values were used in all the statistical analyses.Growth rate (G) was estimated employing the best-fit meta-
analysis model RNA/DNAs – T – G obtained by Buckley et al. (2008).
G = 0.145 × T + 0.0044 × T × RNA/DNAs − 0.078
Growth performance (Gpf), the quotient of the observed growthrate and the growth rate achieved by larva under optimal feedingand environmental conditions (Gmax), provides an objective mea-sure of larval condition (Buckley et al., 2008). Due to the lack of aGmax model for E. anchoita, larval growth rates were compared to areference growth rate (Gref). Gref was calculated according to Houdeand Zastrow (1993) who published a multi-species model based on80 marine and estuarine species:
Gref = 0.0106 × T − 0.0203
G estimated using this equation is <Gmax, since this relationshipwas established using larvae in different nutritional stages.
2.6. Statistical analysis
Differences among areas were determined through ANOVA fol-lowed by Tukey’s Honestly Significant Difference (HSD) test. Whentest’s assumptions were not satisfied, statistical comparisons weremade using a non-parametric Kruskal–Wallis analysis of variancefollowed by non-parametric comparisons. An ANCOVA was alsoused to test the differences in log RNA by region with log DNA ascovariate.
Sieg (1998) found a correlation between developmental stagesand length of E. anchoita larvae collected in southern Argentineanwaters. Based on Clemmesen et al. (1997) and Sieg (1998) weassign each larva a developmental stage according to their standardlength. Thus three groups were defined: larvae showing standardlength <4 mm were considered as in DS II, larvae within 5–10 mm SLas III-V and larvae >11 mm SL as in DS VI. The comparison of anchovylarvae nutritional condition between the studied areas was thenbased on these larval developmental stages.
3. Results
3.1. Oceanography
Estuarine area was situated in the mouth of the Río de la Platawith low salinities due to the river influence (Fig. 1). Two mixedwaters areas were found; the first one situated in the outer Ríode la Plata and the second one in the El Rincón area (between39◦S and 41◦S), both were characterized by intermediate salinitiesattributable to the two estuarine frontal systems of the area. Finally,shelf waters area, characterized by high salinity waters (>33.5), waslocated between both mixed waters area from 38◦ to 41◦S.
Temperature and salinity horizontal profiles showed the signifi-cant influence of the Río de la Plata fresh water discharge indicatingthe presence of a thermohaline front (Fig. 3). The Río de la Plata’splume was towards the south-east reaching latitude 38 ◦S and agreat extension of the frontal area could be observed (Fig. 4).
334 M.V. Diaz et al. / Fisheries Research 109 (2011) 330–341
tal pro
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A positive correlation was found between HCI and SL of larvaefrom mixed or shelf waters (Pearson’s correlation coefficient: 0.27and 0.23, p < 0.05, respectively).
Table 3Results from the PCA on E. anchoita larvae, the eigenvectors (Evc), eigenvalues, per-cent of the variance explained of the original data set (r2), and correlation (r) of theoriginal variables with the first two principal components, based upon six morpho-metrical variables: head length (HL), body depth at cleithrum (BDC), body depthposterior to cleithrum (BDPC), body depth at the anus (BDA) eye diameter (ED) andtotal weight (W).
Fig. 3. (A and B) Temperature and salinity horizon
Transect 1 was situated across northern mixed waters area,oastal stations of this transect presented homogeneous watersnd outer stations depths >50 m showed stratified waters. Tran-ect 2 showed a strong influence of the Río de la Plata on thehallower stations, which showed high temperatures, low salin-ties and a strong vertical thermohaline stratification. Transect 3nd 4 showed typical high salinities from shelf waters, exceptingome stations with estuarine influence. External stations of tran-ect 3 presented intermediate salinities due to the Río de la Plataow salinity waters input. Transect 4 also showed some stations
ith intermediate salinities, but in this case from El Rincón estu-rine system. Vertical profiles from the latter transect exhibitedslight thermal stratification towards external stations and alsoslight salinity stratification caused for low salinity waters fromegro and Colorado rivers.
Horizontal distribution of larval densities is depicted in Fig. 5.igher densities were observed close to 50 m depth isobath andetween 39◦S and 41◦S in the region called “El Rincón”. No signifi-ant differences were found between mean larval densities fromhe different areas; the highest density was observed in mixedaters area with mean abundance of 5117 larvae per 10 m2, shelfaters and estuarine areas showed lower densities being 2780 and
313 larvae per 10 m2, respectively.
.2. Morphometrics
A negative correlation was found between FCI and SL (Pear-on’s correlation coefficient: −0.60, p < 0.001). Correlation was alsoignificant within the studied areas (p < 0.001) and the coeffi-ients were: −0.66 in estuarine area, −0.60 in mixed waters and0.61 for larvae collected in shelf waters. Due to the existing rela-
ionship between larval length and FCI, larval size distributionsere studied in each area (Fig. 5). Mixed waters area showed
oth the smallest and biggest larvae. Narrower size distributionas found in estuarine area, where well-developed larvae wereot collected.
No differences were found between mean FCI of early developednchovy larvae collected in the different areas. Mean FCI of larvae
t larvae intermediate developmental stages was significantlyigher in estuarine waters than that estimated with samples fromixed waters area (Tukey, p < 0.05) (Fig. 6). Although, neither of
hem showed significant differences with mean FCI calculatedor larvae from shelf waters (Tukey, p > 0.05). Larvae in advanced
files at 5 m depth obtained within the study area.
developmental stages were absent in estuarine waters and nodifferences were found between well-developed larvae frommixed and shelf waters.
Multivariate analyses were performed on normalized variables.The first two principal components of the PCA explained 62% oftotal variance (Table 3). PC1 explained the 42% of total varianceand though all variables were correlated to PC1, body depths (BDA,BDPC and BDC) were most closely related with it. PC2 explained the20% of total variance and was closely related to length variables (HLand ED).
The PCA did not show a clear separation of larvae from any of thestudied areas (Fig. 7A), showing the individual variability of larvalcondition in the wild. However, when observations were groupedby their area of origin (Fig. 7B), larvae from mixed waters wherecharacterized by higher body depths; variables that indicate a bet-ter nutritional condition. Therefore, larvae from estuarine and shelfarea would exhibit slender bodies than larvae from mixed watersarea.
M.V. Diaz et al. / Fisheries Research 109 (2011) 330–341 335
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ig. 4. Temperature and salinity vertical profiles obtained in four transects (1–4).stuarine area, M: mixed waters area, S: shelf waters area.
As showed in Table 4, length provides restricted information
bout larval developmental stage since length ranges overlapetween different developmental stages. Therefore, nutritionalondition was compared within larval developmental stagesFigs. 8 and 9).
able 4ean standard length of each developmental stage (DS) of Engraulis anchoita larvae
xpressed in millimetres (mm), standard deviation (SD), maximal and minimumalues. N: number of larvae.
s indicate sampling stations location and letters above its corresponding area, E:
No differences were found between mean HCI of early devel-oped anchovy larvae collected in the different areas. Mean HCIobtained employing larvae showing intermediate developmentalstages was significantly lower in estuarine waters than those esti-mated with samples from mixed or shelf waters (Kruskal–Wallisanalysis of variance, p < 0.001) (Fig. 8). No differences were foundbetween mean HCI of well-developed larvae from mixed and shelfwaters.
Histological techniques allowed us to compare HCI betweenactual developmental stages (described in Table 2) of anchovy lar-vae as depicted in Fig. 9. No differences were found between meanHCI of larvae characterized as in DS II collected in the different areas.
Larvae of stages III and IV collected in the estuarine area showedsignificantly lower HCI than those from mixed and shelf waters.Developmental stages V and VI were not found in estuarine waters.No differences were found between mean HCI of larvae from mixedand shelf waters.
336 M.V. Diaz et al. / Fisheries Research 109 (2011) 330–341
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ig. 5. Distribution and abundance of Engraulis anchoita larvae collected in eachrea, densities are expressed as larvae per 10 m2. Length frequency distributionlasses for anchovy larvae collected in each area, A: estuarine area (E), B: mixedaters area (M), C: shelf waters area (S).
.4. RNA/DNA index
A negative correlation was found between RNA/DNAs indexnd SL, and a positive correlation between RNA/DNAs index andarval density was observed (Pearson’s correlation coefficients0.44 and 0.31, respectively, p < 0.001). Although mean tempera-
ure and salinity differed significantly between areas, no correlationas found between RNA/DNAs index and temperature or salinity
p > 0.05).Due to the limit of nucleic acids concentrations detection of the
rotocol used, larvae larger than 4 mm were employed to deter-ine RNA/DNAs indices. Therefore, no comparisons were made
or early developed larvae (DS I and II). Larval condition mea-ured in terms of RNA/DNAs index resulted slightly better in mixedaters than in shelf area (Fig. 10, Table 6). Mean RNA/DNAs index
stimated employing larvae collected from estuarine area was
ig. 6. Mean values of Fulton’s Condition Index (FCI) and standard error for Engraulisnchoita larvae collected in each studied area. Different letters indicate significantifferences (p < 0.05) among areas, after one-way ANOVA followed by post-hocukey’s (HSD) test. E: estuarine area, M: mixed waters area, S: shelf area. Romanumbers indicate developmental stages according to Clemmesen et al. (1997) andieg (1998).
Fig. 7. Scatterplot of PC2 on PC1 for Engraulis anchoita larvae from the studied areas.PCA based upon normalized morphometrical variables and total weight (indicatedas -N). (A) Factor scores of the cases after PCA, different colour indicate area of
origin; (B) observations grouped by areas and correlations between the variables.E: estuarine area, M: mixed waters area, S: shelf area, HL: head length, BDC: bodydepth at cleithrum, BDPC: body depth posterior to cleithrum, BDA: body depth atthe anus, ED: eye diameter.
intermediate. Still, larval condition in terms of RNA/DNAs index didnot differ significantly between areas at any developmental stage(Tukey, p > 0.05). No larvae of DS VI were found in estuarine waters.
In order to verify differences in larval condition between areas,an ANCOVA was performed to test for the effect of area on log RNA,with log DNA as covariate. The interaction effect of area-log DNAwas significant (Table 5). These results showed that the increase ofRNA is larger per unit DNA for larvae from mixed waters area than
those caught in shelf or estuarine area (Fig. 11).
Growth rate resulted significantly higher in the estuarine area(Table 6) and was positively correlated to temperature (Pear-son’s correlation coefficients 0.37, p < 0.001). Finally, our estimatedgrowth rate resulted 3 times higher than Gref in all the studied areas,
M.V. Diaz et al. / Fisheries Research 109 (2011) 330–341 337
Fig. 8. Mean values of Histological Condition Index (HCI) and standard error forEngraulis anchoita in each studied area. Different letters indicate significant dif-ferences (p < 0.001) among areas, after non-parametric Kruskal–Wallis analysis ofvariance followed by non-parametric comparisons. E: estuarine area, M: mixedwaters area, S: shelf area. Roman numbers indicate developmental stages accordingto Clemmesen et al. (1997) and Sieg (1998).
Fig. 9. Mean values of Histological Condition Index (HCI) and standard error esti-mated for each developmental stage (DS) of Engraulis anchoita larvae in the studiedareas. E: estuarine area, M: mixed waters area, S: shelf area. Different letters indi-capa
smbo
4
4
att
TAa
tional condition indices (FCI, HCI and RNA/DNA) are correlatedto larval size. Therefore, the influence of size due to allometricgrowth should be eliminated to guaranty that indices variationsare only reflecting nutritional status of larvae. We achieved thisby restricting analysis to a size group, making comparisons within
ate significant differences (p < 0.05) among larvae of the same DS from differentreas, after non-parametric Kruskal–Wallis analysis of variance followed by non-arametric comparisons. Roman numbers indicate developmental stages (II–VI)ccording to Sieg (1998).
ince Gpf mean values were 3.350, 3.354 and 3.086 for estuarine,ixed and shelf waters, respectively; no differences were found
etween areas (Table 6) and no dependence with temperature wasbserved (p > 0.05).
. Discussion
.1. Main findings
Anchovy eggs and larvae were more abundant in mixed watersrea characterized by intermediate salinities as a consequence ofhe influence of the Río de la Plata and El Rincón estuarine sys-ems. Our results are coincident with previous studies carried
able 5NCOVA to test the effect of area on log RNA Engraulis anchoita larvae, with log DNAs covariate. **p < 0.001, ns: not significant differences.
df SS MS F
Area 2 3.9 × 10−3 2 × 10−3 0.07nsLog DNA 1 5.85 5.85 208.97**Interaction 3 6.18 2.06 73.65**
Fig. 10. Mean values of standardized RNA/DNAs index and standard error forEngraulis anchoita in each studied area obtained according to Caldarone et al. (2006).E: estuarine area, M: mixed waters area, S: shelf area. Roman numbers indicatedevelopmental stages according to Clemmesen et al. (1997) and Sieg (1998).
out in the study area by Pájaro et al. (2008). However, due tothe high variability observed in eggs and larvae abundances, nosignificant differences were found along the region. Bakun andParrish (1991) and Sánchez and Ciechomski (1995) gave evidencethat adult anchovies find in Northern Argentinean coasts, speciallyduring spring, appropriate conditions for reproduction related toEkmanıs transport towards the coast, water column stability, andtrophic enrichment which characterize the frontal systems previ-ously described in the study area.
Ciechomski et al. (1986) emphasized the importance of estab-lishing criteria to evaluate nutritional condition of anchovy larvaeas essential to understand the effects of poor feeding in larvalsurvival and its relationship with stock-recruitment processes. Fol-lowing this proposal, the present study analysed several nutritionalcondition indices for E. anchoita larvae from areas with differentoceanographic situations.
The results herein presented showed that the employed nutri-
Fig. 11. Engraulis anchoita. Linear relationship by area of log RNA vs log DNA. E:estuarine area, M: mixed waters area, S: shelf area.
338 M.V. Diaz et al. / Fisheries Research 109 (2011) 330–341
Table 6Mean and standard deviation of the water temperature, nauplii density, anchovy eggs and larvae densities, anchovy larvae length, FCI, HCI and RNA/DNAs ratio values for eachof the areas. E: estuarine area, M: mixed waters area, S: Shelf area. See Fig. 1 for site locations. FCI: Fulton’s Condition Index, HCI: Histological Condition Index, RNA/DNAs:Standardized RNA/DNA index according to Caldarone et al. (2006), G: growth rate, Gref: reference growth rate, Gpf: growth performance.
ean values corresponding to the study area during spring 2003 and 2004.a Unpublished data, M.D. Vinas and R. Di Mauro pers. com.
evelopmental stages and by the normalization of variables prioro PCA.
To evaluate whether anchovy larvae from field are starving orot, results of field collected larvae should be compared to val-es obtained in laboratory experiments, where larval condition isnown. Unfortunately, there is no information on these aspectsor E. anchoita larvae. Within the study area, only a few individ-als showed histological condition close to 1 which indicates annhealthy condition. Our lowest estimated RNA/DNAs values wereetween 0.74 and 2.98, but the vast majority of larvae had val-es between 4 and 5. The critical RNA/DNA values, using Ethidiumromide methodologies, have been found to vary between 1.0 and.3 for some clupeoids (Chícharo, 1997; Clemmesen, 1994; Kimurat al., 2000), depending on several variables as, for example, tem-erature or specimen sizes. Our results showed that only 1.4%
ndividuals had RNA/DNAs index close to critical ratio 1–1.3. Sup-orting our results, García et al. (1998) in wild Engraulis encrasicolusound a mean RNA/DNA ratio of 4.49 (ranging from 1.09 to 7.79) andust 1.1% under 1.2. These high RNA/DNA values may be a result ofredators in the field selectively removing slower growing indi-iduals in a cohort situation that is not encountered in laboratorytudies.
However, due to methodological differences, direct compar-sons cannot be done without knowing the ratios of the slopes of thetandards calibration curves, and temperatures in those previoustudies. Nevertheless, the relatively high RNA/DNAs obtained here,ogether with the good HCI observed in most of the larvae, indicateshat condition values in the majority of the larvae can be qualifieds healthy. This suggests that E. anchoita finds a favourable envi-onment for larval growth and survival within the studied area.owever, it is unlikely to find heavily starved larvae in the field,
ince they probably died much earlier than they do in captivity,ue to the higher predation pressure.
Results of the several analysed condition indices were fairlyoincident, indicating that larvae from the mixed waters area weren better condition than larvae from shelf and estuarine areas,
hich could be due to the effect of different environments onarval condition. Temperature and appropriate food availabilityre the main environmental variables that usually correlate witharval growth rates (Buckley et al., 1999). Temperature rules chem-cal processes and sets the rhythm of metabolic needs, digestiverocesses and growth rate (Fry, 1971). On the other hand, food
vailability is related to the oceanographic processes that produceater enrichment and concentration, and could be reflected inigher nutritional condition indices.
The difference observed between the slopes of log RNA versusog DNA regressions of the three areas showed that the increase in
0.1295 ± 0.038 <0.0013.086 ± 0.978 ns
RNA per unit DNA was higher for larvae from the mixed waters area.A higher rate of increase in RNA per unit DNA indicates a highermetabolic rate, hence higher rates of protein synthesis. It is knownthat appropriate prey availability, in terms of quality and quantity,guaranty a high growth rate and a privileged nutritional conditionof fish larvae (Buckley, 1984; Canino, 1994). Thus, the fact that lar-vae from the three areas were in a good nutritional condition mightbe reflecting that within the study area food does not represent alimiting factor for anchovy larvae growth and survival.
The lack of anchovy larvae in advanced developmental stagesin the estuarine area, together with the low histological conditionof the small larvae collected there, indicated that this area wouldbe less proper for anchovy larvae survival and growth. Supportingthis idea, Diaz (2010) showed that this area was, in comparisonwith the other areas, characterized by lower densities of potentialfeeding items as copepod nauplii (Table 6) in combination withhigher potential predators or competitors of anchovy larvae such ashydromedusae and chaetognaths. Larval fish may be vulnerable toboth predation and competition by the same organism (Chícharo,1998; Purcell, 1989; Purcell and Grover, 1990). Baier and Purcell(1997) concluded that chaetognaths were not important predatorsof fish larvae, but may consume substantial amounts of the copepodpopulations, which are shared by fish larvae. In addition, Purcell andGrover (1990) found severe levels of predation of hydromedusae onherring larvae, and pointed out that predation was a major sourceof larval mortality.
Larval RNA/DNA and food availability are highly correlated in awide variety of species in both laboratory and field studies (Buckley,1980, 1984; Buckley and Lough, 1987; Chícharo et al., 1998a, 1998b;Theilacker et al., 1996). Diaz (2010), while studying densities ofpotential prey and predators of anchovy larvae in the study area,found that densities of nauplii larvae (main food item for anchovylarvae), were higher in mixed waters area and in almost every stud-ied station close or higher than 5 individuals per litre (5000 ind/m3).Zenitani et al. (2007) demonstrated that larvae of Engraulis japon-icus showed higher survival with higher nauplii densities up to anasymptote of approximately 89% when densities reached 5 ind/l.
Growth rate in estuarine area was higher than in mixed and shelfwaters, being 0.54, 0.47 and 0.40, respectively. As expected, growthrate was different between areas and was temperature dependant.On the other hand, no trends in the RNA/DNAs or Gpf with tempera-ture were observed. These results could support the idea expressed
by Buckley et al. (2008) who suggested that the increase in G withtemperature accomplished solely through an increase in activityrather than an increase in the amount of RNA. Supporting this idea,Malzahn et al. (2003) observed in larval corengonid fish that theeffect of rearing temperature had a significant effect on growth but
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less remarkable response in nucleic acids contents. Buckley et al.2008) observed that RNA/DNAs obtained from well-fed larvae didot depend on rearing temperature. Thus, when larvae are not food
imited, direct comparison of the RNA/DNA values could be madeegardless of the environmental temperature.
Finally, Buckley et al. (2006) suggested that the size-dependentNA/DNAmax approaches a RNA/DNAs value of about 4.4 for bothild Atlantic cod and haddock. Our mean RNA/DNAs ranged
etween 4.38 and 4.93 (Table 6) which could support the idea that. anchoita is not food limited in any of the studied areas.
.2. Comparison of the condition methods
Multivariate analysis is one of the best techniques that allowhe study of several variables from individuals as a whole to deter-
ine affinities between those individuals. McGurk (1985) statedhat only PCA satisfies the requirements of the ideal morphometricactor: size-independence; biological meaning and orthogonality.nother advantage of this technique is that it opens the possibil-
ty of assessing wild larvae where nutrition and development isnknown (Cunha et al., 2003). Although the main drawback of thisechnique is the influence of size due to allometric growth, in ordero guaranty that shape variations are only reflecting nutritional sta-us of larvae, in the present study data were normalized accordingo Lleonart et al. (2000) method, and comparisons were restrictedo a small size range. The main advantage of using morphometri-al variables and weight is that they are simple and easy to assess;owever, the individual variability of morphometry in wild larvaeade difficult to establish boundaries between healthy and starved
arvae.Nowadays, histological techniques represent valuable tools to
cquire reliable information on larval nutritional condition. Histo-ogical study can be focused on tissues that better reflect variationsn larval condition of a certain species, or on developmental stageshat are more appropriate for the temporal scale according to thearticular objectives of the study.
One of the main problems of this methodology, apart from theuge amount of time required to be performed, is the low objectiv-
ty since measures are mainly qualitative (McFadzen et al., 1997;’Connell, 1980) and the variability introduced by the scientist’sxperience (Ferron and Leggett, 1994). Recently, digital image anal-sis is becoming popular in nutritional condition studies. Theseools permit accurate quantification (for example, cellular areas orolumes) that allows statistical analysis. However, Catalán (2003)ompared quantitative and qualitative measurements on larvaerom experimental starvation treatments and stated that qualita-ive indices allow a correct classification of the individuals in the
ajority of the cases and in different developmental stages. Theseesults were explained on the basis that qualitative studies take intoonsideration more cellular or tissular attributes, than quantitativenes.
Many studies have shown evidence that the RNA/DNA ratio isne of the best indicators of the nutritional condition and growthf several marine organisms (e.g. Bailey et al., 1995; Clemmesen,994; Folkvord et al., 1996); and to date, it is the most widelysed biochemical index to determine larval condition. RNA con-ents and RNA/DNA index are reliable indicators of limiting factors,uch as food for first feeding larvae. The analytical protocol (mainlyype of fluorochrome and standard used), the temperature experi-nced by the larvae, as well as the particular species, made straightomparisons difficult (Berdalet et al., 2005; Caldarone et al., 2006),
articularly with previous publications in which values of thelopes ratios of the standard calibration curves were not given.
The effect of tissue on RNA/DNA ratios in larval fish had beennalysed by Olivar et al. (2009) who showed that including headissues produce a reduction in RNA/DNA rates. In the present study
rch 109 (2011) 330–341 339
we used the whole larvae, including the guts, in order to offer com-parable data with other studies on clupeiform larvae (Chícharo et al.1998b; Clemmesen et al., 1997; García et al., 1998). The influence ofthe gut contents does not seem important for the index (Caldaroneet al., 2001; Clemmesen, 1996; Olivar et al., 2009). Moreover, ingeneral anchovy larvae had their guts empty as we could directlyobserve under stereoscope or in histological preparations.
No “single index” is the “best index” and usually a combina-tion must be chosen in order to respond a particular question. Inthe absence of background information, histological, nucleic acidsand protein contents seem to offer the greatest overall return toshow differences between larvae of different levels of nutritionalcondition (Ferron and Leggett, 1994).
4.3. Larval nutritional condition and environment
Within the studied region, and by means of different methodolo-gies, larval condition resulted in general terms high at all stations.However, our results suggest that condition was slightly betterin mixed waters area characterized by water masses with inter-mediate salinities due to the influence of frontal systems (Río dela Plata and El Rincón estuaries), which guaranty stability in thewater column, nutrient enrichment and retention of early devel-opmental stages within favourable environment. This area showeda combination of higher concentration of potential larval prey andlower concentrations of potential competitors. In fact, this area alsoshowed higher densities of anchovy eggs and larvae in compar-ison with estuarine and shelf waters areas. The spatio-temporalco-occurrence of anchovy eggs and larvae and high densities ofsmall-sized copepods have been observed previously in the outerRío de la Plata estuary (Marrari et al., 2004; Vinas et al., 2002).Biological production may be intensified in areas with physical dis-continuities, such as fronts and upwelling processes (Mann, 1993),providing abundant prey for fish. Kiørboe (1991) suggested that theproduction of planktivorous fish, as well as the growth and survivalof fish larvae, depend primarily on mesozooplankton productionat spatio-temporal oceanographic discontinuities. In summary, theavailability of adequate food, a moderate competition and pre-dation, the appropriate thermal and saline ranges, the presenceof physical discontinuities, and the existence of retention mech-anisms make the mixed waters area a suitable environment forsuccessful larval fish development. Probably, adult spawning strat-egy favour those individuals that hatched in zones with appropriateenvironmental properties for growth and survival with respect tothose larvae hatched in a less appropriate environment.
It becomes evident that frontal zones play a key role in ecologicalprocesses of the ocean (Acha et al., 2004), allowing an exceptionallylarge primary production (Carreto et al., 1986), offering adequatefeeding and/or reproductive habitats for nektonic species (Vinaset al., 2002) and acting as retention areas for larvae. Neverthe-less, they also might represent a disadvantageous zone because ofabundance enhancement of potential predators for E. anchoita asctenophores and jellyfish (Alvarez Colombo et al., 2003; Mianzánand Guerrero, 2000).
The present results were also supported by previous studies inother clupeiform larvae. In the Norrhwestern Mediterranean sea,García et al. (1998) (employing nucleic acids contents) found bet-ter nutritional condition of Engraulis encrasicolus larvae in the zoneswith higher productivity associated to the presence of a frontal sys-tem. Employing the same methodology, Huang and Chiu (1998)and Chiu and Huang (1999) studied condition of E. japonicus in four
water masses from the northeastern of Taiwan, and found that lar-vae collected within waters from the Kuroshio Current showed apoor condition in comparison with those of the Kuroshio frontalsystem, characterized by higher nutrients and chlorophyll-a lev-els. Additionally, Chícharo (1998) studied Sardina pilchardus larval
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ondition in southern Portugal (Eastern Central Atlantic) and foundhat sardine larvae were in better nutritional condition in the areahat receives the influx of the enriched upwelled waters, whereigher concentrations of planktonic organisms were found.
The results of the present study showed how larvae of mixingrea are in better condition that those in the estuarine or shelf ones,nd suggest that the frontal system, which is part of this mixingrea, must be an important zone for larval development.
In order to elucidate the influence of frontal systems on E.nchoita larvae nutritional condition further studies taking intoccount both biological and oceanographical parameters at smallerpatial scales, and covering the frontal structure are required. Vinasnd Ramírez (1996) provided evidence indicating that transitionalnd stratified sectors of the Península Valdés tidal mixing frontalystem provide better feeding conditions for survival and growth ofnchovy larvae than non-stratified waters. In this regard, it woulde necessary a detailed vertical sampling to obtain material fromhe different water masses that comprise the frontal systems of theío de la Plata and El Rincón estuaries.
cknowledgments
We wish to thank Marcela De Falco for her valuable commentsn the manuscript and Horacio Bogo (INQUIMAE, Buenos Airesniversity) for the loan of the Sartorius electrobalance. Special
hanks to Ernesto Christiansen for his constant help during histo-ogical procedures, to María Delia Vinas, Roxana Di Mauro, Gabrielenzano and Carolina Rodríguez for providing zooplankton abun-ances. We also would like to thank BIP E. Holmberg personnel forheir help during sample collection. This work was supported by theNIDEP and the CONICET. M.V. Diaz acknowledges MAEC-AECI Fel-owship support that allowed transportation to Barcelona in ordero analyse RNA/DNA contents.
This is INIDEP contribution no. 1638.
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