www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolog

Effects of estuarine conditions and organic enrichment on the

fecundity and hatching success of Acartia clausi

in contrasting systems

Ibon Uriartea, Unai Cotanob, Fernando Villatec,TaLaboratory of Ecology, Department of Plant Biology and Ecology, Faculty of Pharmacy, University of the Basque Country,

E-01006, GasteizbDepartment of Fisheries Resources, AZTI, Herrera Kaia, 20110 Pasaia, Spain

cLaboratory of Ecology, Department of Plant Biology and Ecology, Faculty of Science and Technology,

University of the Basque Country, PO Box 644, 48008 Bilbao, Spain

Received 8 December 2004; received in revised form 21 December 2004; accepted 22 December 2004

Abstract

The fecundity and hatching success of Acartia clausi were analysed at fixed salinity sites (35, 34 and 33 psu) in two nearby

estuaries (Bilbao and Urdaibai, Basque coast, Bay of Biscay) from March to June 1997. Field incubations were conducted to

estimate egg production rates and hatching success, and the size of eggs and experimental females measured. Water temperature

and dissolved oxygen saturation were also determined, as well as seston samples to quantify food abundance and quality.

Between-estuary and within-estuary differences were tested statistically, and correlation and regression analyses were used to

determine relationships between reproductive and environmental variables. Egg production rates were higher in the organically

enriched estuary of Bilbao; this denoting that food supply controls the fertility of A. clausi in these systems. Temporal patterns

of egg production differed between estuaries, and were associated with different nutritional factors in each estuary. Within the

salinity range analysed, egg production reached higher values at intermediate salinity (c34 psu) in both estuaries. This was

interpreted as the result of the interaction between the positive effect of food increase, and the negative effect of

physicochemical conditions with decreasing salinity. Egg size variations mainly occurred temporally in relation to female size,

but no clear trade-off between egg size and egg number was observed in any case. A drop in hatching success in Bilbao, mainly

in waters of b34 psu, was related to the oxygen depletion caused by organic pollution. This indicates that organic enrichment in

Bilbao has opposite effects on the reproductive success, because it enhances egg production but reduces offspring survival.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Acartia clausi; Egg production; Environmental conditions; Estuaries; Hatching success

0022-0981/$ - s

doi:10.1016/j.jem

T Correspondi

E-mail addr

y and Ecology 320 (2005) 105–122

ee front matter D 2005 Elsevier B.V. All rights reserved.

be.2004.12.021

ng author. Tel.: +34 4 460 55 15; fax: +34 4 464 85 00.

ess: gvpvigul@lg.ehu.es (F. Villate).

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122106

1. Introduction

A wide variety of laboratory and field studies on

factors that control copepod fecundity allow the

conclusion that temperature and food availability are

key factors regulating egg production rate within the

range fixed by the physiological characteristics of

each species (e.g. Corkett and McLaren, 1969;

Checkley, 1980a; Ambler, 1985; Bautista et al.,

1994; Calbet and Alcaraz, 1996; Saiz et al., 1997;

Gomez-Gutierrez and Peterson, 1999). However,

when different temporal and spatial scales are

involved, there is still controversy whether temper-

ature or food availability is more important.

Temperature seems to play a major role accounting

for the seasonal variability of copepods fertility

(Ambler, 1986; Kiørboe et al., 1988), whereas food

determines fertility changes on shorter time scales

(Durbin et al., 1983; Ambler, 1986; Kiørboe et al.,

1988; Huskin et al., 2000). In a geographic context,

Huntley and Lopez (1992) consider that food is not

a limiting factor for copepods in marine temperate

areas, and in consequence only physicochemical

factors will influence copepod egg production rate,

temperature being the main factor (Huntley and

Lopez, 1992; Hirst and Sheader, 1997). However,

no relationships between egg production and food

availability or temperature have been also found

over seasonal cycles (Halsband-Lenk et al., 2001),

and several studies have pointed out the influence

of many other factors such as salinity (Ambler,

1985; Stearns et al., 1989), chemical composition of

the seston (White and Dagg, 1989; Jonasdottir et

al., 1995; Gasparini et al., 1999) and specific factors

related to female gonadal maturity and body size

(Campbell and Head, 2000a). Therefore, copepod

egg production can be considered as the final result

of the integrated response of mature females to the

different environmental conditions in which they

have developed, and in consequence the hierarchy

of factors that control copepod fertility may differ

among systems depending on their particular envi-

ronmental properties, and on the range of each

variable.

The reproductive effort (egg production), how-

ever, is not equivalent to the reproductive yield

(survival of the offspring), and the recruitment of

new individuals may be determined by variations in

egg mortality rather than by egg production (Kiørboe

et al., 1988; Ianora and Buttino, 1990; Peterson and

Kimmerer, 1994). A loss of spawned eggs may

depend on several factors: predation, diffusion,

advection, sedimentation (Tang et al., 1998); how-

ever, the result is always predetermined by the

production of viable eggs (Miralto et al., 1998).

The viability of eggs has been related to female

fertility (Jonasdottir and Kiørboe, 1996), remating

(Parrish and Wilson, 1978; Miralto et al., 1995),

female senescence (Ianora et al., 1995), egg size

(Guisande and Harris, 1995), food quality (Miralto et

al., 1995; Jonasdottir and Kiørboe, 1996; Laabir et

al., 1999; Lee et al., 1999) and environmental quality

(Buttino, 1994; Marcus and Lutz, 1994; Jonasdottir

and Kiørboe, 1996; Marcus et al., 1997). All this

indicates that environmental conditions can affect the

hatching success directly as indirectly.

In this study we analyse egg production rates and

hatching success of the calanoid Acartia clausi in two

contrasting estuarine environments to investigate how

the coupled effect of salinity related stress and organic

enrichment affects the fertility and recruitment of this

neritic species in estuaries.

2. Methods

2.1. Study area

Selected estuaries were those of Bilbao (43823VN,38W) and Urdaibai (also called Mundaka) (43820VN,38W), located nearby on the Basque coast (Bay of

Biscay). Consequently both are in the same marine

region and under the same climate regime, but they

offer different environmental conditions to inhabit-

ing populations mainly due to human perturbations.

As shown in Fig. 1, the estuary of Bilbao (23 km

long) is a completely channelled system with its

natural depth increased (now 7 m on average) by

dredging. This modified its circulation pattern

resulting in a highly stratified system. This estuary

reached very high levels of pollution around the

middle of the 20 century due to a considerable

amount of untreated wastewater released into the

estuary over many years. Nowadays, it is in a

process of recovery due to industrial decline and

new wastewater treatment plants. In contrast, the

Kadagua

A N

AbraHarbour

BILBAO

SESTAO

PORTUGALETE

SANTURTZI

LEIOA

ERANDIO

ZIERBENA

Galindo

Asua

Gobelas

Nerbioi-Ibaizabal

GETXO

SUKARRIETA

GERNIKA

KANALA

BUSTURIA

FORUA

B

Oka

Golako

TF

TF

TFTFMS

MS MS

MS

MS

MSMS

MSMS = Marshes and supratidal areas

TF = Tidal flats

Bay ofBiscay

BARAKALDO

35 psu

Mape

34 psu

33 psu

35 psu

33 psu

N

MUNDAKA

31 psu

31 psu

34 psu

1km1km

Basque coast

Fig. 1. Maps of the estuaries of Bilbao (A) and Urdaibai (B), and their geographical location. Arrows indicate approximately the extent of

sampling salinity zones at high tide in each estuary.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 107

estuary of Urdaibai is a 13 km long, on average 2.5

m depth, mixed and relatively undisturbed system

that maintains wide tidal flats and marshes, and

constitutes the main part of the Urdaibai Biosphere

Reserve. In both estuaries, neritic zooplankton

occupies the major part of the system at high tide

because river discharges are usually low in compar-

ison with tidal prisms, and euhaline (N30 psu)

waters penetrate beyond the mid-estuary (Urrutia,

1986; Villate et al., 1989).

2.2. Sampling design

As salinity zonation is highly variable in the

estuaries of Bilbao and Urdaibai, depending on tides

and river runoff, A. clausi was sampled at fixed

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122108

salinity sites of around 35, 34, 33 and 31 psu in each

estuary from March to October 1997. For the purposes

of this study, however, data obtained at the 31 psu site

and data from July to October were not taken into

account because they were too few to do comparisons

with statistical methods owing to the scarcity of adult

females in many cases.

Sampling was carried out monthly but intensified

to fortnightly intervals (I and II in the text) in May and

June. On each occasion, to determine adequate

sampling sites, vertical profiles of salinity and

temperature (WTW LF 197 thermosalinometer), and

oxygen (YSI 55 oxymeter) were performed along the

outer half of estuaries. Oxygen in water was measured

as dissolved oxygen saturation (DOS). Zooplankton

was collected by towing a 200 Am net (mouth

diameter: 0.5 m), equipped with a General Oceanic

flowmeter, at the appropriate site and depth for the

required salinity. Water samples for the determination

of chlorophyll a (Chl a), particulate organic matter

(POM) and proteins, carbohydrates and lipids in

seston were obtained by pumping from the same site

and depth as the zooplankton samples. All samplings

were conducted during the high tide slack to avoid the

effects of tidal currents and associated resuspension

processes.

2.3. Seston analysis

Water samples for seston determinations were

carried to the laboratory in dark carboys and filtered

through precombusted (450 8C, 24 h) Whatman G/FC

glass-fibre filters within 3 h after sampling, and

immediately frozen at �30 8C until analyses were

made. POM was measured gravimetrically on pre-

weighed ash-free filters in a F0.1 Ag precision

microbalance after samples were consecutively dried

(60 8C) and burned (450 8C). The biochemical

soluble components were analysed by spectrophoto-

metrical methods: proteins were determined as in

Lowry et al. (1951), carbohydrates as described by

Dubois et al. (1956), and lipids were extracted

according to Bligh and Dyer (1959) and measured

as described in Marsh and Weistein (1966). Chl a was

determined spectrophotometrically in acetone

extracts, and by using the equation of Lorenzen

(1967). Three replicates were used for the determi-

nation of seston constituents.

Labile food was calculated as the sum of proteins,

carbohydrates and lipids (PCL). Qualitative properties

of food were determined by relating chlorophyll,

biochemical compounds and POM. The contribution

of labile material to the total organic matter was stated

as the PCL/POM ratio; and the contribution of

proteins, carbohydrates and lipids to the labile

material as the percentage of proteins, carbohydrates

and lipids, respectively. The contribution of chlor-

ophyll to total organic matter and labile organic matter

was determined by mean of the Chl a/POM and the

Chl a/PCL ratios, respectively. Food quality variables

were used to relate to the reproductive response of A.

clausi, since digestibility, biochemical composition

and taxonomic composition are recognised as nutri-

tional factors affecting copepod growth and repro-

duction (e.g. Jonasdottir, 1994; Kleppel and Burkart,

1995; Koski et al., 1998).

2.4. Egg production and hatching success

After capture, organisms were immediately trans-

ferred to dark carboys (20 l), previously filled with

water from the same sampling site. Later, the

mesozooplankton was gently concentrated (with a

plastic tube to minimize bubbling and splashing) in a

collector with overflow windows covered with 200

Am meshes. Water for incubations was screened

through a 20 Am mesh to remove metazoan

zooplankton including copepod eggs and nauplii,

and dispensed into twenty 250-ml glass bottles.

Then, a maximum of 5, and usually no less than 3,

undamaged and actively swimming adult females of

A. clausi were sorted using a glass pipette, and

placed into each bottle. Identification and sorting of

females was carried out in Petri disks under a

stereomicroscope.

Incubation bottles were placed into coarse mesh

bags and suspended at buoys in 1.5 m depth at the

same estuary sites. Twenty replicate bottles were

incubated, when possible, per salinity site. The

incubation period was 24 h, which reduces the bottle

effect and cannibalism (Kimmerer, 1984; Runge,

1985) but integrates nycthemeral variations in egg

production (Stearns et al., 1989; Cervetto et al., 1993;

Landry et al., 1994). After incubation, the contents of

the bottles were filtered through 200 Am meshes to

remove females. Females that did not move (b1%)

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 109

were discarded. The content of half of the bottles were

filtered through 45 Am meshes to remove and quantify

eggs and nauplii. The other half of the bottles

remained incubated with eggs and nauplii only for

another 24 h. This was made once again, so that

incubations periods of approximately 24, 48 and 72 h

were obtained. The number of eggs and nauplii

recorded at each time-interval was used to estimate

hatching success and hatching time.

Daily egg production rate (EPR) was calculated as

EPR=[(E+N)/F](24/T), where EPR=eggs female�1

day�1, E=number of eggs,N=hatched nauplii, F=num-

ber of females and T=incubation time (h). Egg

cannibalism was not observed in experimental bottles,

because empty shells corresponded to hatched nauplii.

Specific egg production rate (SEPR) was calculated

from estimated egg carbon (egg-C) and female body

carbon (female-C) as SEPR=(We/Wf)(24/T), where

SEPR=egg-C female-C�1 day�1, We=egg carbon

weight (Ag C), Wf=female carbon weight (Ag C) and

T=incubation time (h). Egg hatching success (EHS)

was expressed as a percentage of hatching eggs, as

EHS=(N/E)d 100, where EHS=the percentage of eggs

hatched,N=number of appeared nauplii and E=number

of eggs. bAbnormalQ nauplii were not distinguished

(Poulet et al., 1995; Miralto et al., 1998), and therefore

viability might be lightly overestimated.

2.5. Size, weight and carbon in eggs and females

Egg diameter (ED) and female cephalothorax

length (FCL) were measured under an inverted

microscope equipped with a micrometer eyepiece.

Female dry weight (FDW) was measured from three

replicate groups of 30 individuals for each salinity

site. After a brief rinse with distilled water to remove

salt and formalin, samples were dried at 60 8C for 24

h, and then weighed with an electrobalance. Dry

weights were corrected by 30% for weight loss due to

preservation (Durbin and Durbin, 1978; Landry,

1978). Body carbon weight of females was estimated

from female dry weight by assuming a carbon content

of 45% (Uye, 1982a; Bamstedt, 1986; Pagano and

Saint-Jean, 1993; Kiørboe and Nielsen, 1994). Egg

carbon was estimated from egg diameter by assuming

a perfect sphericity of eggs and a carbon/volume

content of 0.14 10�6 Ag m�3 (Kiørboe et al., 1985;

Huntley and Lopez, 1992).

2.6. Population abundance

The abundance data for A. clausi were obtained

from the N200 Am net samples once individuals

(mainly adults and late copepodid stages) were

identified and counted under a binocular microscope.

Results were expressed as density values in individ-

uals per unit volume (ind. m�3), the samples volume

being calculated from data obtained with the flow-

meter installed in the net.

2.7. Data analysis

Because EPR, SEPR and ED values showed non-

normal distributions (even after logarithmic trans-

formation) we used non-parametric tests. Thus,

between-salinity site differences within estuaries were

tested using the Kruskal–Wallis H-test. Multiple

comparisons between salinity sites were carried out

with the test of Tukey (Zar, 1984). Between-estuary

differences for the total data set, and between-estuary

differences by salinity sites, were performed with the

U of Mann–Whitney test.

Spearman correlation coefficient and stepwise

regression analysis were used to identify between-

variables relationships, and to determine the influence

of environmental factors on the reproductive variables

(EPR, SEPR, ED and EHS), respectively. Likewise,

stepwise regression analysis was applied to biological

variables (EPR, SEPR, ED, EHS, FCL and FDW) after

subtracting the corresponding mean from each value in

order to eliminate temporal variability.

3. Results

3.1. Temporal variability in environmental conditions

As shown in Fig. 2, water temperature increased 4–5

8C from March to June, and DOS did not show clear

temporal trends in either estuary. POM and PCL

temporal patterns differed between estuaries, since

both showed increasing trends in Urdaibai but no clear

trends in Bilbao. Temporal Chl a variations of also

were different between estuaries, because Chl a peaked

in April and late June in Bilbao, and in early May and

early June in Urdaibai. Among seston quality variables

(Fig. 3), PCL/POM, Chl a/POM and Chl a/PCL ratios

10

15

20

25

30

35

400.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Chl

a/P

CL

(10

0 µg

µg-1

)

Chl

a/P

OM

(µg

mg-1

)

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

Car

bohy

drat

es (

%)

Lip

ids

(%)

Prot

eins

(%

)PC

L/P

OM

(µg

mg-1

)

80

100

120

140

160

180

200

220

240

0.0

0.5

1.0

1.5

2.0

2.5

10

15

20

25

30

35

40

45

50

20

30

40

50

60

70

80

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

Fig. 3. Temporal variation of PCL/POM, Chl a/POM and Chl a/PCL ratios, and of protein, carbohydrate and lipid percentages. Values averaged

for the 33, 34 and 35 salinity sites in Bilbao (filled circles) and Urdaibai (open circles). Vertical lines show standard error.

PCL

(µg

l-1)

DO

S (%

)

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

30

40

50

60

70

80

90

100

110

100

200

300

400

500

600

700

800

900

0

1

2

3

4

Chl

a (

µg l-1

)

Tem

pera

ture

(°C

)

13

14

15

16

17

18

19

20

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

1

2

3

4

5

POM

(µg

l-1)

Fig. 2. Temporal variation of temperature, dissolved oxygen saturation (DOS), particulate organic matter (POM), particulate biomolecules

(PCL) and chlorophyll a (Chl a). Values averaged for the 33, 34 and 35 salinity sites in Bilbao (filled circles) and Urdaibai (open circles).

Vertical lines show standard error.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122110

Table 1

Minimum, maximum and meanFstandard deviation (S.D.) values of salinity, temperature, dissolved oxygen saturation (DOS), chlorophyll a

(Chl a), particulate organic matter (POM), particulate biomolecules (PCL), PCL/POM ratio, Chl a/POM ratio, Chl a/PCL ratio and percentages

of protein, carbohydrate and lipid in the estuaries of Bilbao and Urdaibai, and p-value of the Mann–Whitney test for between-estuary differences

Estuary of Bilbao Estuary of Urdaibai p-value

Min Max MeanFS.D. Min Max MeanFS.D.

Salinity (PSU) 32.80 35.20 33.91F0.83 32.80 35.40 34.32F0.81 0.0927

Temperature (8C) 13.10 18.50 15.59F1.96 13.40 19.70 16.15F2.19 0.3624

DOS (%) 14.30 101.70 67.09F22.79 89.40 110.90 98.94F5.68 b0.0001

POM (mg l�1) 1.53 6.11 3.39F1.11 1.05 4.29 2.11F0.97 0.0003

PCL (Ag l�1) 201.56 980.78 579.9F190.91 168.06 563.23 316.53F131.60 b0.0001

Chl a (Ag l�1) 0.33 5.69 2.16F1.42 0.47 3.89 1.86F1.04 0.6482

PCL/POM 89.43 264.28 175.23F39.98 104.07 225.38 157.06F39.28 0.1532

Chl a/POM 0.07 3.49 0.76F0.69 0.28 1.77 0.94F0.50 0.0948

Chl a/PCL 0.08 1.65 0.45F0.43 0.17 1.35 0.63F0.35 0.0416

% Protein 25.58 72.28 50.15F11.93 20.13 73.69 47.78F13.96 0.5286

% Carbohydrate 7.57 36.91 20.06F7.85 10.47 42.01 24.86F9.09 0.0972

% Lipid 19.72 47.73 29.77F7.36 13.82 54.25 27.34F9.43 0.2567

p-values in bold indicate significant differences at a=0.05.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 111

showed no clear trends (except the decreasing trend of

PCL/POM in Urdaibai), and different patterns of

variation between estuaries. The percentages of pro-

teins and carbohydrates, however, showed decreasing

and increasing trends, respectively, in both estuaries,

while lipid percentages did not show clear trends in

either estuary.

PCL

(µg

l-1)

Tem

pera

ture

(°C

)

Salin

ity (

PSU

)PO

M (

mg

l-1)

00

1

2

3

4

5

** * **

10020030040050060070080090013

14

15

16

17

18

19

20

32

33

34

35

36

35Salin

35 34 33Salinity site

Fig. 4. Mean values (bars) and standard error (lines) of salinity, water te

matter (POM) and chlorophyll a (Chl a) at the salinity sites of 33, 34 and 35

Significant between-estuary differences by salinity site are indicated as *

3.2. Environmental variability between estuaries and

within estuaries

Between-estuary differences in environmental

conditions are summarized in Table 1, and shown

by salinity sites in Figs. 4 and 5. Salinity and

temperature did not show significant differences, but

DO

S (%

)C

hl a

(µg

l-1)

0

1

2

3

4** *

20

40

60

80

100

120 ** **

35 34 33Salinity site

34 33ity site

mperature, dissolved oxygen saturation (DOS), particulate organic

psu in the estuaries of Bilbao (filled bars) and Urdaibai (open bars).

for pb0.05 and ** for pb0.01.

PCL

/PO

M (

µg m

g-1)

Prot

eins

(%

)

Lip

ids

(%)

Car

bohy

drat

es (

%)

Chl

a/P

OM

(µg

mg-1

)

Chl

a/P

CL

(10

0 µg

µg-1

)

0

10

20

30

40

*

10

20

30

40

50

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

100

120

140

160

180

200

220

30

40

50

60

700.0

0.5

1.0

1.5

2.0

35 34 33Salinity site

35 34 33Salinity site

35 34 33Salinity site

Fig. 5. Mean values (bars) and standard error (lines) of PCL/POM, Chl a/POM and Chl a/PCL ratios, and of protein, carbohydrate and lipid

percentages at the salinity sites of 33, 34 and 35 psu in the estuaries of Bilbao (filled bars) and Urdaibai (open bars). Significant between-estuary

differences by salinity site are indicated as * for pb0.05 and ** for pb0.01.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122112

DOS was significantly lower in Bilbao. As DOS

decreased drastically with decreasing salinity in this

estuary, but remained near saturation at all salinity

sites in Urdaibai, between-estuary differences

Table 2

p-values of the Kruskal–Wallis H-test for differences within estuaries ( p-va

the test of Tukey for multiple comparisons between salinity sites of salinity,

a), particulate organic matter (POM), particulate biomolecules (PCL), PCL

protein, carbohydrate and lipid in the estuaries of Bilbao and Urdaibai

Estuary of Bilbao

p-value Between-s

difference

Salinity (PSU) b0.0001 35N34N33

Temperature (8C) 0.9829 34=35=33

DOS (%) b0.0001 35N34N33

POM (mg l�1) 0.0107 33=34=35

PCL (Ag l�1) 0.0152 33=34=35

Chl a (Ag l�1) 0.2709 35=34=33

PCL/POM 0.6673 34=35=33

Chl a/POM 0.0132 35N34=33

Chl a/PCL 0.0240 35N34=33

Proteins (%) 0.0709 34=35=33

Carbohydrates (%) 0.6017 35=33=34

Lipids (%) 0.0067 33N34=35

Values decrease from the left located salinity to the right located salini

significant differences between consecutive salinities; underlined salinities

increased with decreasing salinity. POM and PCL

decreased with salinity in both estuaries and were

significantly higher in Bilbao, but the differences

between estuaries were strongest at higher salinity.

lues in bold indicate significant differences at a=0.05), and results oftemperature, dissolved oxygen saturation (DOS), chlorophyll a (Chl

/POM ratio, Chl a/POM ratio, Chl a/PCL ratio and percentages of

Estuary of Urdaibai

alinity

s

p-value Between-salinity

differences

0.0006 35N34N33

0.3140 33=34=35

0.2620 35=34=33

0.0023 33=34=35

0.0092 33=34=35

0.0442 33=34=35

0.5663 35=33=34

0.9748 34=33=35

0.7463 34=35=33

0.8683 35=34=33

0.3907 33=34=35

0.7853 35=34=33

ty. N: significant differences between consecutive salinities; =: no

: significant differences between them.

Den

sity

(in

d m

-3)

1

10

100

1000

10000

35 34 33Salinity site

31

* **

Fig. 7. Mean values (bars) and standard error (lines) of Acartia

clausi abundance at the salinity sites of 31, 33, 34 and 35 psu in the

estuaries of Bilbao (filled bars) and Urdaibai (open bars)

Significant between-estuary differences by salinity site are indicated

as * for pb0.05 and ** for pb0.01.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 113

Chl a did not show significant differences between

Bilbao and Urdaibai, but showed opposite trends:

Chl a increased with salinity in Bilbao, but

decreased in Urdaibai. The PCL/POM ratio and the

percentages of proteins, carbohydrates and lipids did

not show significant differences between estuaries, in

general. Comparisons by salinity sites, however,

showed that carbohydrate percentages were signifi-

cantly lower, and lipid percentages significantly

higher in Bilbao at the 33 psu site. Chl a/POM

and Chl a/PCL ratios were significantly higher in

Urdaibai, in general, between-estuary differences

increasing at lower salinity. This was due to the fact

that Chl a-related ratios decreased drastically from

the 35 psu site to the 34 psu site in Bilbao while

they remained fairly constant with salinity in

Urdaibai.

The statistical analysis of environmental differ-

ences within estuaries (Table 2) showed that POM and

PCL differed significantly with salinity in both

estuaries, while chlorophyll differed significantly with

salinity only in Urdaibai, and DOS, Chl a-related

ratios and lipid percentages differed significantly with

salinity only in Bilbao.

FDW

(µg

)

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

73

74

75

76

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II

FPL

(µm

)

ED

(µm

)

EPR

(eg

gs f

emal

e-1 d

ay-1)

50

60

70

80

90

100

1100

4

8

12

16

20

780

820

860

900

940

980

7

8

9

10

11

12

13

EH

S (%

)

SEPR

(da

y-1)

Mar

chA

pril

May

IM

ay I

IJu

ne I

June

II0

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Fig. 6. Temporal variation of egg production rate (EPR), specific egg production rate (SEPR), egg diameter (ED), egg hatching success (EHS)

female cephalothorax length (FCL) and female dry weight (FDW). Values averaged for the 33, 34 and 35 salinity sites in Bilbao (filled circles

and Urdaibai (open circles). Vertical lines show standard error.

.

3.3. Temporal changes in A. clausi reproductive

parameters and female size

As shown in Fig. 6, EPR and SEPR showed no

clear temporal trends of variation. In both estuaries the

lower mean values were obtained in March and the

higher mean values in late June, except for SEPR in

Urdaibai where the higher mean values were recorded

in late May. EHS neither showed clear temporal trend

,

)

Table 3

Minimum, maximum and meanFstandard deviation values of egg production rate (EPR), specific egg production rate (SEPR), egg diameter

(ED), egg hatching success (EHS), female cephalothorax length (FCL) and female dry weight (FDW) in the estuaries of Bilbao and Urdaibai,

and p-value of the Mann–Whitney test for between-estuary differences

Estuary of Bilbao Estuary of Urdaibai p-value

Min Max MeanFS.D. Min Max MeanFS.D.

EPR (eggs female�1 day�1) 0.50 21.83 9.49F5.48 0.45 12.31 5.69F3.74 0.0224

SEPR (day�1) 0.004 0.196 0.068F0.045 0.002 0.067 0.037F0.021 0.0135

ED (Am) 72.19 75.67 73.93F0.89 73.00 75.84 74.30F0.84 0.1662

EHS (%) 21.60 100.00 76.54F18.67 66.70 100.00 92.46F8.74 0.0009

FCL (Am) 790.24 954.78 878.66F50.00 793.11 956.04 879.70F51.69 0.9678

FDW (Ag) 6.85 12.05 9.08F1.39 6.89 12.88 9.84F1.68 0.1268

p-values in bold indicate significant differences at a=0.05.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122114

in any estuary, but values were lower from March to

May and higher from May to June in Bilbao, while in

Urdaibai remained high most of the time. ED, FCL

and FDW showed clear decreasing trends over the

study period in both estuaries.

3.4. Variability in A. clausi abundance, reproductive

parameters and female size between estuaries and

within estuaries

Within the euhaline region of these estuaries, A.

clausi was significantly more abundant in Bilbao (U

35Sali

SEPR

(da

y-1)

35 34 33Salinity site

0

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

*

EPR

(eg

gs f

emal

e-1 d

ay-1

)

ED

(µm

)

40

50

60

70

80

90

100

110

120

72

73

74

75

76

02468

1012141618

EH

S (%

)

Fig. 8. Mean values (bars) and standard error (lines) of egg production rate

hatching success (EHS), female cephalothorax length (FCL) and female

estuaries of Bilbao (filled bars) and Urdaibai (open bars). Significant betw

and ** for pb0.01.

of Mann–Whitney test, pb0.001), and decreased

significantly with decreasing salinity (ANOVA,

pb0.0001 in Bilbao and p=0.0002 in Urdaibai) by

an order of magnitude on average, from 35 psu to 31

psu in both estuaries (Fig. 7).

Between-estuary differences in A. clausi EPR,

SEPR, EHS, ED, FCL and FDW over the study

period are summarized in Table 3. The range of

variation for all reproduction variables was larger in

Bilbao, but EPR and SEPR were significantly higher

in Bilbao, whereas EHS was significantly lower. ED,

FCL and FDW showed higher mean values in

34 33nity site

FDW

(µg

)

35 34 33

FPL

(µm

)

Salinity site

6

7

8

9

10

11

12

13

* **

800

820

840

860

880

900

920

940

960

(EPR), specific egg production rate (SEPR), egg diameter (ED), egg

dry weight (FDW) at the salinity sites of 33, 34 and 35 psu in the

een-estuary differences by salinity site are indicated as * for pb0.05

Table 4

p-values of the Kruskal–Wallis H-test for differences within estuaries ( p-values in bold indicate significant differences at a=0.05), and results ofthe test of Tukey for multiple comparisons between salinity sites of egg production rate (EPR), specific egg production rate (SEPR), egg

diameter (ED), egg hatching success (EHS), female cephalothorax length (FCL) and female dry weight (FDW) in the estuaries of Bilbao and

Urdaibai

Estuary of Bilbao Estuary of Urdaibai

p-value Between-salinity

differences

p-value Between-salinity

differences

EPR (eggs female�1 day�1) 0.4828 34=35=33 0.1718 34=33=35

SEPR (day�1) 0.1910 34=35=33 0.3361 34=33=35

ED (Am) 0.8510 34=33=35 0.0550 35=34=33

EHS (%) 0.0882 34=35=33 0.3857 33=34=35

FLC (Am) 0.9346 33=35=34 0.2543 35=34=33

FDW (Ag) 0.2323 33=34=35 0.2977 34=35=33

Values decrease from the left located salinity to the right located salinity. N: significant differences between consecutive salinities; =: no

significant differences between consecutive salinities; underlined salinities: significant differences between them.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 115

Urdaibai but differences were not significant. The

analysis of between-estuary differences by salinity

sites (Fig. 8) showed that differences in EPR and

SEPR increased with salinity, while the opposite was

observed for EHS (not significant at 35 psu; highly

significant at 33 psu). As shown in Fig. 8, higher

values of EPR and SEPR were recorded at intermedi-

ate salinity (34 psu site) in both estuaries, but EPR and

SEPR declined more drastically at 33 psu than at 35

psu in Bilbao. EHS showed a clear decrease at 33 psu

Bilbao, and ED diminished with decreasing salinity in

both estuaries. However, none of these variables, nor

FPL or FDW were found to differ significantly within

estuaries (Table 4).

Table 5

Results of the stepwise forward multiple regression analysis for egg produ

(ED) and egg hatching success (EHS) in the estuaries of Bilbao and Urda

Variables Regression

Estuary of Bilbao EPR 5.34+1.91 Chl a

SEPR 0.032+0.032 Ch

ED 61.8+0.01 FCL+

EHS 49.5+0.41 DOS

Estuary of Urdaibai EPR 12.2�0.04 PCL

SEPR n.s.

ED 48.6+0.01 FCL+

EHS 189�55.3% Lip

Both estuaries EPR 4.43+3.20 Chl a

SEPR 0.033+0.026 Ch

ED 56.6+0.01 FCL+

EHS 49.8+0.41 DOS

n.s.: not significant.

3.5. Relationships of reproductive variables with

environmental and female-size related variables

The stepwise regression analyses (Table 5)

selected the positive effect of Chl a to account for

the EPR and SEPR variability in Bilbao, and in both

estuaries taken together. The analyses added the

negative effect of the Chl a/PCL on SEPR in Bilbao,

and the negative effect of the Chl a/POM ratio on

EPR and SEPR for both estuaries together. In

Urdaibai only the negative effect of the PCL/POM

ratio was selected to explain EPR variability (but

approached the non-significant level, p=0.0493), and

none of the variables was selected to explain SEPR

ction rate (EPR), specific egg production rate (SEPR), egg diameter

ibai, and in both estuaries taken together

r2 p

0.262 0.0038

l a�6.81 Chl a/PCL 0.315 0.0060

0.20 Chl a 0.596 b0.0001

0.236 0.0075

/POM 0.266 0.0493

– –

0.39 Salinity 0.683 0.0010

id�0.82 DOS 0.523 0.0118

�3.70 Chl a/POM 0.306 0.0005

l a�32.56 Chl a/POM 0.239 0.0011

0.23 FDW+0.2 Salinity 0.614 b0.0001

0.319 b0.0001

Table 6

Results of the stepwise forward multiple regression analysis for egg

production rate (EPR), specific egg production rate (SEPR), egg

diameter (ED) and egg hatching success (EHS) in the estuaries of

Bilbao and Urdaibai, and in both estuaries taken together, once

temporal variability was eliminated

Variables Regression r2 p

Estuary of EPR n.s. – –

Bilbao SEPR n.s. – –

ED n.s. – –

EHS �19.6+0.3 DOS 0.163 0.0330

Estuary of EPR n.s. – –

Urdaibai SEPR n.s. – –

ED �9.3+0.3 Sal 0.336 0.0235

EHS n.s. – –

Both EPR n.s. – –

estuaries SEPR n.s. – –

ED 0.4�0.14 POM 0.126 0.0169

EHS n.s. – –

n.s.: not significant.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122116

variability. ED variability was mostly explained by

the positive effect of female size (FCL) in every

case. Secondarily the analysis selected the positive

effect of Chl a in Bilbao, the positive effect of

salinity in Urdaibai, and the positive effects FDW

and salinity in both estuaries taken together. Only the

positive effect of DOS was selected to account for

the EHS variability in Bilbao and in both estuaries

taken together, while the negative effects of the

lipids contribution (% Lipids) and DOS were

selected in Urdaibai.

Once the temporal variability was eliminated (Table

6), the analysis did not choose any factor to account for

the EPR and the SEPR variability in any case.

However, the positive effect of DOS was newly

selected to account for the EHS variability in Bilbao.

Finally, the positive effect of salinity and the negative

effect of POM were selected to explain the variability

of ED in Urdaibai and in both estuaries taken together,

respectively.

4. Discussion

4.1. Environmental scenario

According to the sampling design, corroborated

by the analysis of measured salinity data, salinity

might be responsible for differences in reproductive

variables within estuaries, but not for differences

between estuaries. Dissolved oxygen, however,

showed strong differences between Bilbao and

Urdaibai, as well as throughout the salinity gradient

in the estuary of Bilbao, denoting the effect of

organic inputs to this system. Deoxygenating of

waters is a well-known feature in organically

enriched estuaries such as the estuary of Bilbao,

where anoxic conditions predominate in the inner

half of the estuary (personal observations). The

values obtained at the 33 psu site during this study

indicate a situation of frequent hypoxia, which could

perturb the physiological and behavioural functions

of organisms (Diaz and Rosenberg, 1995; Wu,

2002).

Results on POM and biochemical compounds

confirm that Bilbao was organically richer than

Urdaibai. However, the between-estuary differences

in total and labile food availability were more evident

at high salinity, while those of oxygen availability

were more evident at low salinity. This may be

explained in part by the inverse distribution of

phytoplankton and organic materials with salinity in

Bilbao, because it indicates that phytoplankton and

autotrophic processes are more important at higher

salinity, while detritus and microbial activities respon-

sible for oxygen depletion are more important at lower

salinity. In Bilbao, light seems to limit phytoplankton

development at lower salinity while conditions are

more favourable at higher salinity due to a decrease of

turbidity and high concentrations of nutrients (Agirre,

2000). In contrast, phytoplankton and seston organic

compound concentrations are associated, both

decreasing with salinity throughout the euhaline zone

of Urdaibai. In this estuary, a peak of phytoplankton

biomass usually develops in waters around 30 psu

(Ruiz et al., 1998), and the decrease of phytoplankton

biomass seawards is attributed to dilution and nutrient

limitation (Franco, 1994).

Overall food availability for A. clausi females was

higher in Bilbao. However, phytoplanktonic food was

more abundant in Bilbao at higher salinity, but at

lower salinity in Urdaibai. Within estuaries, food

characteristics were more homogenous in Urdaibai,

while in Bilbao they differed significantly in phyto-

plankton and detritus contributions but less in the

contribution of labile compounds and biochemical

composition. This indicates that nutritional properties

Mar

ch

Apr

il

May

I

May

II

June

I

June

II

July

Aug

ust

Sept

embe

r

Oct

ober

1

10

100

1000

10000

Den

sity

(in

d m

-3)

Fig. 9. Temporal variation of Acartia clausi abundance. Values

averaged for the 33, 34 and 35 salinity sites in Bilbao (filled circles

and Urdaibai (open circles). Vertical lines show standard error.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 117

of organic particles were similar in both estuaries and

within the salinity range studied. Taking into account

the values of the PCL/POM ratio and the proportion

of biomolecules, with values of protein:lipid:carbohy-

drate between 1:1:1 and 4:3:1 in both estuaries, we

may consider that the food available was appropriate

for the nutritional requirements of filter-feeding

organisms (Scott, 1980; Poulet et al., 1986).

4.2. A. clausi populations

The higher abundance of A. clausi in the estuary of

Bilbao at intermediate salinity sites corroborates the

opportunist character of this species, and its high

capacity to develop in eutrofizied and polluted areas

(e.g. Gaudy, 1985; Siokou-Frangou and Papathanas-

siou, 1991; Zaitsev, 1992). Although the available

literature shows that A. clausi can tolerate a broad

range of salinities, and particular populations may

show optimal salinity ranges bellow 30 ups (e.g.

Cervetto et al., 1995; Gaudy et al., 2000), our results

indicate that salinity decrease or co-varying environ-

mental factors had an unfavourable effect on the A.

clausi populations of Bilbao and Urdaibai even into

the short salinity range analysed. This agrees with that

observed in other estuarine systems of the European

Atlantic coast, where A. clausi populations reach

maxima atN 33 ups salinity (e.g. Alcaraz, 1977;

D’Elbee, 1984).

4.3. Egg production

Different relationships of egg production with

temperature and chlorophyll have been reported for

A. clausi depending on the studied area (Landry,

1978; Uye, 1982b; Ianora and Scotto di Carlo, 1988;

Ianora and Buttino, 1990; Gomez-Gutierrez et al.,

1999). Our results confirm the inconsistency of such

relationships at least in the temporal context in

which they were analysed, since the relationship

between egg production and temperature was not

clear, and relations to chlorophyll differed between

estuaries. Although Bilbao and Urdaibai are in the

same marine area and have the same climatic

conditions, no parallel variations of chlorophyll were

observed in these estuaries, and egg production

related to chlorophyll concentration only in Bilbao.

In conclusion, the reproductive activity of A. clausi

did not respond similarly to changes in chlorophyll

in nearby estuaries with similar ranges of variation in

chlorophyll content. As the relationships with other

environmental factors were inconsistent, we assume

that no measured factors were also involved control-

ling the reproduction of A. clausi. Similarly, in a

previous study on the congeneric species A. bifilosa

in the estuary of Urdaibai, we found seasonal and

year-to-year differences in egg production that were

not adequately explained by the environmental

variables measured (Uriarte et al., 1998). Campbell

and Head (2000a) consider that egg production is a

function of average population gonad maturity and

female mean size, and effects of temperature and

food on egg production are easier to detect in

individuals belonging to the same population than

for individuals belonging to different populations. A

plausible explanation for the inconsistency of rela-

tionships between egg production and environmental

variables, in small tidal estuaries such as Bilbao and

Urdaibai, is the exchange of populations by tides

(Villate et al., 1989; Villate, 1997). The fact that the

seasonal dynamics of A. clausi differed significantly

between Bilbao and Urdaibai over our study period

(see Fig. 9 and Table 7) also corroborates that this

species has a different population development in

each estuary.

Egg production was always higher in Bilbao, as

well as total and labile organic matter. Since food

quality did not show such clear differences between

estuaries, we conclude that population fertility was

)

Table 7

Results of the two-way ANOVA test for months and estuaries of the

abundance of Acartia clausi in the N200 Am fraction

df Sum of

squares

Mean

square

F-value p-value

Months 9 36.428 4.048 12.662 b0.0001

Estuaries 1 1.554 1.554 4.862 0.0304

Month*Estuaries 9 7.498 0.833 2.606 0.0111

Residual 24.614 0.320 0.320

p-values in bold indicate significant differences at a=0.05.

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122118

primarily determined by food abundance. As

phytoplankton availability was similar in both

estuaries, we can infer that higher fertility was

supported by higher availability of non-phytoplank-

tonic food in Bilbao, since Acartia species utilize a

wide variety of food items of the heterotrophic

microbial food chain (Lonsdale et al., 1979;

Wiadnyana and Rassoulzadegan, 1989; Ederington

et al., 1995). The between-estuary comparisons by

salinity confirmed the relationship of A. clausi

fecundity with total and labile food supply, because

differences in egg production were more evident at

higher salinity, where differences in POM and PCL

were most significant. The major role of non-

phytoplanktonic food enhancing copepod reproduc-

tion in Bilbao has also been found for the egg-

carrying copepod Euterpina acutifrons , which

showed clutch size and brood volume to be related

to the abundance of total labile food (PCL) rather

than to the abundance of phytoplankton (Dıaz et

al., 2003).

As egg production was higher at intermediate

salinity of around 34 psu in both estuaries, but was

not related to any environmental factor, such a pattern

could result from the coupled effect of nutritional and

physicochemical conditions along estuarine gradients.

Our results suggest that the positive effect of increas-

ing food abundance on egg production might be

counteracted by the associated negative effect of

decreasing salinity in waters below 34 psu. Likewise,

the increase of turbidity at lower salinities could also

have a negative effect on egg production (Burdloff et

al., 2000), as well as the decrease of oxygen (Stalder

and Marcus, 1997) in the case of Bilbao. Therefore,

optimal conditions for egg production in neritic

copepods would not coincide with optimal nutritional

conditions within the euhaline region of estuaries. In

the open sea, however, increasing reproductive rates

towards the coast have been reported for some

copepod species along an ocean-coast gradient,

relating to an increase of food abundance (Calbet et

al., 2002).

4.4. Egg size

ED differed seasonally and between estuaries, but

it was not directly related to environmental or nutri-

tional variables. The positive effect of female size was

selected by the stepwise regression analysis as the

main factor accounting for the ED variability in every

case. Therefore, if temperature or nutritional factors

influenced egg size, this was via female size. It is

known that individual size in calanoid copepods is

dependent on temperature and food availability

(Mauchline, 1998), and the relationship between egg

size and female size is well documented in the

literature (e.g. Uye and Shibuno, 1992; Kiørboe and

Sabatini, 1995; Uriarte et al., 1998).

No inverse relationships between egg size and

egg production were found. Thus, a reproductive

strategy of investing more energy in larger eggs

instead of higher egg numbers when food availability

declines (Guisande et al., 1996) was not observed.

However, once the temporal effect was eliminated,

egg size appeared inversely related to salinity and

food abundance, in agreement with the hypothetical

reduction of egg size in nutritionally rich environ-

ments. In the analysis of the reproductive response of

E. acutifrons in these estuaries, the trade-off between

clutch size and egg size was consistent in Urdaibai,

but not in Bilbao (Dıaz et al., 2003). Thus, the

results obtained for A. clausi confirm that the

reduction of egg number in order to increase egg

size is not a common reproductive feature in food-

rich environments.

4.5. Hatching success

The viability of A. clausi eggs in both estuaries

was generally high, compared to other studies

(Burkart and Kleppel, 1998; Gomez-Gutierrez and

Peterson, 1999), and did not show clear relationships

with nutritional variables. This is not surprising, since

the nutritional quality of food met the requirements of

A. clausi in both estuaries. Shin et al. (2003)

I. Uriarte et al. / J. Exp. Mar. Biol. Ecol. 320 (2005) 105–122 119

demonstrate that Acartia omorii egg viability is

affected by food quality, and according to Jonasdottir

and Kiørboe (1996) egg hatching success is expected

to be high when eggs receive a favourable balance of

required nutrients but low when essential nutrients are

missing. Nevertheless, between-estuary differences in

EHS were evident, mainly due to the fall in egg

hatching at b34 psu waters of Bilbao. The stepwise

analysis indicates that EHS variability was primarily

attributable to the oxygenation of waters. Hypoxic

conditions such as registered in Bilbao may cause

failure in hatching of the copepod eggs (Marcus and

Lutz, 1994; Jonasdottir and Kiørboe, 1996; Marcus et

al., 1997). In Urdaibai, where oxygen conditions were

good at all salinity sites, EHS was not related to

physicochemical or nutritional variables. In agreement

with other studies, our results indicate that egg

viability is unaffected by salinity (Chen and Marcus,

1997), temperature or chlorophyll (Ianora et al., 1992;

Laabir et al., 1995).

No consistent relationships were found between

egg hatching success and egg production (the only

significant correlation was between SEPR and EHS

in the estuary of Bilbao; Spearman rank correlation:

0.507; p=0.0073), similarly to that observed in the

literature. Tang and Dam (2001) found for Acartia

tonsa that when egg production rate was high

hatching success was also high, and when egg

production was low egg hatching success was highly

variable, and therefore lower in average. However,

Dam and Lopes (2003) report a highly variable

hatching success for Temora longicornis when egg

production was low but only low hatching success

when egg production was high. The apparent

independence of egg hatching and egg production

is likely due to the complexity of mechanisms

involved in the processes of both egg production

and hatching in the field. For example, Lacoste et al.

(2001) demonstrated experimentally that in Calanus

helgolandicus, fecundity and hatching might show

different relationships depending on the diet offered

to spawning females. Our results did not show a

positive relationship between hatching success and

egg size for A. clausi, in contrast to that observed for

C. helgolandicus by Guisande and Harris (1995),

who indicate that larger egg size increased hatching

success. Perhaps this effect is only evident when egg

size differs greatly, since the range of ED variation

reported for C. helgolandicus was 20%, while in our

case it was V3%.

Remarkably, although egg production was signifi-

cantly higher in Bilbao than in Urdaibai, hatching

success was lower. We conclude that organic enrich-

ment in Bilbao causes two different effects on the

reproductive success of A. clausi, since it enhances

egg production by increasing food supply, but

reduces offspring survival due to low oxygen levels

resulting from the decomposition of organic matter.

It is interesting to point out that the reduction of the

hatching success in Bilbao was found to balance the

reproductive success as compared to Urdaibai, since

the recruitment rates calculated from values of egg

production and hatching success did not show

significant differences between estuaries (Bilbao:

7.88F5.13 eggs female�1 day�1; Urdaibai 4.91F3.29 eggs female�1 day�1; U of Mann–Whitney test:

p=0.0661).

Acknowledgements

This study was supported by the University of the

Basque Country UPV118-310-EA018/96 and by the

Department of Education Universities and Research

of the Basque Government. We also thank this

Department for the two grants to I.U. and U.C. [SS]

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