Accepted Manuscript Seasonal variations of the humoral immune parameters of European sea bass (Dicentrarchus labrax L.) Yulema Valero , Alicia García-Alcázar , M. Ángeles Esteban , Alberto Cuesta , Elena Chaves-Pozo PII:
S1050-4648(14)00160-0
DOI:
10.1016/j.fsi.2014.05.011
Reference:
YFSIM 2983
To appear in:
Fish and Shellfish Immunology
Received Date: 2 April 2014 Revised Date:
30 April 2014
Accepted Date: 12 May 2014
Please cite this article as: Valero Y, García-Alcázar A, Esteban MÁ, Cuesta A, Chaves-Pozo E, Seasonal variations of the humoral immune parameters of European sea bass (Dicentrarchus labrax L.), Fish and Shellfish Immunology (2014), doi: 10.1016/j.fsi.2014.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
SHORT COMMUNICATION
2 3
Seasonal variations of the humoral immune parameters of European sea
4
bass (Dicentrarchus labrax L.)
RI PT
5 6
Authors: Yulema Valero1, Alicia García-Alcázar1, M. Ángeles Esteban2, Alberto
7
Cuesta2, Elena Chaves-Pozo1*.
8
1
9
de la Azohía s/n. Puerto de Mazarrón, 30860 Murcia, Spain
SC
Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO), Carretera
10
2
11
of Biology, Campus Regional de Excelencia Internacional “Campus Mare Nostrum”,
12
University of Murcia, 30100 Murcia, Spain
M AN U
Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty
13
Correspondence and reprint requests: *To whom correspondence should be
15
addressed to Centro Oceanográfico de Murcia, Instituto Español de Oceanografía,
16
Carretera de la Azohía s/n. Puerto de Mazarrón, 30860
17
[email protected], fax: +34-968153934, tel: +34-968153339.
TE D
14
18
Murcia, Spain.
Keywords: Season, temperature, protease, antiprotease, complement, bactericidal
20
activity, peroxidase, lysozyme, European sea bass.
EP
19
AC C
21 22
Abstract
23
Seasonal cycles, mainly due to great variations in the light duration and temperature, are
24
important and modulate several aspects of the animal behavior. In the case of
25
poikilotherms animals such as fish this is very relevant. Thus, temperature changes fish
26
immunity and affects disease resistance. We evaluate in this work the season variations
27
of the European sea bass (Dicentrarchus labrax) humoral innate parameters focusing on
28
winter months, at which the culture of this specie is more difficult. Our results showed
29
that not all the innate immune parameters are depressed by low temperatures. Moreover,
1
ACCEPTED MANUSCRIPT 1
some of them are more dependent than others to the season and both temperature and
2
photoperiod are operating together.
3 Seasonality dominates the life cycle of animals, mainly in the case of
5
poikilotherms such as fish. It is now widely demonstrated that season cycles affect to
6
fish physiology including animal behavior, body weight and food intake, reproduction,
7
locomotor activity or immunity [1]. Several natural factors may influence these cycles
8
but photoperiod and temperature are the most important. Focusing on temperature, it is
9
generally accepted that the innate immune response is independent of the temperature
10
whilst the adaptive response is dependent on it. However, low temperature has been
11
associated with an increase in fish susceptibility to disease [2, 3] suggesting that both
12
innate and adaptive immunity are affected by temperature. Thus, the influence of
13
temperature on fish immunity has been described and varied with the fish species [4-6].
14
Otherwise, temperature also represents a crucial factor in the survival and infective
15
ability of several pathogens, as occurs with viral hemorrhagic septicemia virus (VHSV),
16
some alphavirus or some bacteria such as Vibrio sp, Flavobacterium psychorophilum or
17
Moritella viscose which cause disease when water temperature drops [7-11]. All this
18
data highlight the relevance of water temperature in fish health, but little is known in the
19
case of Mediterranean aquaculture species, mainly gilthead seabream (Sparus aurata)
20
and European sea bass (Dicentrarchus labrax). In the case of gilthead seabream, it was
21
reported that the complement and agglutination activities of serum were lowest in
22
winter months whilst the lysozyme activity was similar during all the year [5]. In sea
23
bass, there is only a study reporting the effect of seasonality on cortisol, hematocrit,
24
leucocrit, serum lysozyme activity and total glutathione [12]. Due to the economical
25
importance of the European sea bass in the Mediterranean aquaculture and the season
26
cycles on fish immunity and health, we propose in this study the evaluation of several
27
humoral innate parameters of European sea bass at different seasons but focusing on
28
winter, the most challenging for fish culture.
AC C
EP
TE D
M AN U
SC
RI PT
4
29
Healthy specimens of European sea bass were bred at the Centro Oceanográfico
30
de Murcia with natural conditions of photoperiod, temperature, salinity and aeration.
31
One year old fish were sampled once a month (n=10 fish/month) from September
32
(121±13 g body weight (bw)) to May (225±21 g bw). The specimens were bred with a
33
commercial pellet dry diet (Skretting) ad libitum. Temperature, mortality and food 2
ACCEPTED MANUSCRIPT intake were recorded daily. Mean temperature in each month was represented. The
2
specimens were anesthetized with 40 µl L-1 of clove oil before handling. The blood was
3
obtained from the caudal peduncle and the serum samples, obtained by centrifugation
4
(10,000 g, 1 min, 4 ºC), immediately frozen in liquid nitrogen and stored at -80 ºC until
5
use. Protease activity was determined as the percentage of hydrolysis of azocasein by 2
6
mg/ml of proteinase K [13]. Total anti-protease activity was determined as the
7
percentage of inhibition of the hydrolysis of azocasein by 2 mg/ml of proteinase K [14].
8
Haemolytic activity of complement was assayed using sheep red blood cells (SRBC;
9
Biomedics) as targets [15]. The results were expressed in ACH50 units, as the titre at
10
which 50 % haemolysis is produced [16]. Lysozyme activity was measured according to
11
a turbidimetric method that uses the lysis of Micrococcus lysodeikicus for determination
12
of the lysozyme activity using hen egg-white lysozyme as standard [17]. Serum
13
antibacterial activity was determined by evaluating the inhibition on the bacterial
14
growth of Vibrio harveyi curves [16]. The peroxidase activity was measured with a
15
method previously described [18]. Data were presented as mean ± S.E.M (n = 10) and
16
significance analysed by ANOVA (p
S1050-4648(14)00160-0
DOI:
10.1016/j.fsi.2014.05.011
Reference:
YFSIM 2983
To appear in:
Fish and Shellfish Immunology
Received Date: 2 April 2014 Revised Date:
30 April 2014
Accepted Date: 12 May 2014
Please cite this article as: Valero Y, García-Alcázar A, Esteban MÁ, Cuesta A, Chaves-Pozo E, Seasonal variations of the humoral immune parameters of European sea bass (Dicentrarchus labrax L.), Fish and Shellfish Immunology (2014), doi: 10.1016/j.fsi.2014.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
SHORT COMMUNICATION
2 3
Seasonal variations of the humoral immune parameters of European sea
4
bass (Dicentrarchus labrax L.)
RI PT
5 6
Authors: Yulema Valero1, Alicia García-Alcázar1, M. Ángeles Esteban2, Alberto
7
Cuesta2, Elena Chaves-Pozo1*.
8
1
9
de la Azohía s/n. Puerto de Mazarrón, 30860 Murcia, Spain
SC
Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO), Carretera
10
2
11
of Biology, Campus Regional de Excelencia Internacional “Campus Mare Nostrum”,
12
University of Murcia, 30100 Murcia, Spain
M AN U
Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty
13
Correspondence and reprint requests: *To whom correspondence should be
15
addressed to Centro Oceanográfico de Murcia, Instituto Español de Oceanografía,
16
Carretera de la Azohía s/n. Puerto de Mazarrón, 30860
17
[email protected], fax: +34-968153934, tel: +34-968153339.
TE D
14
18
Murcia, Spain.
Keywords: Season, temperature, protease, antiprotease, complement, bactericidal
20
activity, peroxidase, lysozyme, European sea bass.
EP
19
AC C
21 22
Abstract
23
Seasonal cycles, mainly due to great variations in the light duration and temperature, are
24
important and modulate several aspects of the animal behavior. In the case of
25
poikilotherms animals such as fish this is very relevant. Thus, temperature changes fish
26
immunity and affects disease resistance. We evaluate in this work the season variations
27
of the European sea bass (Dicentrarchus labrax) humoral innate parameters focusing on
28
winter months, at which the culture of this specie is more difficult. Our results showed
29
that not all the innate immune parameters are depressed by low temperatures. Moreover,
1
ACCEPTED MANUSCRIPT 1
some of them are more dependent than others to the season and both temperature and
2
photoperiod are operating together.
3 Seasonality dominates the life cycle of animals, mainly in the case of
5
poikilotherms such as fish. It is now widely demonstrated that season cycles affect to
6
fish physiology including animal behavior, body weight and food intake, reproduction,
7
locomotor activity or immunity [1]. Several natural factors may influence these cycles
8
but photoperiod and temperature are the most important. Focusing on temperature, it is
9
generally accepted that the innate immune response is independent of the temperature
10
whilst the adaptive response is dependent on it. However, low temperature has been
11
associated with an increase in fish susceptibility to disease [2, 3] suggesting that both
12
innate and adaptive immunity are affected by temperature. Thus, the influence of
13
temperature on fish immunity has been described and varied with the fish species [4-6].
14
Otherwise, temperature also represents a crucial factor in the survival and infective
15
ability of several pathogens, as occurs with viral hemorrhagic septicemia virus (VHSV),
16
some alphavirus or some bacteria such as Vibrio sp, Flavobacterium psychorophilum or
17
Moritella viscose which cause disease when water temperature drops [7-11]. All this
18
data highlight the relevance of water temperature in fish health, but little is known in the
19
case of Mediterranean aquaculture species, mainly gilthead seabream (Sparus aurata)
20
and European sea bass (Dicentrarchus labrax). In the case of gilthead seabream, it was
21
reported that the complement and agglutination activities of serum were lowest in
22
winter months whilst the lysozyme activity was similar during all the year [5]. In sea
23
bass, there is only a study reporting the effect of seasonality on cortisol, hematocrit,
24
leucocrit, serum lysozyme activity and total glutathione [12]. Due to the economical
25
importance of the European sea bass in the Mediterranean aquaculture and the season
26
cycles on fish immunity and health, we propose in this study the evaluation of several
27
humoral innate parameters of European sea bass at different seasons but focusing on
28
winter, the most challenging for fish culture.
AC C
EP
TE D
M AN U
SC
RI PT
4
29
Healthy specimens of European sea bass were bred at the Centro Oceanográfico
30
de Murcia with natural conditions of photoperiod, temperature, salinity and aeration.
31
One year old fish were sampled once a month (n=10 fish/month) from September
32
(121±13 g body weight (bw)) to May (225±21 g bw). The specimens were bred with a
33
commercial pellet dry diet (Skretting) ad libitum. Temperature, mortality and food 2
ACCEPTED MANUSCRIPT intake were recorded daily. Mean temperature in each month was represented. The
2
specimens were anesthetized with 40 µl L-1 of clove oil before handling. The blood was
3
obtained from the caudal peduncle and the serum samples, obtained by centrifugation
4
(10,000 g, 1 min, 4 ºC), immediately frozen in liquid nitrogen and stored at -80 ºC until
5
use. Protease activity was determined as the percentage of hydrolysis of azocasein by 2
6
mg/ml of proteinase K [13]. Total anti-protease activity was determined as the
7
percentage of inhibition of the hydrolysis of azocasein by 2 mg/ml of proteinase K [14].
8
Haemolytic activity of complement was assayed using sheep red blood cells (SRBC;
9
Biomedics) as targets [15]. The results were expressed in ACH50 units, as the titre at
10
which 50 % haemolysis is produced [16]. Lysozyme activity was measured according to
11
a turbidimetric method that uses the lysis of Micrococcus lysodeikicus for determination
12
of the lysozyme activity using hen egg-white lysozyme as standard [17]. Serum
13
antibacterial activity was determined by evaluating the inhibition on the bacterial
14
growth of Vibrio harveyi curves [16]. The peroxidase activity was measured with a
15
method previously described [18]. Data were presented as mean ± S.E.M (n = 10) and
16
significance analysed by ANOVA (p
