Nutritional Condition of Fish Larvae in South African Estuaries As a Proxy to Assess Their Mortality Under Different Environmental Conditions

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Nutritional condition of fish larvae in South Africanestuaries: an appraisal of three biochemical methodsD Costalagoa, N Strydoma & C Frostb

a Department of Zoology, Nelson Mandela Metropolitan University, Port Elizabeth, SouthAfricab Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University,Port Elizabeth, South AfricaPublished online: 30 Oct 2014.

To cite this article: D Costalago, N Strydom & C Frost (2014) Nutritional condition of fish larvae in South Africanestuaries: an appraisal of three biochemical methods, African Journal of Marine Science, 36:3, 377-386, DOI:10.2989/1814232X.2014.957349

To link to this article: http://dx.doi.org/10.2989/1814232X.2014.957349

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Estuaries are highly productive aquatic ecosystems (Day 1980) and, given their direct connection with both rivers and the ocean, are exposed to a very high level of environ-mental variability (Day and Grindley 1981; Kennish 1986). This variability is a key factor in the structure and functioning of estuarine food webs. Changes in dissolved oxygen and turbidity, and especially in temperature and salinity, place considerable physiological demands on estuarine fishes (Harrison and Whitfield 2006; Wasserman and Strydom 2011).

In South Africa, estuarine systems are exposed to a number of both global and regional environmental pressures and threats (Kennish 2002; Turpie et al. 2004), resulting in many South African estuarine systems becoming function-ally and structurally altered (Whitfield 1992). The extent to which fish are affected by these alterations is still to be fully understood, despite studies on the ecological role of fish assemblages in South African estuaries (see Harrison and Whitfield 2012).

The estuarine round herring Gilchristella aestuaria is a clupeoid species that occurs from Namibia, through South Africa, to Mozambique (Smith 1965). It spends its entire life cycle in the upper and middle reaches of permanently open estuaries (Haigh and Whitfield 1993). Breeding takes place throughout the year, peaking in spring and early summer (Cyrus et al. 1993). The species attains maturity generally within the first year, after reaching c. 30 mm standard length (SL) (Talbot 1982). In all estuaries studied, G. aestuaria

feeds predominantly on copepods during all life stages (Whitfield 1980; Blaber et al. 1981; Coetzee 1982; Talbot and Baird 1985; Cyrus et al. 1993; Whitfield and Harrison 1996).

Blaber et al. (1981) found that G. aestuaria in the relatively productive St Lucia Estuary (in northern KwaZulu-Natal) exhibited significant morphological differences compared with individuals from other South African estuaries, having a greater body depth and body length/head length ratio than in other less productive estuaries. The authors suggested that these intraspecific anatomical disparities could be due to differences in the body condition of the fish in relation to the availability or the quality of the food.

Given that these discrepancies are not yet well under-stood, a study of how the trophic dynamics and physio-logical condition of fish vary between different estuaries is required. The generally abundant G. aestuaria, the ecology of which renders it relevant to such a study (Harrison 2005), is an appropriate candidate species for an examination of the ecological links between estuarine fishes and their environment. Some of the most widely used approaches to a quantitative evaluation of how fish are influenced by their environment are based on biochemical methods of assess-ment of the nutritional condition of their larvae (Ferron and Leggett 1994). Whereas morphometric indices take into account only the size and weight of the individuals, and also take a relatively long time to reflect – via the condition status of individuals – the effects of food intake (Catalán et

Nutritional condition of fish larvae in South African estuaries: an appraisal of three biochemical methods

D Costalago1, N Strydom1* and C Frost2

1 Department of Zoology, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa2 Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa* Corresponding author, e-mail: [email protected]

Estuaries are exposed to a number of threats and many South African estuarine systems are functionally and structurally altered. The extent to which fish are affected by these alterations is not fully understood. The estuarine round herring Gilchristella aestuaria is an appropriate species to use when examining such effects. The most widely used approaches to evaluating the nutritional condition of fish larvae are based on biochemical methods. In order to identify the most suitable technique for assessing body condition of fish larvae in estuaries, the nutritional condition of post-flexion larvae of G. aestuaria from the Sundays and Kariega estuaries on the south-east coast of South Africa was evaluated using three different biochemical techniques: (i) total lipid content analysis and the triacylglycerol/cholesterol (TAG/CHOL) ratio; (ii) protein content analysis; and (iii) the RNA/DNA ratio. Results from techniques (ii) and (iii) revealed that G. aestuaria larvae from the Kariega Estuary were in better condition than larvae from the Sundays Estuary, probably due to a recent plankton bloom. It is concluded that the individual RNA/DNA ratio can provide a reliable, sensitive and cost-effective method to assess the immediate effects of environ-mental changes on the nutritional condition of estuarine fish larvae.

Keywords: estuarine ecology, Gilchristella aestuaria larvae, lipid content, protein content, RNA/DNA ratio, round herring

Introduction

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al. 2007), indices based on biochemical components, such as lipids, proteins or nucleic acids, are generally considered a more effective method of evaluating the condition status of fish larvae (Ferron and Leggett 1994; Suthers 1998; Lloret et al. 2005).

Lipids play an essential role during fish larval stages and are used extensively as energy sources during develop-ment. Some studies have demonstrated that, during starva-tion, there is a general and rapid decrease of lipid content in fish larvae, which suggests that lipids can be used as indicators of nutritional condition (see review of Rainuzzo et al. 1997).

The technique of measuring the total lipid content of fish, however, does not discriminate between energy reserve lipids and structural lipids, so results are often difficult to interpret in terms of nutritional condition. The triacylglycerol/cholesterol index (TAG/CHOL) has been the most widely used lipids-based indicator in studies of the nutritional status of early developmental stages of fishes (Fraser 1989; Håkanson 1989, 1993; Suthers 1992; Norton et al. 2001; Costalago et al. 2011; Giraldo et al. 2013).

Fish larvae are known to mobilise protein quickly from body muscle to meet metabolic demands (Pedersen 1997). Protein turnover has been correlated positively with the speed at which fish larvae can respond to environmental shifts (Conceição et al. 2001; Valente et al. 2013), and a reduced protein turnover may lead to reduced plasticity in case of nutritional or environmental challenges (Kiørboe et al. 1987; Conceição et al. 2001). Therefore, if an appropriate proxy for size is used (e.g. SL, as in the present study), protein content should also be a good indicator of condition and growth under environmental fluctuations.

The concentration of DNA is considered constant in somatic tissues, and is directly related to the numbers of cells of an individual. However, the amount of RNA is directly related to the cell’s protein synthesis and is highly dependent on food quantity (Catalán et al. 2007). Thus, RNA/DNA is an index of the cell’s protein-synthetic capacity and has been proven to be a useful indicator of nutritional condition (Buckley 1984; Clemmesen 1994; Ferron and Leggett 1994; Catalán et al. 2007). Moreover, in several fish species, RNA/DNA ratios have been shown to be related to food density and somatic growth (Buckley 1984; Clemmesen 1994; Rooker and Holt 1996).

To determine the most appropriate technique for assessing body condition of fish larvae in estuaries, the nutritional condition of the post-flexion larvae of G. aestuaria was evaluated using three different and commonly used biochemical techniques: (i) total lipid content analysis and the TAG/CHOL ratio; (ii) protein content analysis; and (iii) the RNA/DNA ratio. We also assessed the influence of the presence of the viscera in the analysis of the protein content and the RNA/DNA ratio of fish larvae within each estuary.

The ecological and hydrographical traits of the Kariega and Sundays estuaries, which were selected for this study, have been thoroughly studied: Kariega – Hodgson (1987), Grange and Allanson (1995), Froneman (2000), Sutherland et al. (2013); Sundays – Emmerson (1989), Jerling and Wooldridge (1995), Scharler and Baird (2003); both – Whitfield (1992), Strydom et al. (2002), Wasserman

and Strydom (2011). Typically, the Kariega system has been regarded as the more freshwater-deprived of the two, but the system has been characterised by heavy rains and catchment run-off over the past seven or eight years (Vorwerk et al. 2008a, 2008b).

Larval condition can be interpreted as a proxy for growth rate and, ultimately, recruitment success. The aim of the study was to elucidate, in terms of reliability, sensitivity, and cost-effectiveness, which of the three abovementioned biochemical methods is most appropriate to assess and compare the nutritional condition of G. aestuaria fish larvae in two estuarine systems. Additionally, this study will provide a baseline that will enable a future comparative study on G. aestuaria larval condition in the Kariega Estuary under a freshwater-deprived condition, i.e. when a reversed salinity gradient returns to the estuary, as will expectedly happen at some stage in the future (Grange et al. 2000).

Material and methods

Study areasThe Sundays and Kariega estuaries are located in the Eastern Cape province of South Africa at 33°43ʹ S, 25°51ʹ E and 33°41ʹ S, 26°42ʹ E, respectively (Figure 1). Both systems are temperate-warm, permanently open estuaries (Whitfield 1992). We chose them as contrasting systems – in terms of product ivity and fish abundance (Strydom et al. 2002; Wasserman and Strydom 2011) – in which to evaluate possible differences in the nutritional status of G. aestuaria larvae.

The Sundays system drains a catchment area of 20 063 km2 and has high nutrient levels on account of intense agricul-tural activity along its course (Emmerson 1989) and continuous freshwater inflow, which is a result of an interbasin-water-transfer scheme (Jerling and Wooldridge 1995). The Kariega system is typically more oligotrophic, with a relatively small catchment area (688 km2) and the presence of several impoundments along its course (Hodgson 1987). As a result, zooplankton abundance is generally lower in this estuary than in the Sundays Estuary (Froneman 2000; Grange et al. 2000), although the species composition is very similar in both systems (Froneman 2000; Sutherland et al. 2013).

Field samplingSampling was conducted during three consecutive nights (two in the Sundays and one in the Kariega system) during 21–23 May 2013. Fish larvae were collected after sunset and at a similar time to avoid diel variations in RNA content (Bergeron 1997). Collection was from subsurface waters in the upper reaches of the estuaries by means of two modified WP2 plankton nets (570 mm mouth diameter and 0.2 mm mesh aperture), which were lowered simultaneously from two short (1.5 m) booms that were fixed on either side of the bow of a single-hulled boat. Towing speed was approxi-mately 2 knots (1 m s–1) and tow duration was 5–10 min.

For analysis of proteins and nucleic acids, individual larvae were preserved individually in vials containing RNAlater® (Sigma-Aldrich). For lipid analysis, vials containing approxi-mately 10 larvae each were kept on ice. All samples were frozen at 80 °C in the laboratory.

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African Journal of Marine Science 2014, 36(3): 377–386 379

Lipid extraction and analysisThe wet weight of each larva was measured to the nearest 0.001 mg. Between eight and 10 larvae were pooled together into one sample, with 50 samples per estuary. Lipid extraction followed the method of Folch et al. (1957), modified by Costalago et al. (2011). Briefly, 5 ml of chloroform:methanol (2:1 C:M) was added to each sample in 22 ml glass vials. The samples were vortexed for 1 min and then sonicated for 1 min. Another 5 ml of C:M solution was added, followed by 2.5 ml of 0.88% KCl. The vials were centrifuged at 1 500 rpm for 5 min in an Eppendorf 5804R centrifuge. To extract the chloroform layer containing the lipids, the upper layer was removed and the remaining content of the vials filtered through Whatman paper filters into test tubes. Samples were placed in a Savant SpeedVac SC100 rotor vacuum evaporator until the solvent was totally evaporated. The lipids were then redissolved with C:M and transferred to pre-weighed 2 ml glass vials, the solvent was once again evaporated and the total lipid weighed.

The analysis of lipid classes included the estimation of the TAG/CHOL ratio (Håkanson 1993). Lipid classes were analysed quantitatively using high-performance thin-layer chromatography (HPTLC), following Olsen and Henderson (1989). Lipids standard (TLC Mix 40 by Larodan) was dissolved in C:M to a concentration of 25 mg ml–1. After redissolving the samples in 1 ml of C:M with 0.88% butylated hydroxytoluene, a 10 μl Hamilton syringe was used to load 2 μl each of standard and samples to 1 cm above the edge of silica gel pre-coated HPTLC aluminium plates (Macherey-Nagel) that had been washed previously with hexane:diethyl ether and activated in a ventilated oven at 110 °C for 30 min. Chromatographic tanks were equili-brated with the eluent vapour (hexane:diethyl ether:acetic acid, 80:20:1) before developing the plates. The plates were developed until the solvent front reached approxi-mately 8 cm from the base. Plates were dried and stained

with 3% copper acetate in 8% orthophosphoric acid (Fewster et al. 1969), followed by charring at 160 °C for 20 min.

Quantification of lipid classes was performed by densi -tometry and compared with lipids standard, using a HP Scanjet G3010 scanner linked to the Image-J software with the Densitometry 1 Channel plug-in.

Protein and nucleic acid extractionOnce thawed, the SL of 80 larvae from each estuary was measured to the nearest 0.01 mm with a Mitutoyo digital calliper. The viscera were removed from half of the larvae. Individual samples were freeze-dried and then homogenised using a modified protocol of Caldarone et al. (2001). To each RNAse-DNAse-free vial containing the larvae, 1.50 ml of sarcosil Tris-EDTA buffer at pH 8.0 (containing 0.1% of N-lauroylsarcosine sodium salt in 0.05 M Tris, 0.01 M EDTA, and 0.1 M NaCl) and three glass beads (3 mm diameter) were added. After rehydration of the larvae for 5–6 min, vials were vortexed in a balanced box for 10 min, sonicated for 1 min at 4 °C and then vortexed for another 20–30 min. After total homogenisation, samples were centrifuged at 4 000 rpm in a cooled Eppendorf 5804R centrifuge at 4 °C for 20 min. Two aliquots (600 μl each) obtained from the supernatant were stored separately at 80 °C for protein and nucleic acid analysis, respectively.

Protein content analysisProtein determinations of each sample were completed using a Pierce BCA protein assay kit. Bovine serum albumin was used to generate a calibration curve. Duplicates of 50 μl of samples, standards and 0.01% sarcosil Tris-EDTA buffer (used as a blank), respectively, were pipetted into transparent 96-well plates (Greiner Bio-One). BCA reagent (200 μl) was then added to each well using a multichannel pipette, and the plates were left to incubate for 30 min

AFRICA

SouthAfrica

SOUTHAFRICA

ALGOA BAY

27° E26° E34° S

0 30 60 km

Port Alfred

Kenton on Sea

Sundays RiverKariega

River

Port Elizabeth

Eastern Cape

Figure 1: Map of South Africa showing the location of the Sundays and Kariega estuaries

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at 37 °C. Plates were read with a BioTek PowerWave XS microplate reader at 540 nm, and Gen5 2.0 Data Analysis software was used for the analysis.

Nucleic acid quantificationRNA and DNA quantification was completed using the procedure described by Berdalet et al. (2005). Standards (DNA from herring sperm and RNA from baker’s yeast) and SYBR Green II fluorochrome were obtained from Sigma-Aldrich and Lonza, respectively. Standards were diluted at six different concentrations, ranging from 0.38 μg ml–1 to 6.06 μg ml–1, to obtain the standard curve. Enzymes RNAse and RNAse-free DNAse were acquired from Quiagen.

As above, 50 μl of sample, standard and blank, respect-ively, were pipetted in duplicate into a black 96-well plate. SYBR Green II (50 μl) was added to each well and the fluorescence wavelengths (Ex492nm:Em520nm) were measured. In a second plate, 7.5 μl RNAse per well was added, and in a third plate, 5 μl DNAse per well. These two plates were incubated for 30 min at 37 °C, after which 50 μl of SYBR Green II was added to each well and the fluorescence was measured with a BioTek PowerWave XS microplate reader. Readings were analysed with Gen5 2.0 Data Analysis software.

Statistical analysisAfter testing for normality with the Kolmogorov–Smirnov test, t-tests were performed to evaluate differences in the nutritional condition of the two larval assemblages and, within each population, between the three types of biochemical analysis. Also, t-tests were performed within each estuary in order to assess whether the removal of the viscera affected the results of the protein content analysis and the analysis of the RNA/DNA ratio. All the analyses were performed using the statistical platform R (v. 2.15.3), and the significance level was set at p < 0.05.

Results

Total lipid content and TAG/CHOL ratioThe mean weight of larvae was higher in samples from the Sundays than the Kariega Estuary (93.88 and 66.47 mg, respectively) (Table 1). Total lipid content per larva was also higher in samples from the Sundays Estuary, but not significantly (Table 1). The amount of total lipid per unit weight of sample, however, was significantly higher in the Kariega (16.62 mg lipid g–1 of sample) than in the Sundays Estuary (14.38 mg lipid g–1) (Figure 2a). The TAG/CHOL ratio was statistically higher in the Sundays than in the Kariega Estuary (Table 1; Figure 2b).

Protein content analysisThe average SL of post-flexion larvae from the Sundays Estuary was significantly larger than that from the Kariega Estuary (20.35 mm and 18.99 mm SL, respectively) (Table 2). Higher protein concentrations were found in the larvae that did not have their viscera removed, although the differences were not statistically significant (Table 2, Figure 3a). Protein concentrations averaged 0.17 mg ml–1 (whole)

and 0.14 mg ml–1 (eviscerated) in the Sundays Estuary, and 0.16 mg ml–1 (whole) and 0.13 mg ml–1 (eviscerated) in the Kariega Estuary (Figure 3a) (Table 2).

Overall, the protein concentration in samples from the Sundays Estuary (0.15 mg ml–1) did not differ significantly from that in the Kariega Estuary (0.14 mg ml–1). Length-specific protein content of larvae was slightly higher, albeit not significantly, in the Kariega Estuary (0.76 mg protein mm–1) than in the Sundays Estuary (0.74 mg protein mm–1) (Table 2; Figure 3b).

Sundays Kariega

0.1

0.2

0.3

0.4

0.5

0.6

5

10

15

TOTA

L LI

PID

CO

NTE

NT

(mg

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g1 s

ampl

e)TA

G/C

HO

L R

ATI

O

(a)

(b)

ESTUARY

Figure 2: (a) Total lipid content and (b) TAG/CHOL ratio of G. aestuaria larvae from the Sundays and Kariega estuaries. Error bars denote SE; TAG/CHOL ratios differed significantly between estuaries (p < 0.05)

Table 1: Wet weight and lipid content of G. aestuaria larvae for the Sundays and Kariega estuaries. Standard deviations are shown in parentheses; n 50 in both estuaries, except for TAG/CHOL

Estuary Larva weight (mg)

Lipid content per larva (mg) TAG/CHOL

Sundays 93.88 (60.113)

1.25 (0.45)

0.55 (0.26); n 40

Kariega 66.47 (13.75)

1.10 (0.33)

0.33 (0.20); n 47

Signifi cance * **p < 0.05

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Nucleic acid quantificationIn samples from the Sundays Estuary, the RNA/DNA ratio was significantly higher in eviscerated larvae (1.37) than in whole larvae (0.95; Table 2, Figure 4a). In the Kariega Estuary, the ratio was higher in whole larvae (1.61) than in eviscerated larvae (1.11), although the difference was not statistically significant. In both estuaries, the concentration of DNA was higher in eviscerated larvae, but the differ-ences were not significantly different (Table 2). Similarly, the concentration of RNA was higher in eviscerated larvae in both estuaries, but in this case the difference was signifi cant in the Sundays Estuary (Table 2).

Overall, the RNA/DNA ratio was higher (but not signifi-cantly so) in the Kariega larvae than in the Sundays larvae (1.41 and 1.15 respectively; Table 2, Figure 4b). The concentrations of both DNA and RNA were significantly higher in the Sundays (5.08 and 5.80 μg ml–1 respectively) than in the Kariega Estuary (2.32 and 1.65 μg ml–1 respect-ively) (Table 2).

Discussion

The nutritional condition of a fish larva is an expression of both the biotic and the abiotic parameters that impinge on the individual. While morphometric indices take into account only the size and weight of the individuals, and also take a relatively long time to show the effects of food intake in the condition of individuals (Catalán et al. 2007), indices based on biochemical components such as lipids, proteins or nucleic acids are generally considered a more effective method of evaluating condition (Ferron and Leggett 1994; Suthers 1998; Lloret et al. 2005). Biochemical techniques are also widely used because they are relatively quick to perform and are considered more sensitive to variations in critical environmental factors, such as food availability or temperature (Ferron and Leggett 1994; Catalán et al. 2007).

The presence of the viscera did not have a significant influence on the protein content of G. aestuaria larvae, nor, in the Kariega Estuary, on the RNA/DNA ratio, indicating

Estuary Sample type

Standard length (mm)

[Protein] (mg ml–1)

Protein content per larva

(mg proteins mm–1 SL larva)

[RNA] (μg ml–1)

[DNA] (μg ml–1)

RNA/DNA mDNA/

mRNA

Sep. Comb. Sep. Comb. Sep. Comb. Sep. Comb. Sep. Comb. Sep. Comb.

Sundays (n 76)

Whole (n 37)

20.75 (0.95) 20.35

(1.14)

0.1669 (0.0858) 0.1510

(0.0838)

0.80 (0.40) 0.74

(0.40)

4.31 (1.38) 5.80

(10.37)

4.86 (2.10) 5.08

(2.21)

0.95 (0.26) 1.15

(0.40)

0.8269

Eviscerated (n 39)

19.94 (1.17)

0.1367 (0.0790)

0.67(0.39)

7.37 (3.82)

5.31 (2.34)

1.37 (0.42) 1.6194

Kariega (n 64)

Whole(n 39)

18.91 (1.41) 18.99

(1.58)

0.1579 (0.0745) 0.1439

(0.0716)

0.82 (0.48) 0.76

(0.30)

1.42 (0.96) 1.65

(1.17)

1.75(2.18) 2.32

(2.45)

1.61 (1.67) 1.41

(1.53)

1.2264

Eviscerated (n 25)

18.87 (1.75)

0.1272 (0.0667)

0.68 (0.39)

2.02 (1.40)

2.33 (1.12)

1.11 (1.26) 3.8308

Signifi-cance *

* Sundays

only* *

* Sundays

only*p < 0.05

Table 2: Mean (standard deviation) (separate [Sep.] and combined [Comb.]) of the standard length and of the protein and nucleic acid content of G. aestuaria larvae, whole and eviscerated, from the Sundays and Kariega estuaries. Square brackets indicate concentration. The ratios of the slopes mDNA/mRNA (slope of DNA standard curve/slope of RNA standard curve) are presented for ease of comparison between this and other studies

WholeEviscerated

Sundays Kariega

0.2

0.4

0.6

0.8

Sundays Kariega

PR

OTE

IN C

ON

TEN

T (m

g pr

otei

n m

m la

rva

SL)

0.04

0.08

PR

OTE

IN C

ON

CE

NTR

ATI

ON

(mg

ml

)

(a) (b)

ESTUARY ESTUARY

Figure 3: (a) Total protein content of G. aestuaria larvae, whole and eviscerated, from the Sundays and Kariega estuaries, and (b) length-specific protein content. Error bars denote SE

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that the prey signal did not have a significant effect. This may be a result of observed low stomach fullness (not quantified in our study), which was probably because the time of sampling (during the night) was not coincident with the time of peak feeding of G. aestuaria, which is in the afternoon (Blaber 1979; Froneman and Vorwerk 2003).

Although Olivar et al. (2009) maintained that the removal of the viscera from larvae might have an important effect on the individual RNA/DNA ratio because of the relatively high RNA concentration in the liver, Mustafa and Mittal (1982) showed that the quantity of RNA in the liver, unlike in other tissues such as the brain, exhibited high fluctua-tions following changes in food availability. Hypothetically, larvae from the Sundays Estuary, if in poor condition, may have had a relatively low quantity of RNA in the liver, and hence the removal of the viscera wouldn’t necessarily have lowered the RNA/DNA ratio of individuals. In fact, the ratio was higher in eviscerated larvae than in whole larvae. The significant difference in the ratios in the Sundays Estuary, and the absence of a difference in the Kariega Estuary, may reflect diel variations in RNA content (Bergeron 1997). Potentially, if the larvae happened to have eaten at slightly different times in the two estuaries, the contrasting results between larvae from the two estuaries may reflect some influence of prey in the stomachs of the larvae from the Sundays Estuary. Irrespective of the underlying cause, however, the significant difference found in the Sundays larvae using the nucleic acids method, which contrasts with the non-significant difference found using the protein content analysis, suggests that the nucleic acids method is more sensitive than the protein method, which concurs with the findings of other studies (Ferron and Leggett 1994; Catalán et al. 2007).

Other than the TAG/CHOL index, results from this study have indicated that G. aestuaria larvae from the Kariega Estuary are in better nutritional condition than those from the Sundays Estuary. This might be regarded as unexpected, given that the Sundays Estuary generally is more productive than the Kariega Estuary (Wasserman and Strydom 2011). Several explanations for this, based

on previous studies, are possible. The catchment of the Sundays Estuary is used extensively for agriculture, and the supply of fresh water to estuaries in agricultural areas can enhance the potential for eutrophication (Scharler and Baird 2005). If this is the case for the Sundays Estuary, the food available to G. aestuaria larvae could be below the levels expected in a freshwater-rich estuary. In addition, the resumption, after a flood event, of freshwater inflow into the estuary of a river that is usually subject to excessive water abstraction may result in enhanced productivity (Grange et al. 2000; Vorwerk et al. 2008a). During our study, sampling was carried out after several days of heavy rains, which were especially strong around the Kariega River catchment area (Figure 5). Thus it is likely that a bloom occurred in this estuary, resulting in increased concentration of nutrients, to which the plankton would be expected to react rapidly (Strydom and Whitfield 2000).

Other possible ecological explanations for the different nutritional conditions of fish larvae in the two estuaries under study might be different predation pressures (Harrison and Whitfield 2012) and the influence of the average maternal age (Berkeley et al. 2004). With regard to predation pressure, and given the different hydrological characteristics of these two estuaries, the cues that attract marine fish into the estuaries will be different, and hence the numbers and types of predators entering the estuaries may also be different (Harrison and Whitfield 2012). With regard to maternal age, the average age of the spawning adults in a particular estuary might depend upon several ecological and abiotic parameters (Berkeley et al. 2004). These authors found that larvae from older females had faster growth rates and higher rates of survival after starvation. These aspects, however, require further research in southern African estuaries.

Håkanson (1993) determined that TAG/CHOL values below 0.2–0.3 indicated poor nutritional condition in larval anchovy Engraulis mordax. In the current study, the TAG/CHOL ratios in the larvae from both estuaries fell above this range, suggesting an adequate nutritional condition. The fact that the TAG/CHOL index indicated better larval condition in the Sundays Estuary, whereas the other

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Figure 4: (a) Mean RNA/DNA ratios in whole and eviscerated G. aestuaria larvae, respectively, and (b) in all larvae analysed, from the Sundays and Kariega estuaries. Error bars denote SE; ratios differed significantly between ‘whole’ and ‘eviscerated’ in the Sundays Estuary (p < 0.05)

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Figure 5: (a) Rainfall for May 2013 in South Africa, expressed as a percentage of normal rainfall for the month of May during the period 1971–2000, and (b) the total rainfall (in mm) during that month. S and K indicate the locations of the Sundays and Kariega estuaries, respectively. Maps were obtained, and modified, from the South African Weather Service

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nutritional indices suggested the reverse, is unexplained. The TAG/CHOL index is known to be influenced heavily by the quality of food, rather than quantity (Ferron and Leggett 1994). In the Sundays Estuary, the copepod Pseudodiaptomus hessei provides more than 50% of the food for larvae and juveniles of G. aestuaria (Blaber 1979; Whitfield and Harrison 1996; Sutherland et al. 2013). In the Kariega system, however, individuals between 18 and 25 mm SL have been shown to have a more diverse diet (Froneman and Vorwerk 2003). In addition, G. aestuaria has been described as an opportunistic feeder, depending on prey availability (Blaber 1979; Coetzee 1982; Bennett 1989; Cyrus et al. 1993). Ostracods (Bennett and Branch 1990), macruran and molluscan larvae (Whitfield 1988), chironomid larvae (Coetzee 1982; Sutherland et al. 2013), mysids (Blaber 1979) and diatoms (Talbot and Baird 1985) have been reported as prey for G. aestuaria, especially when the density of copepods is low. Some of these zooplankters are commonly found in the Kariega Estuary (Froneman 2000) and some of them have a lower TAG content than copepods (Fraser 1989; Olsen and Henderson 1989; Parrish 1999).

Experimental studies have shown that a diet with a high lipid content might decrease food consumption and thereby reduce growth rate (Abbass 2007). Hence a potentially different diet in each of the two estuaries might explain the TAG/CHOL indices obtained.

Although the TAG/CHOL index represents a compara-tively cost-effective method to assess the nutritional condition of fish larvae, it has an important disadvantage in that variability between individuals is lost through the need to combine small fish larvae to obtain the minimum amount of sample required. Additionally, the method is laborious and time-consuming compared to other biochemical methods. Thus, it is not an ideal method for routine assessments of the nutritional condition of G. aestuaria larvae. Moreover, although markedly quicker than morphometric indices, the time response (i.e. the time lapse between an environmental trigger and its effect on larval condition, as reflected in lipid content) is too long (up to several days) for this method to be used as an immediate indicator of larval response in terms of nutritional condition (Ferron and Legget 1994; Buckley et al. 1999; Catalán et al. 2007).

The nucleic acid and protein methods have been shown here, as well as by Ferron and Legget (1994), to be more consistent and reliable than the lipid method in generating indicators of food availability and growth rate. The methods are also less dependent on the specific composition of the diets. In this study the protein analysis has been shown to be the most cost-effective, but the influence of the viscera on the analysis of protein and nucleic acid content demonstrated that the RNA/DNA method was more sensitive than the protein method. Moreover, RNA/DNA ratios have been related successfully to food density and somatic growth in several fish species (Buckley 1984; Clemmesen 1994; Rooker and Holt 1996).

In conclusion, although the choice between the three methods of assessment of nutritional condition of fish larvae in estuaries depends greatly on the primary aim of the investigation, the RNA/DNA ratio provides a reliable and sensitive method that requires a comparatively small

sample size to assess and compare the immediate effects of environmental change on the nutritional condition of larvae of estuarine fish.

Acknowledgements — We thank Sabina Kaiser-Phillips and all members of the Frost laboratory for their help and assistance during sampling and analyses. DC was supported by postdoctoral fellowships from Nelson Mandela Metropolitan University and from the National Research Foundation of South Africa.

References

Abbass FE. 2007. Effect of dietary oil sources and levels on growth, feed utilization and whole-body chemical composition of common carp, Cyprinus carpio L. fingerlings. Journal of Fisheries and Aquatic Sciences 2: 140–148.

Bennett BA. 1989. A comparison of the fish communities in nearby permanently open, seasonally open and normally closed estuaries in the south-western Cape, South Africa. South African Journal of Marine Science 8: 43–55.

Bennett BA, Branch GM. 1990. Relationships between production and consumption of prey species by resident fish in the Bot, a cool temperate South African estuary. Estuarine, Coastal and Shelf Science 31: 139–155.

Berdalet E, Roldán C, Olivar MP. 2005. Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part B. Quantification in natural samples. Scientia Marina 69: 17–30.

Bergeron JP. 1997. Nucleic acids in ichthyoplankton ecology: a review, with emphasis on recent advances for new perspectives. Journal of Fish Biology 51: 284–302.

Berkeley SA, Chapman C, Sogard SM. 2004. Maternal age as a determinant of larval growth and survival in a marine fish, Sebastes melanops. Ecology 85: 1258–1264.

Blaber SJ. 1979. The biology of filter feeding teleosts in Lake St Lucia, Zululand. Journal of Fish Biology 15: 37–59.

Blaber SJM, Cyrus DP, Whitfield AK. 1981. The influence of zooplankton food resources on the morphology of the estuarine clupeid Gilchristella aestuarius (Gilchrist, 1914). Environmental Biology of Fishes 6: 351–355.

Buckley LJ. 1984. RNA-DNA ratio: an index of larval fish growth in the sea. Marine Biology 80: 291–298.

Buckley L[J], Caldarone E, Ong TL. 1999. RNA–DNA ratio and other nucleic acid-based indicators for growth and condition of marine fishes. In: Zehr JP, Voytek MA (eds), Molecular ecology of aquatic communities. Hydrobiologia 401: 265–277.

Caldarone EM, Wagner M, St Onge-Burns J, Buckley LJ. 2001. Protocol and guide for estimating nucleic acids in larval fish using a fluorescence microplate reader. Northeast Fisheries Science Center Reference Document 01-11. Woods Hole, Massachusetts: National Marine Fisheries Service.

Catalán IA, Berdalet E, Olivar MP, Roldán C. 2007. Response of muscle-based biochemical condition indices to short-term variations in food availability in post-flexion reared sea bass Dicentrar chus labrax (L.) larvae. Journal of Fish Biology 70: 391–405.

Clemmesen C. 1994. The effect of food availability, age or size on the RNA/DNA ratio of individually measured herring larvae: laboratory calibration. Marine Biology 118: 377–382.

Coetzee DJ. 1982. Stomach content analyses of Gilchristella aestuarius and Hepsetia breviceps from the Swartvlei system and Groenvlei, southern Cape. South African Journal of Zoology 17: 59–66.

Conceição LEC, Skjermo J, Skjåk-Bræk G, Verreth JAJ. 2001. Effect of an immunostimulating alginate on protein turnover of turbot (Scophthalmus maximus L.) larvae. Fish Physiology and Biochemistry 24: 207–212.

Costalago D, Tecchio S, Palomera I, Álvarez-Calleja I,

Dow

nloa

ded

by [

Nel

son

Man

dela

Met

ropo

litan

Uni

vers

ity]

at 0

5:23

19

Mar

ch 2

015

African Journal of Marine Science 2014, 36(3): 377–386 385

Ospina-Álvarez A, Raicevich S. 2011. Ecological understanding for fishery management: condition and growth of anchovy late larvae during different seasons in the Northwestern Mediterranean. Estuarine, Coastal and Shelf Science 93: 350–358.

Cyrus DP, Wellmann EC, Martin TJ. 1993. Diet and reproductive activity of the estuarine roundherring Gilchristella aestuaria in Cubhu, a freshwater coastal lake in northern Natal, South Africa. South African Journal of Aquatic Sciences 19: 3–13.

Day JH. 1980. What is an estuary? South African Journal of Science 76: 198–198.

Day JH, Grindley JR. 1981. The estuarine ecosystem and environmental constraints. In: Day JH (ed.), Estuarine ecology with particular reference to southern Africa. Cape Town: AA Balkema. pp 345–372.

Emmerson WD. 1989. The nutrient status of the Sundays River estuary South Africa. Water Research 23: 1059–1067.

Ferron A, Leggett WC. 1994. An appraisal of condition measures for marine fish larvae. Advances in Marine Biology 30: 217–303.

Fewster ME, Burns BJ, Mead JF. 1969. Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent. Journal of Chromatography A 43: 120–126.

Folch J, Lees M, Sloane-Stanley GH. 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226: 497–509.

Fraser A. 1989. Triacylglicerol content as a condition index for fish, bivalve and crustacean larvae. Canadian Journal of Fisheries and Aquatic Sciences 46: 1868–1872.

Froneman PW. 2000. Feeding studies on selected zooplankton in a temperate estuary, South Africa. Estuarine, Coastal and Shelf Science 51: 543–552.

Froneman PW, Vorwerk P. 2003. Predation impact of juvenile Gilchristella aestuaria (Clupeidae) and Atherina breviceps (Atherinidae) on the zooplankton in the temperate Kariega estuary, South Africa. African Journal of Aquatic Science 28: 35–41.

Giraldo C, Mayzaud P, Tavernier E, Irisson JO, Penot F, Becciu J, Chartier A, Boutoute M, Koubbi P. 2013. Lipid components as a measure of nutritional condition in fish larvae (Pleuragramma antarcticum) in East Antarctica. Marine Biology 160: 877–887.

Grange N, Allanson BR. 1995. The influence of freshwater inflow on the nature, amount and distribution of seston in estuaries of the Eastern Cape, South Africa. Estuarine, Coastal and Shelf Science 40: 403–420.

Grange N, Whitfield AK, De Villiers CJ, Allanson BR. 2000. The response of two South African east coast estuaries to altered river flow regimes. Aquatic Conservation of Marine and Freshwater Ecosystems 10: 155–177.

Haigh EH, Whitfield AK. 1993. Larval development of Gilchristella aestuaria (Gilchrist, 1914) (Pisces: Clupeidae) from southern Africa. African Zoology 28: 168–172.

Håkanson JL. 1989. Condition of larval anchovy (Engraulis mordax) in the Southern California Bight, as measured through lipid analysis. Marine Biology 102: 153–159.

Håkanson JL. 1993. Nutritional condition and growth rate of anchovy larvae (Engraulis mordax) in the California Current: two contrasting years. Marine Biology 115: 309–316.

Harrison TD. 2005. Ichthyofauna of South African estuaries in relation to the zoogeography of the region. Smithiana 6: 2–27.

Harrison TD, Whitfield AK. 2006. Temperature and salinity as primary determinants influencing the biogeography of fishes in South African estuaries. Estuarine, Coastal and Shelf Science 66: 335–345.

Harrison TD, Whitfield AK. 2012. Fish trophic structure in estuaries, with particular emphasis on estuarine typology and zoogeography. Journal of Fish Biology 81: 2005–2029.

Hodgson AN. 1987. Distribution and abundance of the macrobenthic

fauna of the Kariega estuary. South African Journal of Zoology 22: 153–162.

Jerling HL, Wooldridge TH. 1995. Feeding of two mysid species on plankton in a temperate South African estuary. Journal of Experimental Marine Biology and Ecology 188: 243–259.

Kennish MJ (ed.). 1986. Ecology of estuaries: physical and chemical aspects. Boca Raton, Florida: ACRC Press.

Kennish MJ. 2002. Environmental threats and environmental future of estuaries. Environmental Conservation 29: 78–107.

Kiørboe T, Munk P, Richardson K. 1987. Respiration and growth of larval herring Clupea harengus: relation between specific dynamic action and growth efficiency. Marine Ecology Progress Series 40: 1–10.

Lloret J, Galzin R, Gil de Sola L, Souplet A, Demestre M. 2005. Habitat related differences in lipid reserves of some exploited fish species in the north-western Mediterranean continental shelf. Journal of Fish Biology 67: 51–65.

Mustafa S, Mittal A. 1982. Protein, RNA and DNA levels in liver and brain of starved catfish Clarias batrachus. Japanese Journal of Ichthyology 28: 396–400.

Norton EC, MacFarlane RB, Mohr MS. 2001. Lipid class dynamics during development in early life stages of shortbelly rockfish and their application to condition assessment. Journal of Fish Biology 58: 1010–1024.

Olivar P, Diaz MV, Chícharo MA. 2009. Tissue effect on RNA: DNA ratios of marine fish larvae. Scientia Marina 73: 171–182.

Olsen RE, Henderson RJ. 1989. The rapid analysis of neutral and polar marine lipids using double-development HPTLC and scanning densitometry. Journal of Experimental Marine Biology and Ecology 129: 189–197.

Parrish CC. 1999. Determination of total lipid, lipid classes, and fatty acids in aquatic samples. In: Arts MT, Wainman BC (eds), Lipids in freshwater ecosystems. New York: Springer. pp 4–20.

Pedersen BH. 1997. The cost of growth in young fish larvae: a review of new hypotheses. Aquaculture 155: 259–269.

Rainuzzo JR, Reitan KI, Olsen Y. 1997. The significance of lipids at early stages of marine fish: a review. Aquaculture 155: 103–115.

Rooker JR, Holt GJ. 1996. Application of RNA: DNA ratios to evaluate the condition and growth of larval and juvenile red drum (Sciaenops ocellatus). Marine and Freshwater Research 47: 283–290.

Scharler UM, Baird D. 2003. The influence of catchment management on salinity, nutrient stochiometry and phytoplankton biomass of Eastern Cape estuaries, South Africa. Estuarine, Coastal and Shelf Science 56: 735–748.

Smith JLB. 1965. The sea fishes of southern Africa. Cape Town: Central News Agency.

Strydom NA, Whitfield AK. 2000. The effects of a single freshwater release into the Kromme Estuary. 4: Larval fish response. Water SA 26: 319–328.

Strydom NA, Whitfield AK, Paterson AW. 2002. Influence of altered freshwater flow regimes on abundance of larval and juvenile Gilchristella aestuaria (Pisces: Clupeidae) in the upper reaches of two South African estuaries. Marine and Freshwater Research 53: 431–438.

Sutherland K, Wooldridge TH, Strydom NA. 2013. Composition, abundance, distribution and seasonality of zooplankton in the Sundays Estuary, South Africa. African Journal of Aquatic Sciences 38: 79–92.

Suthers IM. 1992. The use of condition indices in larval fish. In: Hancock DA (ed.), Larval biology. Australian Society for Fish Biology workshop, 20 August 1991, Hobart. Bureau of Rural Resources Proceedings No. 15. Canberra: Australian Government Publishing Service. p 49.

Suthers IM. 1998. Bigger? Fatter? Or is faster growth better? Considerations on condition in larval and juvenile coral-reef fish. Australian Journal of Ecology 23: 265–273.

Dow

nloa

ded

by [

Nel

son

Man

dela

Met

ropo

litan

Uni

vers

ity]

at 0

5:23

19

Mar

ch 2

015

Costalago, Strydom and Frost386

Talbot MMJ-F. 1982. Aspects of the ecology and biology of Gilchristella aestuarius (G & T) (Pisces: Clupeidae) in the Swartkops estuary. MSc thesis, University of Port Elizabeth, South Africa.

Talbot MF, Baird D. 1985. Feeding of the estuarine round herring Gilchristella aestuarius (Stolephoridae). Journal of Experimental Marine Biology and Ecology 87: 199–214.

Turpie JK, Adams JB, Joubert A, Harrison TD, Colloty BM, Maree RC, Whitfield AK, Wooldridge TH, Lamberth SJ, Taljaard S, Niekerk LV. 2004. Assessment of the conservation priority status of South African estuaries for use in management and water allocation. Water SA 28: 191–206.

Valente LM, Moutou KA, Conceição LE, Engrola S, Fernandes JM, Johnston IA. 2013. What determines growth potential and juvenile quality of farmed fish species? Reviews in Aquaculture 5: 16–193.

Vorwerk PD, Froneman PW, Paterson AW, Strydom NA, Whitfield AK. 2008a. Biological responses to a resumption in river flow in a freshwater-deprived, permanently open Southern African estuary. Water SA 34: 597–604.

Vorwerk PD, Froneman PW, Paterson AW, Whitfield AK. 2008b.

Fish community response to increased river flow in the Kariega Estuary, a fresh-water deprived, permanently open southern African estuary. African Journal of Aquatic Science 33: 189–200.

Wasserman RJ, Strydom NA. 2011. The importance of estuary head waters as nursery areas for young estuary- and marine-spawned fishes in temperate South Africa. Estuarine, Coastal and Shelf Science 94: 56–67.

Whitfield AK. 1980. A quantitative study of the trophic relationships within the fish community of the Mhlanga estuary, South Africa. Estuarine, Coastal and Marine Sciences 10: 417–435.

Whitfield AK. 1988. The fish community of the Swartvlei Estuary and the influence of food availability on resource utilization. Estuaries 11: 160–170.

Whitfield AK. 1992. A characterization of Southern African estuarine systems. South African Journal of Aquatic Sciences 18: 89–103.

Whitfield AK, Harrison TD. 1996. Gilchristella aestuaria (Pisces: Clupeidae) biomass and consumption of zooplankton in the Sundays Estuary. South African Journal of Marine Sciences 17: 49–53.

Manuscript received March 2014, revised July 2014, accepted August 2014

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