Condition of larval and early juvenile Japanese temperate bass Lateolabrax japonicus related to spatial distribution and feeding in the Chikugo estuarine nursery ground in the Ariake

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Journal of Sea Research

Condition of larval and early juvenile Japanese temperate bass

Lateolabrax japonicus related to spatial distribution and feeding in

the Chikugo estuarine nursery ground in the Ariake Bay, Japan

Md. Shahidul Islam a,*, Manabu Hibino b, Kouji Nakayama a, Masaru Tanaka a

a Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japanb Aichi Fisheries Promotion Fund, Department of Sea-Farming, 1-3 Ichizanmatu Nakayama Atsumi-cho, Atsumi-gun, Aichi 441-3615, Japan

Received 7 January 2005; accepted 26 August 2005

Available online 28 October 2005

Abstract

The present study investigates feeding and condition of larval and juvenile Japanese temperate bass Lateolabrax japonicus in

relation to spatial distribution in the Chikugo estuary (Japan). Larvae were collected in a wide area covering the nursery grounds of the

species in 2002 and 2003. Food habits of the fish were analysed by examining their gut contents. Fish condition was evaluated by

using morphometric (the length-weight relationship and condition factor) and biochemical (the RNA:DNA ratio and other nucleic

acid based parameters) indices and growth rates. The nucleic-acid contents in individually frozen larvae and juveniles were quantified

by standard fluorometric methods. Two distinct feeding patterns, determined by the distribution of prey copepods, were identified.

The first pattern showed dependence on the calanoid copepod Sinocalanus sinensis, which was the single dominant prey in low-saline

upper river areas. The second pattern involved a multi-specific dietary habit mainly dominated by Acartia omorii, Oithona davisae,

and Paracalanus parvus. As in the gut contents analyses, two different sets of values were observed for RNA, DNA, total protein,

growth rates and for all the nucleic acid-based indices: one for the high-saline downstream areas and a second for the low-saline

upstream areas, which was significantly higher than the first. The proportion of starving fish was lower upstream than downstream.

Values of the allometric coefficient (b) and the condition factor (K) obtained from the length-weight relationships increased gradually

from the sea to the upper river. Clearly, fish in the upper river had a better condition than those in the lower estuary. RNA:DNA ratios

correlated positively with temperature and negatively with salinity.We hypothesise that by migration to the better foraging grounds of

the upper estuary (with higher prey biomass, elevated temperature and reduced salinity), the fish reduce early mortality and attain a

better condition. We conclude that utilisation of the copepod S. sinensis in the upstream nursery grounds is one of the key early

survival strategies in Japanese temperate bass in the Chikugo estuary.

D 2005 Elsevier B.V. All rights reserved.

Keywords: RNA:DNA ratio; Nutritional condition; Protein growth rate; Length-weight relationship; Condition factor; Japanese temperate bass;

Sinocalanus sinensis; Ariake Bay

1385-1101/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.seares.2005.08.004

* Corresponding author.

E-mail address: [email protected] (Md.S. Islam).

1. Introduction

A variety of techniques (morphometric, histological

and biochemical) to diagnose the nutritional condition

of fish larvae and juveniles have been developed and

applied to laboratory-reared as well as wild fishes

55 (2006) 141–155

Md.S. Islam et al. / Journal of Sea Research 55 (2006) 141–155142

(Buckley, 1979; Yin and Blaxter, 1986; Clemmesen,

1987, 1988; Bailey et al., 1995; Rooker and Holt,

1996; Canino, 1997; Chicharo, 1997, 1998; Chicharo

et al., 1998a,b). Assessment of the nutritional condi-

tion of field-caught larvae would help explain larval

survival and year-class fluctuations (Richard et al.,

1991). Among the biochemical indices, the ratio of

RNA:DNA has proved to be a useful and reliable

indicator of nutritional condition and growth of larval

and juvenile fishes (Buckley, 1980; Robinson and

Ware, 1988; Buckley et al., 1999). The quantity of

DNA in most animal cells is believed to be stable but

RNA quantity varies with physiological status, the

requirement for protein synthesis, and growth (Buck-

ley et al., 1999). Because mRNA, tRNA and rRNA

are essential for the biosynthesis of protein, the quan-

tity of bulk RNA in a cell varies in response to

changes in demand for protein synthesis, and RNA

quantities are high in rapidly growing organisms (Ber-

geron, 1997; Buckley et al., 1999). Any factor pre-

venting or slowing growth is reflected by a reduction in

RNA quantities. Among such factors, nutritional con-

dition seems to be the most studied and the most widely

used; nutritional condition is associated with food qual-

ity and quantity, and feeding success of the fish. There-

fore, variation in the trophic environment is reflected in

their nutritional condition. Since the larval stage of fish

is characterised by rapid exponential growth (i.e., rapid

protein synthesis), RNA:DNA ratio is a good index of

relative growth rate (Buckley et al., 1999).

While the RNA:DNA ratio allows the recent growth

and condition of individual fish to be assessed, the

length-weight relationship allows the condition of a

fish population to be assessed over a relatively broader

time frame. The length-weight relationship is a useful

morphometric tool in fishery assessment that helps pre-

dict weight from length (Garcia et al., 1998). The length-

weight relationship of a particular species allows the

inter-conversion of these parameters. Furthermore, the

length-weight relationship allows fish condition to be

estimated. The allometric coefficient obtained from the

length-weight equation is a useful estimate of the condi-

tion of a fish population and the condition factor is

frequently used for life-history comparisons between

regions (Weatherley and Gill, 1987; Petrakis and Ster-

giou, 1995).

Japanese temperate bass Lateolabrax japonicus, one

of the most abundant fish species of the Ariake Bay, is

important for commercial fishery and a highly promis-

ing species for sea farming in winter (Matsumiya et al.,

1982). It is also an ecologically important species be-

cause, in the Ariake Bay, it is endemic and regarded as a

dcontinental relict speciesT (because closely related spe-

cies occur in China and the Korean peninsula). It has

nursery grounds in estuarine waters and later migrates

upriver to reach freshwater. The species is suitable for

the study of larval ecophysiology because because of

these significant habitat changes. In the present study,

we describe the spatial patterns in dietary habits of larval

and early juvenile L. japonicus in the estuary and in-

vestigate their growth rates and condition using

RNA:DNA ratio and other nucleic acid-based indices

and the parameters derived from the length-weight rela-

tionships such as the allometric coefficient and condi-

tion factor.

2. Materials and methods

2.1. Study area and sampling

The Ariake Bay, the largest tidal wetland of Japan,

is located in south-western Japan; the Chikugo estuary

is the largest estuary of the Ariake Sea, with the highest

tidal differences in Japan. Seven sampling stations were

set up in the Chikugo estuary (Fig. 1). The sampling

stations are lined along the tideway of the Chikugo

River. Four of the stations (R4-R1) were positioned up

the river. Station R1 is located in the river mouth; R4 is

the uppermost station, 16 km upstream, with little

seawater influence even at spring high tide. The other

three stations (E1-E3) are located on tidal flats in the

estuary. E3 is the most distant station with the highest

salinity.

Larval and juvenile fish were collected at selected

stations during two research cruises in March 2002 and

2003. Samples were taken by surface towing with a

larval ring net (1.3 m mouth diameter; 1 mm mesh size

along the body and 0.33 mm mesh size at the cod end)

for 10 min against the tidal flow. The samples were

sorted and immediately frozen in dry ice on board, then

transported to the laboratory for storage in a deep

freezer at �85 8C. All larvae and juveniles were

counted, total length (TL) was measured to the nearest

0.1 mm with digital slide callipers and wet weight was

determined with a sensitive electronic balance to the

nearest 0.1 mg.

During both cruises, hydrographic data and plankton

samplings were also carried out. Temperature and salin-

ity were recorded at each station by an Environmental

Monitoring System (YSI 650 MDS, YSI Incorporated,

USA). Copepod samples were collected by oblique tow-

ing of a plankton net (45 cm mouth diameter; 0.1 mm

mesh). Copepod samples taken at the sampling stations

were preserved in water-diluted formalin.

Fig. 1. Map of the Ariake Bay and Chikugo River estuary showing the sampling stations.

Md.S. Islam et al. / Journal of Sea Research 55 (2006) 141–155 143

2.2. Analyses of gut contents and copepod populations

A minimum of ten fishes from each station were

randomly selected for gut study. The guts were opened

and food organisms, separated from the oesophagus to

the rectum, were examined under a microscope. Prey

organisms were counted and identified to the lowest

possible taxonomic level. To determine the dry

weights, the gut contents were filtered through pre-

dried and pre-weighed filter papers (Whatman GF/F);

the filter papers were then dried in an oven at 45 8Cfor 24 h. Dry weights of the gut contents were deter-

mined from the difference in weights before and after

filtration and drying.

Copepods were sorted from the suspended particles

and detritus under a binocular stereomicroscope. Qual-

itative and quantitative abundances of copepods were-

determined by identifying and counting the total

number; copepod density was expressed as number

per m3 of water. Copepod dry biomass at each sampling

station was determined by drying samples at 45 8C for

24 h in a thermostat oven and the dry weight was

expressed as mg m�3.

2.3. Determination of RNA, DNA and protein quantity

A total of 135 bass larvae and early juveniles ranging

in length from 14.8 mm to 26.9 mm (mean 20.8F2.3

mm) and weight from 18.1 mg to 180.5 mg (mean

71.0F26.9 mg) were used for nucleic-acid analysis.

Measurements of RNA, DNA and protein contents

were carried out for individually frozen fish samples

and each individual fish was used for RNA, DNA and

protein analyses. Nucleic acids were extracted from the


whole body by homogenizing the sample in ice-cold

Tris-EDTA buffer (0.05M Tris, 0.1M NaCl, 0.01M

EDTA, pH 8.0) using a glass homogenizer and subse-

quently transferred to a mixture of Tris-EDTA buffer,

proteinase-K (pro-K), and sodium dodecyl sulfate

(SDS). The quantity of RNA and DNA in the whole

body was determined by the fluorescence-photometric

technique using a specific nucleic acid fluorescent dye

— ethidium bromide (Nacalai Tesque Co. Ltd, Kyoto,

Japan), as described by Clemmesen (1993) and slightly

modified by Sato et al. (1995). In order to measure the

DNA content of a sample, RNA was enzymatically

digested with RNAase and the remaining DNA was

determined with ethidium bromide. The fluorescence

due to total RNA was calculated as the difference be-

tween total fluorescence (RNA and DNA) and the fluo-

rescence after RNAase treatment, which is assumed to

be due to DNA. Salmon sperm DNA (Wako Pure

Chemical Co. Ltd) and yeast RNA (Kanto Chemical

Co. Ltd) were used as standards. RNA and DNA con-

tents are both expressed as mg fish�1. Total protein

(dissolved in NaOH) was determined by a Bio-Rad

protein kit (Bio-Rad, Tokyo, Japan) using bovine

serum albumin as a standard. Results are expressed as

mg of protein fish�1; the ratio of RNA to protein and

those of protein to DNA content are cited as indices of

protein synthesis capacity and cell size, respectively

(Buckley et al., 1999). The instantaneous protein growth

rate (Gpi) was calculated from the larval growth model

based on the RNA:DNA values and temperature; the

model, proposed by Buckley (1984), was given as

Gpi ¼ 0:93T þ 4:75 RNA:DNAÞ � 18:18ð ð1Þ

where Gpi is the protein growth rate (% d�1) and T is the

water temperature. This equation was manipulated,

according to Robinson and Ware (1988), to calculate

the critical values of RNA:DNA ratio. The critical ratio

is the theoretical ratio where there is no net protein

growth (Gpi=0) in a larval fish at a specified tempera-

ture and can be given by the following equation

R:Dcrit ¼ 18:18� 0:93Tð Þ=4:75 ð2Þ

where R:Dcrit is the critical RNA:DNA ratio and the

other variables are the same as above.

2.4. Length-weight relationships and condition factor

The total length-weight relationship and relative

condition of the fish were calculated as a morphomet-

ric measurement of fish condition. Total length of each

individual fish was measured to the nearest mm and

weight taken to the nearest mg on a sensitive digital

electronic balance. The regression line of length-

weight relationships was drawn by plotting the body

weight data against the total length data. Le Cren’s

widely used formula of W=aLb was used to establish

the relation between the length and the weight, where

W is the weight and L is the length, a is a constant

and b is the allometric factor. The equation W=aLb

produces a curvilinear length-weight relation when

plotted and can also be expressed as logW =a+blogL,

where the length and weight are log transformed and

produces a straight-line relation when plotted; in this

linear equation, daT and dbT are constants estimated by

least square regression. The slope of the regression

line, believed to be an estimate of b (Safran, 1992),

indicates the isometry or allometry of growth and is,

therefore, a useful indicator of the condition of the

fish. The functional regression value b =3 describes

isometric growth and unchanging body form and spe-

cific gravity. The allometry in growth indicates that

the weight of the fish is not in proportion with the

length and is characterised by b values below or

above 3, a lower value indicating negative allometry

and a higher value indicating positive allometry.

The condition factor was used to compare length

and weight; it was calculated separately for individual

fish according to Fulton (1911) as K =W/L3 and mod-

ified by Safran (1992) as K =1000W/L3. The value of

K is believed to be equivalent to the parameter a in

the allometric equation assuming that b =3. The heavi-

er a fish is for a given length, the greater the condition

factor and, by implication, the better the condition.

2.5. Statistical analysis

One-way ANOVA was used separately to examine

the differences between the sampling stations and be-

tween the years. The ANOVAwas followed by a Tukey

test to compare the means and to assign the level of

significance. Effects of fish size (length and weight)

and hydrographical parameters (temperature and salin-

ity) on fish condition parameters were assessed by

simple linear regression analysis. Values were consid-

ered significant at a 5% level of confidence.

3. Results

3.1. Hydrology and fish distribution

Temperatures ranged from 13.5 to 15.0 8C (mean

14.2F0.54 8C) in 2002 and from 15.5 to 15.7 8C(mean 15.6F0.08 C) in 2003 with little spatial variation

Table 1

Hydrographic variables, numbers and corresponding mean lengths and weights of fish collected from seven sampling stations in the Chikugo

estuary during two cruises in 2002 and 2003

Station Temperature (8C) Salinity (PSU) No. of fish collected TL (mm) Weight (mg)

2002 2003 2002 2003 2002 2003 2002 2003 2002 2003

R4 13.5 15.53 0.14 0.12 28 46 24.4F1.2A 23.4F1.7A 118.3F25.3A 112.0F31.6A

R3 14.0 15.54 1.35 1.2 9 22 22.5F1.7AB 23.3F1.6AB 95.01F20.5A 99.3F23.4A

R2 14.0 15.47 20.11 17.43 32 52 19.4F3.4C 21.7F2.0C 61.2F34.6AB 78.0F24.5AB

R1 13.8 15.54 24.23 19.56 14 50 21.3F2.9BC 20.9F1.9BC 74.2F32.4B 71.0F19.1B

E1 14.6 15.67 26.84 22.4 10 27 22.4F3.0AB 20.7F2.1BC 84.0F31.9B 72.6F21.3B

E2 14.7 15.69 26.93 23.2 42 34 19.7F3.6C 21.0F2.5C 64.2F36.1B 74.2F26.4B

E3 15.0 15.54 28.49 27.5 11 25 20.9F2.4BC 20.9F2.4C 64.9F27.8B 71.0F25.8B


(Table 1). Salinity showed a gradual increase from st.

R4 to st. E3 and ranged from 0.13 to 26.2 PSU in

2002 and from 0.15 to 26.7 PSU in 2003 (Table 1).

Temperature showed significant variations between

2002 and 2003, with higher values in 2003, while

salinity did not vary (ANOVA; Pb0.05; Table 2).

Distribution of the fish extended over a wide range

of salinity from about 0 PSU upstream to as high as 28

PSU in the sea. Fish were collected at all seven sta-

tions during both cruises, but there was no clear spatial

pattern (Table 1). A one-way ANOVA showed that

fish abundance was significantly higher in 2003 than

in 2002 (F=5.3; Pb0.05; Table 2). A total of 402 (146

in 2002 and 256 in 2003) juveniles were collected with

a total length ranging from 12.5 to 27.2 (21.1F3.4)

mm in 2002 and from 13.9 to 27.4 (21.8F2.3) mm in

2003 (Table 2) and a body weight from 14.7 to 182.3

(78.2F38.0) mg in 2002 and in 20.0 to 199.2

(84.0F29.8) mg in 2003.

Table 2

Results of one-way analysis of variance (ANOVA) comparing parameters be

Parameters Between stations

2002 2003

MS F P Remark MS F

Temperature

Salinity

TL 18.043 4.878 0.002 s 38.02 1

Weight 2527.90 6.027 0.000 s 5189.7 1

RNA 10.09 51.07 0.000 s 1.78

DNA 0.1011 4.37 0.003 s 0.06

Protein 24.66 8.074 0.000 s 9.73

RNA:DNA 15.54 37.39 0.000 s 1.666

RNA:Protein 0.078 18.17 0.000 s 0.017

Protein:DNA 11.92 3.514 0.010 s 5.635

Gpi 327.16 34.895 0.000 s 36.815

R:Dcrit

Starvation (%)

Fish abundance

3.2. Gut contents and ambient copepod assemblage

A total of 11 food types, mainly copepods, were

recorded and the following prey items were identified:

Sinocalanus sinensis, Acartia omorii, Paracalanus par-

vus, Oithona davisae, Calanus sinicus, Pseudodiapto-

mus marinus, Coryaceous affinis, Harpacticoida sp.,

Copepodite, and decapod mysis (Fig. 2). Nine species

of copepods were recorded; they contributed as much as

97.7% of the total food. The gut contents of the fishes in

low-to-medium saline zones (st. R4-R2) were highly

dominated by a single species of copepod, S. sinensis,

in both years (Fig. 2). In these stations, S. sinensis

constituted nearly 100% of the prey consumption. In

contrast to the mono-specific gut composition in the

low-saline area, a multi-species gut assemblage, domi-

nated by A. omorii, P. parvus and O. davisae, was

observed in the high-saline lower estuary in both years

(Fig. 2).

tween stations and between years (s = significant; ns = not significant)

Between years

P Remark MS F P Remark

6.303 41.725 0.000 s

0.691 0.0057 0.941 ns

2.34 0.000 s 0.588 0.216 0.650 ns

1.83 0.000 s 195.6 0.528 0.482 ns

9.019 0.000 s 2.078 1.963 0.187 ns

4.284 0.002 s 0.002 0.121 0.734 ns

3.716 0.004 s 50.64 19.27 0.001 s

9.425 0.000 s 45.02 26.40 0.000 s

7.247 0.000 s 0.062 7.961 0.015 s

1.057 0.390 ns 14.02 9.995 0.008 s

9.231 0.000 s 153.42 9.085 0.000 s

0.2404 41.618 0.000 s

417.75 2.013 0.206 ns

864.3 5.25 0.041 s

Fig. 2. Gut contents composition (%) of Japanese temperate bass larvae and early juveniles (upper two graphs) and environmental copepod

composition (lower two graphs) along the Chikugo estuary. The low-saline upper estuary was dominated by a single species (Sinocalanus sinensis),

while the high-saline lower estuary had a multi-species assemblage mainly dominated by Acartia omorii and Oithona davisae.


Copepod composition (%) in the water at each

station (Fig. 2) showed that S. sinensis was over-

whelmingly dominant throughout the low-saline

areas, especially in st. R4 and R3 and was the single

dominant copepod species recorded in these two sta-

tions where it constituted 99.6–99.8% in 2002 and

97.7–98.8% in 2003. In contrast to the low-saline

upper estuary, a multi-species assemblage was ob-

served in the highly saline lower estuary (st. R1-E3),

which was dominated by Oithona davisae (50.3–

89.8%) in 2002 and Acartia omorii (39.2–55.5%) in

2003 (Fig. 2). The other species in these regions

included Calanus sinicus, Paracalanus parvus and

Pseudodiaptomus marinus.

Dry weights of gut contents ranged from 0.17 to

0.67 mg per fish; significantly higher gut dry weights


were recorded in st. R4-R2 than in the other stations

(Fig. 3). Copepod densities ranged from 5963 to 34 214

individuals m�3 and showed a general and steady

increase from station R4 seaward. Spatially, st. R4

had significantly lowest and st. E1-E3 the significantly

highest numerical densities (Fig. 3). In contrast, cope-

pod dry biomass, which ranged from 4.89 to 122.2 mg

m�3, was significantly higher in st. R4 and R3 than in

the other stations (Fig. 3).

Fig. 3. Spatial variations in the gut contents dry weight, copepod

density, and copepod dry biomass along the Chikugo estuary. The

letters assigned to each value indicate the significance of difference:

the mean values having different letters were significantly different

from each other. Both the gut contents dry weight and copepod dry

biomass showed their highest values in the upper stations; in contrast,

copepod numerical density increased consistently towards the sea

with the highest values in three lowest stations.

3.3. Biochemical indices

The mean RNA, DNA, protein, RNA:DNA ratio,

RNA:Protein ratio, and Protein:DNA ratio are pre-

sented in Fig. 4 and corresponding mean lengths and

weights of the fish analysed are shown separately for

2002 and 2003 in Table 3. A high degree of spatial

variability was observed in all the parameters, with a

general trend of higher values in upstream stations (st.

R4-R2). The total length and weight of fish used for

biochemical analyses were significantly higher in

upper river areas than in the lower estuaries in both

2002 and 2003 (Table 3) but did not differ signifi-

cantly between the two cruises (Table 2). For RNA,

DNA and protein, two clearly contrasting and highly

significant sets of values were observed: the higher set

in the upper river and the lower set in the estuary (Fig.

4a). The spatial differences are more evident and clear

in 2002 than in 2003 (Table 2; Fig. 4a). Although

RNA and DNA did not show significant annual var-

iation, variation in total protein was significant (Table

2). The RNA:DNA ratio, RNA:protein ratio and pro-

tein:DNA ratios also showed exactly the same pattern,

i.e., significantly higher values in the upper river (st.

R4-R2) than in the lower estuary (Fig. 4b) in 2002.

Spatial variations were not significant in 2003 except

for st. E3, which had the lowest values for RNA:DNA

ratio and RNA:protein ratio. All ratios were signifi-

cantly different between 2002 and 2003 (Table 2).

When the data of 2002 and 2003 were pooled, fish

had significantly higher TL and weight in st. R4 and

R3 than in the other stations (data not shown); in the

pooled data, the amount of RNA and all RNA-based

ratios were significantly higher in the three upstream

stations (st. R4-R2) than that in the stations in the

lower estuary (data not shown).

While the amount of RNA, DNA and protein corre-

lated significantly with fish length and body weight,

RNA:DNA ratios, RNA:protein ratios and pro-

tein:DNA ratios did not have a significant relation

with either fish length or fish body weight (Table 4;

Fig. 5). Mean RNA:DNA ratios had significant nega-

tive relations with temperature (R2=0.64; P=0.019) in

2002 but the relations between these two parameters in

2003 were not significant (R2=0.052; P=0.555) (Fig.

6). RNA:DNA ratios correlated significantly with sa-

linity in 2002 (R2=0.932; P=0.000) as well as in 2003

(R2=0.535; P=0.000) (Fig. 6). Significant negative

relations of RNA:DNA ratios were observed with co-

pepod density, but positive relations were observed

with copepod dry biomass and gut content dry weight

(Fig. 7).

Fig. 4. a. Variations in RNA, DNA, and total protein in individual larval and early juvenile fishes collected from seven sampling stations in the

Chikugo estuary during March 2002 and 2003. Significant spatial variations were observed in all the parameters. Closed and open circles represent

2002 and 2003, respectively. The letters assigned to each value indicate significance of difference: values having different letters were significantly

different; capital letters are assigned to values of 2002 and small letters are assigned to those for 2003. b. Variations in RNA:DNA ratio,

RNA:Protein ratio and Protein:DNA ratio in individual larval and juvenile fishes collected from seven sampling stations in the Chikugo estuary

during March 2002 and 2003. Significant spatial variations were observed in all the parameters. Closed and open circles represent 2002 and 2003,

respectively. The letters assigned to each value indicate significance of difference: values having different letters were significantly different; capital

letters are assigned to values of 2002 and small letters are assigned to those for 2003.


Instantaneous protein growth rate (Gpi, % d�1) varied

between 4.81 (E2) and 24.59 (R2) in 2002 and between

6.03 (E3) and 10.17 (R4) in 2003. Significantly higher

Table 3

Total lengths and weights of fish used for nucleic acid analyses (values in t

Station 2002

TL (mm) Weight (mg)

R4 23.51F1.06A 101.04F17.72

R3 21.85F1.04AB 83.43F15.75

R2 20.33F1.67ABC 65.27F25.05

R1 19.51F2.03ABC 52.82F16.42

E1 20.64F3.36BC 62.78F29.94

E2 17.60F1.67C 35.70F14.23

E3 19.12F1.51BC 43.06F16.83

growth rates were observed in the three upper stations (st.

R4-R2) than in the other stations during both cruises

(Fig. 8), but differences were greater in 2002 than in

he same column with different superscripts are significantly different)

2003

TL (mm) Weight (mg)

A 22.98F1.70A 98.10F28.26A

AB 22.28F1.21A 84.79F17.63A

ABC 20.11F1.62B 61.86F15.15B

BC 19.94F1.96B 64.18F17.55B

ABC 20.23F1.87B 65.32F18.45B

C 20.48F1.90B 66.18F19.75B

BC 19.41F1.85B 55.99F16.74B

Table 4

Results of regression analyses to assess the effects of fish size (TL and weight) on nucleic-acid-based and other fish condition indices

Regression 2002 Regression 2003

R2 P value N R2 P value n

W=10.436TL-149.17 0.857 0.000 35 W=10.98TL-156.85 0.861 0.000 100

RNA=0.3956TL-5.6382 0.502 0.000 35 RNA=0.1978TL-2.4824 0.683 0.000 100

DNA=0.0655TL-0.7065 0.723 0.000 35 DNA=0.0489x-0.4095 0.665 0.000 100

Protein=0.9147TL-12.191 0.759 0.000 35 Protein=0.6296TL-5.7645 0.645 0.000 100

Gpi=1.3395TL-14.752 0.171 0.170 35 Gpi=0.5331TL-2.1914 0.244 0.277 100

Gpi=0.1213W+4.8371 0.178 0.131 35 Gpi=0.0469W+5.5488 0.264 0.000 100

RNA:DNA=0.3021TL-2.4724 0.184 0.283 35 RNA:DNA=0.1126TL+0.3115 0.244 0.464 100

RNA:protein=0.0121TL+0.1157 0.053 0.532 35 RNA:protein=0.0069TL+0.0795 0.078 0.123 100

Protein:DNA=0.5112TL-0.4131 0.332 0.874 35 Protein:DNA=0.1549TL+8.9469 0.022 0.000 100

RNA=0.0383W-0.0104 0.599 0.978 35 RNA=0.0174W+0.3913 0.737 0.000 100

DNA=0.0062W+0.2328 0.828 0.000 35 DNA=0.0042W+0.3046 0.700 0.000 100

Protein=0.0878W+0.871 0.889 0.025 35 Protein=0.0524W+3.5972 0.625 0.000 100

RNA:DNA=0.0276W+1.9316 0.195 0.007 35 RNA:DNA=0.0099W+1.9465 0.264 0.000 100

RNA:protein=0.001W+0.2956 0.050 0.000 35 RNA:protein=0.0007W+0.1721 0.117 0.000 100

Protein:DNA=0.0468W+7.0282 0.355 0.000 35 Protein:DNA=0.0094W+11.506 0.011 0.000 100


2003 (Table 2). Gpi did not have a significant relation

with either length or weight of fish (Table 4). The pat-

terns of relations were consistent during both cruises

(Table 4).

Critical values of RNA:DNA ratios were 0.755–

0.798 in 2002 and 0.891–1.184 in 2003. Spatial scale

of starvation status was determined on the basis of

Fig. 5. Relationships of RNA:DNA ratio with total length (mm) and

weight (mg) of larval and juvenile Japanese temperate bass collected

in the Chikugo estuary. RNA:DNA ratios did not have a significant

relation with either TL or weight of fish. See Discussion for a

description of the outlying values in the dashed circles.

two indices: the critical RNA:DNA values recorded

in the present study and critical values reported in

other studies (Martin et al. (1985) in striped bass and

Robinson and Ware (1988) in Atlantic herring); they

reported critical RNA:DNA values of 2.0 for starving

Fig. 6. Relationships of RNA:DNA ratio with temperature and salin

ity. RNA:DNA ratios showed significant correlations with both tem

perature and salinity in 2002 but the relations were non-significant in

2003.

-

-

Fig. 7. Relationships of RNA:DNA ratio values with gut content dry

weight, prey density and prey dry weight. RNA:DNA ratios had

significant negative relation with prey density but positive relation

with prey dry biomass and gut content dry weight.

ig. 8. Spatial variations in the protein growth rate (Gpi) of individual

rvae and juveniles of Japanese temperate bass collected in the

hikugo estuary. Significantly higher growth rates were observed in

e fish collected in the upper estuary than those collected down-

tream. The letters assigned to each value indicate significance of

ifference; values having different letters were significantly different;

apital letters are assigned to values of 2002 and small letters are

ssigned to those for 2003.


fish for these two species. These two indices pro-

duced two scenarios: based on the calculated critical

values in the present study, no fish was found starv-

ing and the critical values showed significant annual

variations (Table 2). In contrast, based on the

reported critical value of 2.0, on average 10.4% of

the fish were starving: 9% in 2002 and 14.3% in

2003. The starvation rates did not show significant

annual variations (Table 2). However, these two in-

dices produced similar spatial trends; calculated crit-

ical values were higher in the upstream stations than

in the downstream stations (Fig. 9), indicating that

fish are less likely to starve in the upper areas than

in the lower estuary. This pattern is consistent when

the starvation percentages were calculated for each

station on the basis of reported critical values; no

fish were found starving in the upper three stations,

while in the lower estuary, 20–45% were starving in

2002 and 7–40% fish in 2003. Clearly the proportion

of starving fish increased seaward (Fig. 10).

3.4. Length-weight relationships and condition factor

Mean fish lengths and weights showed significant

spatial variations in both 2002 and 2003 (Table 1);

significantly higher lengths and weights were recorded

in st. R4 and R3 than in the other stations (Table 1);

annual variations were not significant (Table 2). The

parameters derived from the length-weight relationships

were a =0.0002–0.0062 in 2002 and 0.0022–0.0119 in

2003 and b =3.1–4.2 in 2002 and 2.9–3.4 in 2003. The

mean values of the condition factor K ranged from 5.92

to 7.96 in 2002 and from 7.43 to 7.97 in 2003. The

allometric factor b and the condition factor K showed

generally higher values in the upstream stations (st. R4-

R2) and decreased towards the sea, indicating that the

fish upstream were in a better condition than those

downstream (Fig. 11).

4. Discussion

The distribution found in the present study is typical

of the early life history of L. japonicus (Matsumiya et

al., 1982, 1985; Hibino et al., 1999). The species was

distributed over wide estuarine areas and, therefore,

showed a high degree of plasticity to salinity. Speci-

mens were collected from all seven stations. They also

F

la

C

th

s

d

c

a

Fig. 9. Spatial patterns in the critical values (values of RNA:DNA

ratios where protein growth rate is zero) of RNA:DNA ratios (calcu-

lated according to Robinson and Ware, 1988) of Japanese sea bass

larvae and juveniles in the Chikugo estuary. Higher critical

RNA:DNA values indicate better condition of fish upstream.

Fig. 11. Variation in the allometric factor b derived from the length

weight equation W =aLb and in the condition factor K (calculated as

K =1000 W/L) of larval and juvenile Japanese temperate bass in the

Chikugo estuary. The gradually decreasing values of both dbT and dKindicate that larval and juvenile fishes collected in the upper estuary

had a better condition than those in the lower estuary.


showed a high degree of plasticity in their food habits.

Such plasticity to environmental changes and feeding

may be an important factor for a wide spatial distribu-

tion. The results of the gut analyses showed S. sinensis

to be the most important food item in the upstream

stations (st. R4-R2) and A. omorii and O. davisae in the

downstream stations. The gut contents exhibited a clear

relationship with food composition in the water, and the

prey organisms in the guts were representative of the

prey types in the water.

To our knowledge, no published information is

available on the RNA:DNA ratio of Japanese temperate

bass describing condition and starvation status of the

fish in the field. Information on the RNA:DNA ratio of

other fish species studied in the field (Canino et al.,

1991; Clemessen, 1996; Lough et al., 1996; Rooker et

al., 1997; Chicharo, 1998; Esteves et al., 2000a,b)

Fig. 10. Starvation status (% fish starving calculated according to

Martin et al., 1985) of the larval and juvenile fish caught from

seven sampling stations in the Chikugo estuary, as determined by

the RNA:DNA ratio, indicating increased numbers of starved fish

downstream.

-

T

suggests that the value of RNA:DNA is species depen-

dent. Chicharo (1998) reported mean RNA:DNAvalues

of sardine (Sardina pilchardus) being 2.3–4.82 with 1.3

as the critical value, below which fish were considered

to be starved, while Lough et al. (1996) reported 5.23–

5.96 for cod and 4.74–5.87 for haddock with a critical

value of 4.1 for both. Martin et al. (1985) reported

RNA:DNA ratios of striped bass ranging from 0.9 to

11.9 in Potomac River estuary, USA, and established a

critical value of 2.0 for the fish in laboratory conditions.

Our RNA:DNA values, which range from 1.29 to 7.82

(2.93F0.99), are in strong agreement with Martin et al.

(1985). No fish was found starving when critical

RNA:DNA values were calculated according to Robin-

son and Ware (1988) based on growth data at a specific

temperature; however, when calculated on the basis of

Martin et al. (1985), 10% of the fish analysed were

identified as starving. Calculations of starvation status

from published literature on different species should be

used with extreme caution because, as mentioned ear-

lier, critical values of RNA:DNA ratios are species-

specific and show spatio-temporal and regional vari-

abilities. Therefore, species calibration by laboratory


experiment is needed to have concrete information on

the critical RNA:DNA values during starvation, which

is absent for Japanese sea bass. Although the calcula-

tion used in the present study is not an absolutely

reliable estimate of starvation, we report a reliable

scale for between-station comparison which showed

that a higher proportion of the fish is starving in the

estuary than in the river (Fig. 10); this spatial trend was

consistent with both calculations. Therefore, it can be

clearly stated on the basis of all nucleic-acid-based

indices that fish in the upper regions were in a better

condition than in the estuary.

The parameters derived from the length-weight rela-

tionships are considered important indicators of fish

growth and condition in a specified environment. Var-

iation in such parameters is expected to indicate varia-

tion in growth and fish condition. Therefore, these

values have been used widely to assess the growth

condition of many fish species (Safran, 1992; Gon-

calves et al., 1997; Lobo and Erzini, 2001; Andrade

and Campos, 2002; Stergiou and Karpouzi, 2003).

Values of these parameters (b and K), as plotted in

Fig. 11, showed clear variation among stations, indicat-

ing that fish upstream were in a better condition than

those downstream. Clearly, we have identified two

different sets of conditions: fish in the river have a

better condition than those in the sea. Such variation

in condition could be explained by: (1) changes in the

hydrological parameters; (2) changes in fish morphol-

ogy such as length and weight; (3) changes in food

quantity and food quality.

Among the hydrological variables that play a poten-

tial role in fish condition, temperature is believed to be

the most prominent due to its direct involvement in both

foraging behaviour and food utilisation. In our study,

mean RNA:DNA ratios correlated negatively with tem-

perature in 2002; an absence of correlation in 2003might

be due to the narrow range of temperature recorded.

Several authors have found a negative relationship be-

tween RNA:DNA ratio and temperature (Buckley, 1982;

Ferguson andDanzmann, 1990). These studies show that

fish acclimated to cold waters have a higher RNA con-

tent and RNA:DNA ratio than fish acclimated to warm

waters. Goolish et al. (1984) reported that the increase in

the RNA:DNA ratio at lower temperatures is due to a

compensatory mechanism for lower RNA activity,

which produces a higher RNA concentration. It has

been suggested that a temperature difference of around

2 8C is necessary to produce a significant effect on the

RNA:DNA ratio (Buckley et al., 1999). In the present

study, the temperature difference was 1.5 8C in 2002

which is only slightly lower than the temperature sug-

gested by Buckley et al. (1999). Therefore, in 2002, the

effect of temperature could have shown some influence

on the spatial difference in the RNA:DNA ratio which

produced a significant relation between RNA:DNA ratio

and temperature in 2002. In contrast, the temperature

difference was only 0.22 8C in 2003, producing no

significant relation with RNA:DNA.

The RNA:DNA ratio correlated negatively with sa-

linity which is consistent with the findings of Jurss et al.

(1986, 1987) and Imsland et al. (2002). The osmotic

concentration of plasma in euryhaline teleosts is known

to be relatively little affected by salinity changes. How-

ever, the energetic cost of osmoregulation can be high

due to osmo- and ionoregulatory enzymatic mechan-

isms present in gills, gut and kidney, resulting in con-

siderable influence on growth. Although much work

has been done on salinity influences on growth in a

number of fish species covering a relatively wide sa-

linity range (see the review by Boeuf and Payan, 2001),

relationships between salinity and RNA:DNA ratio in

larval fishes have not been comprehensively documen-

ted. However, there is some evidence that the ability to

adjust to levels of salinity ranging from freshwater to

seawater is related to significant increase of rRNA due

to the function of osmoregulatory enzymatic mechan-

isms. Consequently, the RNA:DNA ratio would be

even lower in the highly saline lower estuary in the

present study if the total RNA concentrations could be

corrected for osmoregulatory mechanisms. However,

the energetic cost of osmoregulatory mechanisms and

subsequent growth and condition of fishes is extremely

species-specific (Boeuf and Payan, 2001) and depends

on biological and ecological factors as well as the

species’ adaptability to salinity changes; therefore, spe-

cies calibration should be made before comprehensive

conclusions on this subject are possible.

Although Clemmesen (1994) and Canino et al.

(1991) reported significant correlations between length

and RNA:DNA ratio of marine larval fish, field studies

have not consistently indicated such a correlation

(Lough et al., 1996; Chicharo et al., 1998a,b). Buckley

(1982, 1984) showed that the relations between

RNA:DNA ratio, growth and temperature were unaf-

fected by the size or age of the larvae. They found that

when RNA:DNA ratio increased with age, a

corresponding increase in growth rate followed. In

our study, fish lengths and weights in the upstream

stations were significantly higher than in other stations

and are expected to have resulted in higher condition

indices. However, the RNA:DNA values, when plotted

against TL and weight (Fig. 5), produced no significant

relations with either TL or weight of fish (Table 4).


Therefore, fishes having the same length gave variable

values of RNA:DNA ratio, suggesting that the varia-

tions in fish condition were not related to fish size and,

therefore, other factors were responsible for the differ-

ence in fish condition within the same size groups.

Although higher numerical prey densities were

recorded in the downstream stations than in the upstream

stations, a completely contrasting scenario was found in

the copepod dry mass which was several times higher at

the two uppermost stations than at the other stations,

indicating that the upstream areas are richer in prey

biomass and provide a better foraging environment for

fish. This was reflected in the gut content dry weight.

Therefore, the third hypothesis regarding the spatial

variability in fish condition cannot be ruled out: poor

condition of fish was recorded in areas where a lower

mass of copepod prey was recorded and vice versa,

indicating that fish condition was related to prey bio-

mass. A number of studies have reported poor fish

condition in areas with sufficient prey densities. McGurk

(1986), for example, found larvae in poor condition at

sites with sufficient prey available and they suggested

that the larvae were unable to take advantage of this prey

because they were still learning to forage. An absence of

significant relationship between RNA:DNA ratio and

potential prey density was reported by Chicharo

(1998). We also observed negative relations between

prey density and RNA:DNA ratio; however, positive

relations between RNA:DNA ratio and copepod dry

biomass clearly indicate spatial variations in copepod

size. Therefore, the relation between prey quantity and

fish condition can be better explained by prey biomass

than by prey numbers.

Several authors have suggested that changes in con-

dition of larval and juvenile fishes may be brought about

by particular prey types. Martin et al. (1985), for exam-

ple, reported that the condition of striped bass in the

Potomac River estuary was highly influenced by cladoc-

eran Bosmina abundance; they reported that the lowest

level of starvation and better condition of the fish was

associated with the highest abundance of this prey spe-

cies. Canino et al. (1991) studied the feeding and condi-

tion of walleye pollock inside and outside a plankton

patch in Shelikof Straight of the Gulf of Alaska and

found that the distribution and condition of the fish

was strongly associated with the concentration of cope-

pod nauplii. They reported reduced feeding intensity and

RNA:DNA ratio in areas of lower nauplier concentration

outside the plankton patch than in areas of higher nau-

plier concentration inside the plankton patch. We also

think that the spatial variations in fish condition in the

present study were, among other things,caused by food

quality, particularly of S. sinensis, which was abundant

in the upper region; this is also evident from the com-

parison of the gut contents of the fish between the two

regions. We suggest that the better quality of the fish in

the upper river can be attributed to this particular cope-

pod species.

The results of the present study indicate that starva-

tion may be a significant source of mortality during early

life stages of the fish in the lower estuary of Chikugo;

there the condition of the fish as indicated by the

RNA:DNA ratios, growth rates and length-weight para-

meters results in suboptimal growth and extended dura-

tion of the stage where their size make them vulnerable to

predation loss. Evidence of starvation in the lower estu-

ary can be seen in Fig. 5, in some outlying values. We

assume that these values indicate that some animals

survived a poor feeding period reflected in a low K

(Fig. 11) and that subsequently they encountered rich

feeding grounds and had compensatory growth enhanc-

ing the RNA:DNA ratio. We hypothesise that the upriver

migration of the fish is an adaptation to reduce such early

mortality by utilising better foraging grounds that have a

higher prey biomass and favourable physical environ-

ment (temperature and salinity), leading to a better fish

condition. It could even be hypothesised that utilisation

of S. sinensis in the upstream nursery grounds is one of

the key early survival strategies in Japanese temperate

bass in the Chikugo estuary.

Acknowledgment

This research was supported by the research grant

provided by the Japanese Government Ministry of Edu-

cation, Culture, Sports, Science and Technology (Mon-

bukagakusho,MEXT); the first author acknowledges the

financial support provided by the dMonbukagakushoT(through dMonbukagakusho ScholarshipT) during his

stay in Japan. We thankfully acknowledge the assistance

of the graduate students of Kyoto University during field

samplings.

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Documents

Condition of larval and early juvenile Japanese temperate bass Lateolabrax japonicus related to spatial distribution and feeding in the Chikugo estuarine nursery ground in the Ariake