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High carotenoids content can enhance resistance of selected Pinctada fucata families to high temperature stress
Accepted Manuscript High carotenoids content can enhance resistance of selected Pinctada fucata families to high temperature stress Zihao Meng, Bo Zhang, Baosuo Liu, Haimei Li, Sigang Fan, Dahui Yu PII:
S1050-4648(16)30794-X
DOI:
10.1016/j.fsi.2016.12.032
Reference:
YFSIM 4373
To appear in:
Fish and Shellfish Immunology
Received Date: 27 September 2016 Revised Date:
6 December 2016
Accepted Date: 23 December 2016
Please cite this article as: Meng Z, Zhang B, Liu B, Li H, Fan S, Yu D, High carotenoids content can enhance resistance of selected Pinctada fucata families to high temperature stress, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2016.12.032. 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.
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High carotenoids content can enhance resistance of selected
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Pinctada fucata families to high temperature stress
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Zihao Menga,b，Bo Zhanga，Baosuo Liua, Haimei Lia,b, Sigang Fana, Dahui Yua*
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a
Key Laboratory of South China Sea Fishery Resources Exploitation &
Utilization, Ministry of Agriculture; South China Sea Resource
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Exploitation
Protection
Collaborative
Innovation
Center
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and
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(SCS-REPIC); South China Sea Fisheries Research Institute, Chinese
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Academy of Fishery Sciences, Guangzhou 510300, China
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b
College of Fisheries and Life Science, Shanghai Ocean University,
Shanghai 201306, China
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Running title: Resistance to high temperature stress in Pinctada fucata
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*Corresponding author:
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Dr. D.H. YU
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South China Sea Fisheries Research Institute, Chinese Academy of
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Fishery Sciences, Guangzhou 510300, China
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E-mail: [email protected]
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Tel: +86-20-89103420; Fax: +86-20-84451442
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ABSTRACT
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Carotenoids are a class of natural antioxidants widely found in aquatic, and
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they have significant effects on the growth, survival, and immunity of these
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organisms. To investigate the mechanisms of carotenoids in high temperature
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resistance, we observed the immune response of selected pearl oyster Pinctada
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fucata (Akoya pearl oyster) families with different carotenoids contents to high
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temperature stress. The results indicated that the survival rate (SR) of P. fucata
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decreased significantly with increase in temperature from 26 °C to 34 °C and
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with the decrease of total carotenoids content (TCC); when the TCC was higher,
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the SR tended to be higher. TCC and total antioxidant capacity (TAC)
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decreased significantly at 30 °C with increasing stress time. Correlation
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analysis indicated that TAC was positively and linearly correlated with TCC,
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and SR was S-type correlated with TCC and TAC. Immune analysis indicated
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that
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malondialdehyde (MDA) in selected families (with higher TCC) under
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temperature stress (at 30 °C) were generally significantly lower than in the
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control group (with lowest TCC) and from 0 to 96 h, the levels of each of these
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substances varied significantly. Levels of SOD, CAT, and MDA within each
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family first rose from 0 to 3 h, then decreased to their lowest point after 24 h, and
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then rose again to their highest levels at 96 h. When TCC was higher, the levels of
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SOD, CAT, and MDA tended to be lower. These findings indicated that
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carotenoids play an important role in improving survival rates of P. fucata
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under high temperature stress by enhancing animals’ antioxidant system, and
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could serve as an index for breeding stress-resistant lines in selective breeding
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practices.
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Keywords: Pinctada fucata families; carotenoids; high temperature stress;
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immune response; antioxidants
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1. Introduction As an economically important shellfish for the production of
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seawater pearls in China, pearl oysters Pinctada fucata are mainly
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cultured on the Guangxi, Guangdong, and Hainan coasts [1]. In recent
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years, farming of P. fucata has been facing severe challenges owing to
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dramatic environmental changes such as rising temperatures [2]. As one of
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the most important environmental factors in aquaculture, temperature has
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significant effects on the survival and immunity of shellfish [3].
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Temperature changes, particularly rising summer temperatures, can cause
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the organism to generate a number of reactive oxygen species (ROS) and
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free radicals, and die as a result. Accumulation of ROS and free radicals
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can cause lipid peroxidation, cell and tissue damage, and substantial
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decreases in organisms’ immunocompetence [4-5]. The antioxidant system
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is one of the most important immune systems in shellfish [6] and its most
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important role is to remove the ROS and free radicals using intracellular
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antioxidant enzymes and/or non-enzymatic natural antioxidants [7].
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Superoxide dismutase (SOD) and catalase (CAT) are among the
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most important antioxidant enzymes, whose activities are closely
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associated with shellfish immunity [8]. Malondialdehyde (MDA) is the
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final decomposition product of lipid peroxidation and its accumulation
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severely damages protein and enzyme structure thereby influencing their
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biological functions. Therefore, MDA is an important index for 3
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measuring the peroxidation level in an organism [9]. Currently, research on
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various types of marine shellfish indicates that temperature could
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influence their survival, malondialdehyde content, and the activity of their
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antioxidant enzymes [10-13]. Thus, once an organism encounters adverse
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environments, wounds, or infection, its antioxidant enzymes, like SOD
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and CAT, will first respond to remove the ROS and free radicals.
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Carotenoids are a class of important bioactive substances, i.e.
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non-enzymatic natural antioxidants, widely found in the tissues of aquatic
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animals [14]. Their molecules contain multiple conjugated double bonds,
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which are able to quench the ROS and remove the free radicals efficiently
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. Carotenoids can strengthen organisms’ antioxidant ability [16].
Relevant results have been found in aquatic species such as Cottus gobio [17]
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, Litopenaeus vannamei [18], Anadara inaequivalvis [19], but to date,
reports of carotenoids in P. fucata are rare. In our research, we have found
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that some P. fucata individuals of selective breeding families exhibit color
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polymorphism from white to orange in the adductor muscle. Further
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analysis indicated that the color polymorphism was attributable to
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differences in carotenoids levels. For this reason, we have selected and
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bred the lines with golden yellow adductor muscles. In the southern
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region of China, particularly on Hainan Island, the seawater temperature
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generally reaches more than 30 °C in summer, thus leading to a high
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mortality of P. fucata in culture practice. During the selection and
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breeding process, we have preliminarily discovered that families with a
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darker yellow colored adductor muscle have significantly higher survival
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rates during the high temperature period. Thus, we conducted research on
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the survival rate and immune responses of individuals with different
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carotenoids contents under high temperature stress to provide a scientific
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basis for selection and breeding of high-temperature-resistant lines.
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2. Materials and Method
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2.1 Experimental animals
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In a previous study, we found that the colors of adductor muscles in P.
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fucata varied gradually from white to orange, and further study showed
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that the color differential was caused by variation in carotenoids contents
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(data not published). Thus, selective breeding for high carotenoids
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content was performed, and 15 families were constructed by artificial
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fertilization according to a partial factorial mating design [20-21] with five
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males and five females selected from previously constructed families with
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colors from white to orange, with two duplications (thus 30 families in
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total). The families were cultured in Xincun Port of Hainan Province,
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China, with a method described by Li et al [22]. The carotenoids content in
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the adductor muscle of the parents of the 30 families was measured
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subsequently. The families with parental adductor muscles containing a
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high, intermediate, or low level of carotenoids were selected as the
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experimental group. Two families were selected for each level and
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numbered as F1-6, respectively. The average values of carotenoids
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contents in the parents of the six families were as follows: 56.04, 65.28,
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28.90, 22.65, 9.10, and 15.38 mg·g-1 (dry weight). The individuals subject
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to no selective breeding served as the control group (C). A total of 1800
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individuals of 7-month age were selected from each family for the
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experiment, and were of similar body size with regard to shell length
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(20.97 ± 2.92) mm, shell height (21.94 ± 3.14) mm, shell width (8.15 ±
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1.47) mm, and wet weight (2.17 ± 0.85) g. They were cultured in a
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concrete pond for one week before the experiment with a temperature of
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around 26 °C and salinity around 30‰. Filtered seawater was exchanged
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twice a day before feeding microalgae Platymonas subcordiformis and
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Chlorella vulgaris (1:1) at 9:00 and 18:00 every day.
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2.2 Temperature tolerance of individuals with different carotenoids
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contents
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For the temperature tolerance experiment, the 7-month-old
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individuals were cultured in five 1-m3 concrete ponds that maintained
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temperatures of 26, 28, 30, 32, and 34 °C, respectively. Three biological
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replicates were employed for each temperature and one hundred
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individuals were used for each replicate, totaling 1500 individuals. The
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surviving individuals were counted after 96 h. The survival rate was
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calculated using the following formula:
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Survival Rate = (number of the surviving individuals at the end of
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the experiment / total number of individuals at the beginning of the
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experiment).
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2.3 Immune responses of the individuals with different carotenoids
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contents under high temperature stress
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One hundred juvenile pearl oysters were taken from each family and control group, respectively, and were cultured in a seawater pond at 30 °C.
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Three replicates were performed. Four individuals were sampled from
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each replicate after 0, 3, 6, 12, 24, 48, and 96 h. Subsequently, the soft
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parts (including pallium, gill, etc.) were removed after dissecting, and
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were preserved in liquid nitrogen for detection of total antioxidant
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capacity (TAC), activities of superoxide dismutase (SOD) and catalase
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(CAT), and the malondialdehyde (MDA) content and total carotenoids
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content (TCC).
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2.4 Determination of immune indexes and total carotenoids content
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2.4.1 Determination of immune indexes
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Four individuals of each sample were processed. About 0.50 g of
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each sample was placed in a 5 ml homogenate tube. Normal saline was
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added to the ratio of mass (g): volume (ml) = 1:9. The sample was fully
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homogenized on ice, transferred to a centrifugal tube, and centrifuged for
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15 min at 4000 r·min-1. The supernatant was used to prepare about 5 ml
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of 10% tissue homogenate, from which 50 µl, 100 µl, and 40 µl were 7
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taken to determine the contents of TAC, SOD, and MDA, respectively.
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For the CAT test, 1 ml of 10% tissue homogenate was diluted to 2%
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tissue homogenate with saline, from which 50 µl was taken to detect the
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CAT activity. All immune indexes were determined with the kits
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manufactured by the Nanjing Jiancheng Bioengineering Research
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Institution in accordance with the kit instructions.
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2.4.2 Determination of total carotenoids content (TCC)
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TCC was determined in accordance with the method proposed by
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Yanar et. al [23]. The main process was as follows: approximately
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0.20–0.30 g of the aforementioned ground sample was taken. Equal
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amounts of anhydrous sodium sulfate and 5 ml of acetone (analytically
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pure) were fully homogenized with the ground sample, and 5 ml of
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acetone (analytically pure) was used to wash the homogenizer twice,
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which was transferred to a 10 ml brown EP tube with a plug, preserved
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for 3 d at 25 °C in the dark, and centrifuged for 5 min at 5000 r·min-1.
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The supernatant was taken to be scanned in a UV-Vis recording
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spectrophotometer (UV2501PC, SHIMADZU, Japan) from 400 to 700
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nm. Finally, TCC (µg·g-1) was calculated at the absorption value of 480
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nm. Its computational formula was as follows:
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TCC (µg·g-1) = A λ=480 nm× K × V/ (E ×G),
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Where A λ= 480 nm is the absorbance value at λ = 480 nm; K is a
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constant (104); V is the volume of the extracting solution (ml); E is the
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extinction coefficient (1900); and G is the sample mass (g).
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2.5 Statistical analysis
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The results were subjected to one-way analysis of variance (ANOVA) and data was given as mean ± standard error (SE). Additionally,
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correlations among the survival rate, TCC, and TAC were analyzed using
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Pearson correlation. All statistical analyses were done on SPSS Software
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for Windows (SPSS, 21.0, IBM, USA) and significance for all analyses
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was set to P < 0.05 unless noted otherwise.
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3. Results
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3.1 Effects of high temperature stress on the survival rate of selected P.
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fucata families with different carotenoids levels
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The survival rates of selected P. fucata families with different
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carotenoids contents are shown in Table 1. The survival rates of all
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families declined with increasing temperature. The survival rates of F1-6
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and the control group were significantly different (P < 0.05) at all
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temperatures. Compared with the control group, the F1-6 families had a
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much higher survival rate (P < 0.05). The families with higher
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carotenoids contents had higher survival rates. There were remarkable
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differences among various families at different TCC levels (P < 0.05).
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Differences in survival rates among various families with the same TCC
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level, however, were not substantial (P > 0.05).
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3.2 Effects of high temperature stress on TCC and TAC of selected P.
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fucata families with different carotenoids levels
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Figure 1 shows the variation of TCC and TAC in selected P. fucata
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families with stress time at temperatures of 30 °C. TCC in the control
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group and all the 6 experimental groups (F1-6) declined significantly with
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increasing stress time from 0–96 h (P < 0.05), as indicated by lowercase
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letters in Fig. 1A, and reached a minimum as follows: 2.017, 8.469, 8.549,
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3.391, 3.259, 2.829, and 2.866 µg·g-1 after 96 h, respectively. TCC levels
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in the experimental group were significantly higher than those in the
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control group at the same corresponding points in time (P < 0.05). The
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differences between families with different carotenoids levels were
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significant at the same points in time as indicated by bars having different
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capital letters in Fig. 1A, but the differences between two families with
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the same carotenoids level (including high (F1-2), intermediate (F3-4)
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and low (F5-6) levels) were not significant (P > 0.05) at some points in
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time as indicated by bars having the same capital letters in Fig. 1A.
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Similar to TCC, TAC in the experimental group and the control
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group declined significantly with increasing stress time and reached a
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minimum as follows: 0.225, 1.238, 1.238, 0.559, 0.559, 0.267, and 0.266
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U·mgprot-1 after 96 h (Fig. 1B), respectively. TAC was much higher in 10
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the experimental group than in the control group after the same stress
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time (P < 0.05). There were remarkable differences in TAC between the
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selected P. fucata families with different carotenoids levels (P < 0.05).
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The difference in TAC between the two families with the same TCC level
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was not significant (P > 0.05).
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3.3 Effects of high temperature stress on the activity of antioxidant
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enzymes in selected P. fucata families with different carotenoids levels
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Figure 2 shows the variation of SOD and CAT activity of the
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selected P. fucata families with the stress time. As seen in Fig. 2A and 2B,
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the SOD and CAT activity levels in the control group increased gradually
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with increasing stress time, while the experimental group exhibited a
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varying trend of increase-decrease-increase, reaching the first peak after 3
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h, then decreasing to its lowest levels after 24 h, and then reaching its
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highest after 96 h. Generally, the activities of SOD and CAT were lower
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in selected families than in the control; when TCC was higher, the SOD
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and CAT activity levels were generally lower, especially for CAT. The
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enzyme activities were significantly different between families at the
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same TCC level at the same point in time as indicated by the capital
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letters in Fig. 2. However, the enzyme activities in some families with the
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same carotenoids level were not significantly different at the same stress
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times as indicated by lowercase letters in Fig. 2.
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3.4 Effect of high temperature stress on MDA in selected P. fucata
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families with different carotenoids levels As shown in Fig. 3, the MDA in the control group and selected
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families increased gradually with increasing stress time. Similar to CAT,
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MDA content in selected families was significantly lower than in the
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control (P < 0.05); when TCC was higher, MDA content was generally
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lower. The MDA content was significantly different between families
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with the same TCC level at the same point in time as indicated by the
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capital letters in Fig. 3. However, the MDA content in some families with
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the same carotenoids level was not significantly different at the same
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stress times as indicated by lowercase letters in Fig. 3.
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3.5 Correlations among survival rate, TAC, and TCC in selected P. fucata
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with different carotenoids levels
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Pearson correlation coefficients among the survival rate (SR), TAC,
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and TCC in P. fucata are listed in Table 2, indicating that they exhibited
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positive correlations (P < 0.05). The survival rate exhibited S-like
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relationships with both TCC and TAC (Fig. 4A and 4B). The inflection
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points for SR-TCC and SR-TAC curves were at 56.05 µg·g-1 and 3.66
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U·mgprot-1, respectively. The equation for survival rate plotted against
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TCC was y = 24.34ln(x) + 2.00 and the equation for survival rate plotted
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against TAC was y = 33.04ln(x) + 57.17. TAC exhibited a linear
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relationship with TCC, with its linear equation being y = 0.06x + 0.27, as
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shown in Fig. 4C.
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4 Discussion
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4.1 Resistance of selected P. fucata families with different carotenoids
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levels to high temperature stress
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Shellfish are poikilotherms, which means that their physiological
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activities are influenced by the surrounding temperature [24]. In our
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research, the survival rate of carotenoids-enriched P. fucata decreased
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slowly with temperatures rising from 26 °C to 34 °C. A lower carotenoids
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content led to a substantially decreased survival rate (Table 1). When the
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temperature was higher than 32 °C, the survival rate in the control group
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was only 0.5%–1% while the survival rates in the experimental groups
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ranged from 10% to 50% (Table 1). However, the survival rates in test
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groups were over 30% when temperature was lower than 28 °C, with the
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highest being 98.4% in the family of the highest carotenoids content. The
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resistance to high temperature stress in the experimental group was much
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higher than that in the control group (P < 0.05). The survival rate of the
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selectively bred P. fucata families with high, intermediate, and low
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carotenoids levels cultured in Xincun Port, Hainan Island were 80.25%,
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67.25%, and 60.25%, respectively, while the survival rates of the previous
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selectively bred high-growth lines were only 30%–40% in September and
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October (the seawater temperature is usually high during these months)
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(data not published), indicating that carotenoids can enhance resistance of
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P. fucata to high temperatures. Similar situations have also been found in Haida Golden Scallop
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Patinopecten yessoensis [25-26]. The survival rate of “Haida Golden
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Scallop” with higher carotenoids content was 86.7% while that of the
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ordinary P. yessoensis is only 60%. In addition, the survival rates of
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Haliotis discus hannai Ino [27] and Lutraria sieboldii [ 28] also decrease
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substantially under high temperature stress. High temperature stress can
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aggravate the oxidative stress, which severely damages the tissue or cells
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of shellfish and significantly influences their survival. Carotenoids can
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remove the ROS and free radicals produced by oxidative stress as a
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means of reducing the damage to the organism [29]. This study found that
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TCC exhibits a significant decreasing trend with increasing stress time (P
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<0.05) (Fig. 1), indicating that carotenoids were consumed significantly
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with increasing temperature. It also indicated that carotenoids play a
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positive role for shellfish under high temperature stress. The correlation
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analysis further indicates that TCC and the survival rate exhibit a
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curvilinear correlation (Fig. 4), which means that the survival rate will
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rapidly increase with the increasing TCC to a plateau of 56.05 µg·g-1, an
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inflection point that can be used as a selective breeding index.
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4.2 Immune responses of selected P. fucata with different carotenoids
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levels to high temperature stress
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The antioxidant system is an important regulatory mechanism of the
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oxidization/reduction balance in organisms. The antioxidant defensive
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system automatically maintains the balance between oxidization and
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reduction by utilizing antioxidant components including enzymes and
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non-enzymes, which are able to eliminate the ROS and free radicals [7]. As
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an overall measure of the antioxidant level in an organism, TAC
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represents the antioxidant ability, including all enzymes and non-enzymes,
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within the organism. In our research, TAC, both in the control group and
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in the experimental group, decreases with increasing stress time (Fig. 2),
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indicating that antioxidant ability is gradually consumed under high
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temperature stress. The data from different points in stress time shows
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that a higher TCC would lead to a higher TAC, displaying a linear
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correlation. Research on Chlamys nobilis by Zheng [30] et al. and Zhang
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et al. has also shown that TAC and TCC exhibit a significant positive
correlation. TAC and the survival rate (SR) (at 30 °C) exhibit a
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curvilinear positive correlation (Fig. 4), indicating that SR will reach an
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inflection point with increasing TAC. Thus, the survival rate tends to be
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stable with the increase in TAC. It can reach a limit value, which is
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consistent with similar changes in TCC (Fig. 4). Therefore, the TAC at
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the inflection point of SR (3.66 U·mgprot-1) can also serve as a threshold
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index for selective breeding of stress-resistant lines.
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SOD and CAT are important antioxidant enzymes in organisms [32-33]. In our research, the SOD and CAT activities in the control group
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constantly increased with increasing stress time (Fig. 2), indicating that
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they responded positively and rapidly in normal individuals under high
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temperature stress. A similar situation has also been observed in
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Chamelea gallina [34] in which the SOD activity increased significantly
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when the temperature rose from 20 °C to 30 °C. However, the SOD and
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CAT activities in the selected families with higher TCC are significantly
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lower than those in the control group (P < 0.05), and when TCC was
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higher, the SOD and CAT activity levels were generally lower, indicating
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that TCC is responsible in part for high temperature stress. Thus, SOD
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and CAT showed low responses at the presence of carotenoids. It has also
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been observed that the SOD activity (176.59 U·g-1) in the experimental
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group is lower than that in the control group (328.37 U·g-1) after
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Hyriopsis cumingii [35] were fed with β-carotenoid in different
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concentrations. In addition, research on Anadara inaequivalvis by
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Gostyukhina [19] et al. has also found that a higher carotenoids content in
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the organism tissue would lead to a lower activity of the key antioxidant
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enzymes such as SOD and CAT. The research indicates that carotenoids
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can take the place of SOD and CAT in antioxidant system of shellfish to
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some extent [36].
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Malondialdehyde (MDA) is the product of peroxidation of lipids in
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organisms under stress, reflecting the influence of high temperature stress
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on individuals. A higher MDA level indicates greater stress on the
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organism. In this study, the MDA content in the control group increased
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significantly and rapidly and tended to be stable with ongoing stress,
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indicating that the members of the control group generated a stress
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response, enhancing lipid peroxidation. The variation of MDA in the
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experimental group was similar to that of the control group, but the MDA
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levels in the experimental group were significantly lower than in the
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control group (P < 0.05). Higher carotenoids content would lead to a
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lower MDA. There were significant differences among the families with
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different TCC levels (P < 0.05), but no significant differences among the
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families with the same TCC level (P > 0.05). These findings indicate that
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carotenoids are able to resist the peroxidation of lipids in response to high
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temperature stress. It has been reported that the MDA content in
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Adamussium colbecki and Pecten jacobaeus containing 813.76 µg·g-1 and
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454.93 µg·g-1 carotenoids, respectively, increased significantly when the
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temperature rose from 0 °C to 25 °C. The MDA content in P. jacobaeus
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was nearly twice as high as in A. colbecki [37], indicating that shellfish
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with higher carotenoids levels will have lower MDA levels in response to
373
high temperature stress. In other words, carotenoids can protect
374
organisms in adverse environments.
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4.3 Concluding remarks In summary, high temperature resistance, as an essential objective
377
for breeding shellfish like P. fucata, has a direct bearing on the survival of
378
shellfish in summer. The results obtained from our study suggest that the
379
antioxidant system, including enzymes and non-enzymes, participated in
380
maintaining the oxidization/reduction balance when P. fucata was being
381
exposed to high temperature stress. Furthermore, a more interesting and
382
significant result in the present study is that there were significantly
383
positive correlations among the survival rate, TAC, and TCC in P. fucata,
384
which demonstrated that carotenoids, as a kind of antioxidant active
385
substance, are an important part of the antioxidant system in P. fucata.
386
That may be the reason why carotenoids play a positive role in
387
strengthening the ability of the pearl oyster to resist adverse environments.
388
Thus, the P. fucata with high carotenoids content or high total antioxidant
389
capacity can be selectively bred for stress-resistant lines in the future.
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Competing interests
398
The authors declare that they have no competing interests.
399
Acknowledgments
401
This work was supported by the Earmarked Fund for China Agriculture
402
Research System (grant no. CARS-48), Special Fund for Marine
403
Fisheries Research and Extension of Guangdong Province (Z2014006,
404
Z2015006,
405
Agro-scientific Research in the Public Interest (2015TS08) and Special
406
Fund by Sanya Government (2014KS04).
and
407
409
415 416 417
for
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Table list
525
Table 1
526
Resistance to high temperature stress in the pearl oyster Pinctada fucata
527
with different carotenoids contents
528
Note：Different capital letters indicate significant differences ( P＜0.05)
529
in different families with same temperature, the same capital letter
530
indicates no significant difference (P＞0.05); Different lowercase letters
531
indicate significant differences (P＜0.05) in different temperatures with
532
same family, the same lowercase letter indicates no significant difference
533
(P＞0.05)
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Table2
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Correlations between survival rate, TAC and TCC in the pearl oyster
537
Pinctada fucata
538
Note: ** every significant at 0.01 level (P＜0.01)
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Figure legends
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Figure.1. Effects of stress time on TCC and TAC in the pearl oyster
544
Pinctada fucata. (A), (B), represented the changes of TCC and TAC in
545
the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃.
546
The bars represented the mean±SD (n=3). Different capital letters
547
represented significant differences (P＜0.05) in different families with
548
same stress time, the same capital letter indicates no significant difference
549
(P ＞ 0.05) and different lowercase letters represented significant
550
differences (P＜0.05) in different stress times with same family ,the same
551
lowercase letter indicates no significant difference (P＞0.05).
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Figure.2. Effects of stress time on SOD and CAT in the pearl oyster
554
Pinctada fucata. (A), (B), represented the changes of SOD and CAT in
555
the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃.
556
The bars represented the mean±SD (n=3). Different capital letters
557
represented significant differences (P＜0.05) in different families with
558
same stress time, the same capital letter indicates no significant difference
559
(P＞0.05) and different lowercase letters represented significant
560
differences (P＜0.05) in different stress times with same family, the same
561
lowercase letter indicates no significant difference (P＞0.05).
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562 563
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Figure.3. Effect of stress time on MDA in the pearl oyster Pinctada
565
fucata after 96 h stress at 30 ℃. The bars represented the mean±SD (n=3).
566
Different capital letters represented significant differences (P＜0.05) in
567
different families with same stress time, the same capital letter indicates
568
no significant difference (P ＞ 0.05) and different lowercase letters
569
represented significant differences (P＜0.05) in different stress times with
570
same family, the same lowercase letter indicates no significant difference
571
(P＞0.05).
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Figure. 4. Correlations between the survival rate, TCC and TAC in the
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pearl oyster Pinctada fucata
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ACCEPTED MANUSCRIPT Table 1 Resistance to high temperature stress in the pearl oyster Pinctada fucata with different carotenoids contents Survival rate /%
Total carotenoids Family
Temperature /
content -1
(TCC)/µg·g F1
26 98.4±1.67A a
60.81
28
30
32
34
86.6±0.12A b
80.8±0.31A c
48.4±0.14A d
34.3±2.75A e
73.53
98.4±0.86A a
86.7±0.02A b
80.2±0.13A c
47.8±0.03A d
34.3±2.75A e
F3
35.58
90.2±0.24B a
59.9±0.01B b
55.4±.013B c
23.5±0.38B d
12.1±0.74B e
F4
27.01
90.1±0.01B a
59.9±0.02B b
55.3±0.14B c
22.9±0.94B d
12.1±0.17B e
F5
15.18
80.8±0.17C a
36.5±0.03C b
27.0±0.00C c
12.3±0.19C d
0.5±0.00C e
F6
21.29
80.6±0.04C a
36.5±0.01C b
27.0±0.03C c
11.9±0.62C d
0.5±0.00C e
13.53
Da
Db
Dc
Control/C
45.8±1.36
30.6±0.05
20.4±0.34
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F2
1.2±0.07
Dd
0.5±0.13C d
Note：Different capital letters indicate significant differences ( P＜0.05) in different families with same temperature, the same capital
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letter indicates no significant difference (P＞0.05); Different lowercase letters indicate significant differences (P＜0.05) in different temperatures with same family, the same lowercase letter indicates no significant difference (P＞0.05)
Table 2 Correlations among survival rate, TAC, and TCC in the pearl oyster Pinctada fucata Survival rate (SR)
TAC
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Survival rate (SR)
0.809
Total antioxidant capacity (TAC)
0.809**
Total carotenoids content (TCC)
0.739**
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Note: **. very significant at 0.01 level (P＜0.01)
**
0.951**
TCC 0.739** 0.951**
C F1
B a
B b
60
F2
A b
F3 F4
AA cc
50
F5 F6
C a
40
D a
30
F a
a
B c
D b E b F b G c
E a
20 G
AA dd
C b
G b
10
C c
AA e e BB dd
E Fc E c d
C Dd d D e
0 0
3
6
12
A a
4
A b A b
B a
A c A c
B a
BB bb
C Ca a
D a
B c
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2
1
A d
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3
C Cab b
D b
AC C
Total antioxidant capacity (TAC)/nmol· mgprot
-1
A a
AA BB gg BBCC f f CC D f f gggg g
D f
48
96
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AA f f
24
Time/h
B
BB e e CC ee
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70
A a
SC
80
-1
A Total carotenoids content (TCC) /µg· g )
ACCEPTED MANUSCRIPT
F2 F3 F4 F5
A d
F6 A e
A e
A e
A f
B c
A f
B d
B C e Cb c D c
C F1
B dB e C Cc d D e
D d
C Cd e
BB ee C Ce D f f
D e
A g BB f f C Cf f
0
0
3
6
12
24
48
96
Time/h
Figure.1. Effects of stress time on TCC and TAC in the pearl oyster Pinctada fucata. (A), (B), represented the changes of TCC and TAC in the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃. The bars represented the mean±SD (n=3). Different capital letters
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represented significant differences (P＜0.05) in different families with same stress time, the same capital letter indicates no significant difference (P ＞ 0.05) and different lowercase letters represented
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significant differences (P＜0.05) in different stress times with same family, the same lowercase letter indicates no significant difference (P
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＞0.05).
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A e
120 100
A f
AB A b CD b e bb
CC gf
F5
F6
A a A c
A d
DD cc BC DD e f ee
EE cd
BB DD f e f e
EE de
0 6
48
24
12
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EE aa
EE cc
EE de
20
0
CD aa
BB cc
CD dd
DD ef
BB aa
A b
EE bb
60 40
F4
BB dd
BB gf
80
F2
F3
SC
Superoxide dismutase(SOD)/nmol· mgprot
140
C F1
RI PT
160
-1
A
96
Time/h
B
90
70
40
f
F3 F4 F5 F6
BB aa
BB bb
30 A
BB de
DD bb
AC C
CC ef
BB bb
CC aa
BB cc
CC bb
DD f f
C F1 F2
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50
10
A d
A e
60
20
A b
A c
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Catalase(CAT)/nmol· mgprot
-1
80
A a
A b
BB dd
CC dd DD dd
DD dd
CC ee
DD ee
CC ee
CC cc
BB de DD cc
DD aa
0
0
3
6
12
24
48
96
Time/h
Figure.2. Effect of stress time on SOD and CAT in the pearl oyster Pinctada fucata. (A), (B), represented the changes of SOD and CAT in the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃. The bars represented the mean±SD (n=3). Different capital letters
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represented significant differences (P＜0.05) in different families with same stress time, the same capital letter indicates no significant difference (P＞0.05) and different lowercase l letters represented significant
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differences (P＜0.05) in different stress times with same family, the same
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F2
F3
F4
F5
F6
A b
A c
A c
A d
A e
A a
B Ba a
20
15
10 A f
5
CC cc
A Af Bf Be e
DD bb
B Bc c
B Bd d
B Bd Cd Cc c
DD dd
B Be e
C Ce f D deD d
C Ce f
0 3
6
12
DD ee
24
C Ca a
C Cb b
DD bb
48
DD aa
96
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0
C Cd d
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B Bb b
SC
Malonic dialdehyde(MDA)/nmol· mgprot
-1
30
C F1
Time/h
Figure.3. Effect of stress time on MDA in the pearl oyster Pinctada fucata after 96 h stress at 30 ℃. The bars represented the mean±SD
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(n=3). Different capital letters represented significant differences (P＜ 0.05) in different families with same stress time, the same capital letter indicates no significant difference (P＞0.05) and different lowercase
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times with same family, the same lowercase letter indicates no significant difference (P＞0.05).
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A
100
60
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Survival rate(SR)/%
80
y=24.34ln(x)+2.00 2 R =0.775
40
SC
20
0 15
30
45
60
75
M AN U
0
90
-1
Total carotenoids content(TCC)/µg· g
B
100
40
TE D EP
60
y= 33.04ln(x)+57.17 2 R =0.821
20
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Survival rate(SR)/%
80
0
0
1
2
3
4 -1
Total antioxidant capacity(TAC)/U· mgprot
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4
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3
2
y=0.27+0.06x 2 R =0.905 1
0 15
30
45
60
M AN U
0
SC
Total antioxidant capacity(TAC)/U· mgprot
-1
C
75
90
-1
Total carotenoids content(TCC)/µg· g
Figure. 4. Correlations between the survival rate, TCC and TAC in the
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Highlights
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Indexes to selectively breed high temperature resistance in shellfish are proposed. Antioxidant enzymes had low activity in the presence of carotenoids. High temperature stress in Pinctada fucata might be mitigated by carotenoids.
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• • •
S1050-4648(16)30794-X
DOI:
10.1016/j.fsi.2016.12.032
Reference:
YFSIM 4373
To appear in:
Fish and Shellfish Immunology
Received Date: 27 September 2016 Revised Date:
6 December 2016
Accepted Date: 23 December 2016
Please cite this article as: Meng Z, Zhang B, Liu B, Li H, Fan S, Yu D, High carotenoids content can enhance resistance of selected Pinctada fucata families to high temperature stress, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2016.12.032. 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
High carotenoids content can enhance resistance of selected
1 2
Pinctada fucata families to high temperature stress
3 4
Zihao Menga,b，Bo Zhanga，Baosuo Liua, Haimei Lia,b, Sigang Fana, Dahui Yua*
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a
Key Laboratory of South China Sea Fishery Resources Exploitation &
Utilization, Ministry of Agriculture; South China Sea Resource
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Exploitation
Protection
Collaborative
Innovation
Center
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and
SC
8
10
(SCS-REPIC); South China Sea Fisheries Research Institute, Chinese
11
Academy of Fishery Sciences, Guangzhou 510300, China
13
b
College of Fisheries and Life Science, Shanghai Ocean University,
Shanghai 201306, China
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Running title: Resistance to high temperature stress in Pinctada fucata
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*Corresponding author:
18
Dr. D.H. YU
19
South China Sea Fisheries Research Institute, Chinese Academy of
20
Fishery Sciences, Guangzhou 510300, China
21
E-mail: [email protected]
22
Tel: +86-20-89103420; Fax: +86-20-84451442
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ABSTRACT
25
Carotenoids are a class of natural antioxidants widely found in aquatic, and
26
they have significant effects on the growth, survival, and immunity of these
27
organisms. To investigate the mechanisms of carotenoids in high temperature
28
resistance, we observed the immune response of selected pearl oyster Pinctada
29
fucata (Akoya pearl oyster) families with different carotenoids contents to high
30
temperature stress. The results indicated that the survival rate (SR) of P. fucata
31
decreased significantly with increase in temperature from 26 °C to 34 °C and
32
with the decrease of total carotenoids content (TCC); when the TCC was higher,
33
the SR tended to be higher. TCC and total antioxidant capacity (TAC)
34
decreased significantly at 30 °C with increasing stress time. Correlation
35
analysis indicated that TAC was positively and linearly correlated with TCC,
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and SR was S-type correlated with TCC and TAC. Immune analysis indicated
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that
38
malondialdehyde (MDA) in selected families (with higher TCC) under
39
temperature stress (at 30 °C) were generally significantly lower than in the
40
control group (with lowest TCC) and from 0 to 96 h, the levels of each of these
41
substances varied significantly. Levels of SOD, CAT, and MDA within each
42
family first rose from 0 to 3 h, then decreased to their lowest point after 24 h, and
43
then rose again to their highest levels at 96 h. When TCC was higher, the levels of
44
SOD, CAT, and MDA tended to be lower. These findings indicated that
45
carotenoids play an important role in improving survival rates of P. fucata
46
under high temperature stress by enhancing animals’ antioxidant system, and
47
could serve as an index for breeding stress-resistant lines in selective breeding
48
practices.
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Keywords: Pinctada fucata families; carotenoids; high temperature stress;
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immune response; antioxidants
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1. Introduction As an economically important shellfish for the production of
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seawater pearls in China, pearl oysters Pinctada fucata are mainly
56
cultured on the Guangxi, Guangdong, and Hainan coasts [1]. In recent
57
years, farming of P. fucata has been facing severe challenges owing to
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dramatic environmental changes such as rising temperatures [2]. As one of
59
the most important environmental factors in aquaculture, temperature has
60
significant effects on the survival and immunity of shellfish [3].
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Temperature changes, particularly rising summer temperatures, can cause
62
the organism to generate a number of reactive oxygen species (ROS) and
63
free radicals, and die as a result. Accumulation of ROS and free radicals
64
can cause lipid peroxidation, cell and tissue damage, and substantial
65
decreases in organisms’ immunocompetence [4-5]. The antioxidant system
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is one of the most important immune systems in shellfish [6] and its most
67
important role is to remove the ROS and free radicals using intracellular
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antioxidant enzymes and/or non-enzymatic natural antioxidants [7].
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Superoxide dismutase (SOD) and catalase (CAT) are among the
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most important antioxidant enzymes, whose activities are closely
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associated with shellfish immunity [8]. Malondialdehyde (MDA) is the
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final decomposition product of lipid peroxidation and its accumulation
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severely damages protein and enzyme structure thereby influencing their
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biological functions. Therefore, MDA is an important index for 3
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measuring the peroxidation level in an organism [9]. Currently, research on
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various types of marine shellfish indicates that temperature could
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influence their survival, malondialdehyde content, and the activity of their
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antioxidant enzymes [10-13]. Thus, once an organism encounters adverse
79
environments, wounds, or infection, its antioxidant enzymes, like SOD
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and CAT, will first respond to remove the ROS and free radicals.
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Carotenoids are a class of important bioactive substances, i.e.
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non-enzymatic natural antioxidants, widely found in the tissues of aquatic
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animals [14]. Their molecules contain multiple conjugated double bonds,
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which are able to quench the ROS and remove the free radicals efficiently
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. Carotenoids can strengthen organisms’ antioxidant ability [16].
Relevant results have been found in aquatic species such as Cottus gobio [17]
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, Litopenaeus vannamei [18], Anadara inaequivalvis [19], but to date,
reports of carotenoids in P. fucata are rare. In our research, we have found
89
that some P. fucata individuals of selective breeding families exhibit color
90
polymorphism from white to orange in the adductor muscle. Further
91
analysis indicated that the color polymorphism was attributable to
92
differences in carotenoids levels. For this reason, we have selected and
93
bred the lines with golden yellow adductor muscles. In the southern
94
region of China, particularly on Hainan Island, the seawater temperature
95
generally reaches more than 30 °C in summer, thus leading to a high
96
mortality of P. fucata in culture practice. During the selection and
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breeding process, we have preliminarily discovered that families with a
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darker yellow colored adductor muscle have significantly higher survival
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rates during the high temperature period. Thus, we conducted research on
100
the survival rate and immune responses of individuals with different
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carotenoids contents under high temperature stress to provide a scientific
102
basis for selection and breeding of high-temperature-resistant lines.
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2. Materials and Method
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2.1 Experimental animals
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In a previous study, we found that the colors of adductor muscles in P.
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fucata varied gradually from white to orange, and further study showed
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that the color differential was caused by variation in carotenoids contents
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(data not published). Thus, selective breeding for high carotenoids
109
content was performed, and 15 families were constructed by artificial
110
fertilization according to a partial factorial mating design [20-21] with five
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males and five females selected from previously constructed families with
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colors from white to orange, with two duplications (thus 30 families in
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total). The families were cultured in Xincun Port of Hainan Province,
114
China, with a method described by Li et al [22]. The carotenoids content in
115
the adductor muscle of the parents of the 30 families was measured
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subsequently. The families with parental adductor muscles containing a
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high, intermediate, or low level of carotenoids were selected as the
118
experimental group. Two families were selected for each level and
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numbered as F1-6, respectively. The average values of carotenoids
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contents in the parents of the six families were as follows: 56.04, 65.28,
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28.90, 22.65, 9.10, and 15.38 mg·g-1 (dry weight). The individuals subject
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to no selective breeding served as the control group (C). A total of 1800
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individuals of 7-month age were selected from each family for the
124
experiment, and were of similar body size with regard to shell length
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(20.97 ± 2.92) mm, shell height (21.94 ± 3.14) mm, shell width (8.15 ±
126
1.47) mm, and wet weight (2.17 ± 0.85) g. They were cultured in a
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concrete pond for one week before the experiment with a temperature of
128
around 26 °C and salinity around 30‰. Filtered seawater was exchanged
129
twice a day before feeding microalgae Platymonas subcordiformis and
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Chlorella vulgaris (1:1) at 9:00 and 18:00 every day.
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2.2 Temperature tolerance of individuals with different carotenoids
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contents
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For the temperature tolerance experiment, the 7-month-old
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individuals were cultured in five 1-m3 concrete ponds that maintained
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temperatures of 26, 28, 30, 32, and 34 °C, respectively. Three biological
136
replicates were employed for each temperature and one hundred
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individuals were used for each replicate, totaling 1500 individuals. The
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surviving individuals were counted after 96 h. The survival rate was
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calculated using the following formula:
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Survival Rate = (number of the surviving individuals at the end of
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the experiment / total number of individuals at the beginning of the
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experiment).
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2.3 Immune responses of the individuals with different carotenoids
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contents under high temperature stress
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One hundred juvenile pearl oysters were taken from each family and control group, respectively, and were cultured in a seawater pond at 30 °C.
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Three replicates were performed. Four individuals were sampled from
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each replicate after 0, 3, 6, 12, 24, 48, and 96 h. Subsequently, the soft
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parts (including pallium, gill, etc.) were removed after dissecting, and
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were preserved in liquid nitrogen for detection of total antioxidant
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capacity (TAC), activities of superoxide dismutase (SOD) and catalase
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(CAT), and the malondialdehyde (MDA) content and total carotenoids
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content (TCC).
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2.4 Determination of immune indexes and total carotenoids content
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2.4.1 Determination of immune indexes
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Four individuals of each sample were processed. About 0.50 g of
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each sample was placed in a 5 ml homogenate tube. Normal saline was
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added to the ratio of mass (g): volume (ml) = 1:9. The sample was fully
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homogenized on ice, transferred to a centrifugal tube, and centrifuged for
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15 min at 4000 r·min-1. The supernatant was used to prepare about 5 ml
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of 10% tissue homogenate, from which 50 µl, 100 µl, and 40 µl were 7
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taken to determine the contents of TAC, SOD, and MDA, respectively.
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For the CAT test, 1 ml of 10% tissue homogenate was diluted to 2%
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tissue homogenate with saline, from which 50 µl was taken to detect the
165
CAT activity. All immune indexes were determined with the kits
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manufactured by the Nanjing Jiancheng Bioengineering Research
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Institution in accordance with the kit instructions.
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2.4.2 Determination of total carotenoids content (TCC)
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TCC was determined in accordance with the method proposed by
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Yanar et. al [23]. The main process was as follows: approximately
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0.20–0.30 g of the aforementioned ground sample was taken. Equal
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amounts of anhydrous sodium sulfate and 5 ml of acetone (analytically
173
pure) were fully homogenized with the ground sample, and 5 ml of
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acetone (analytically pure) was used to wash the homogenizer twice,
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which was transferred to a 10 ml brown EP tube with a plug, preserved
176
for 3 d at 25 °C in the dark, and centrifuged for 5 min at 5000 r·min-1.
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The supernatant was taken to be scanned in a UV-Vis recording
178
spectrophotometer (UV2501PC, SHIMADZU, Japan) from 400 to 700
179
nm. Finally, TCC (µg·g-1) was calculated at the absorption value of 480
180
nm. Its computational formula was as follows:
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TCC (µg·g-1) = A λ=480 nm× K × V/ (E ×G),
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Where A λ= 480 nm is the absorbance value at λ = 480 nm; K is a
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constant (104); V is the volume of the extracting solution (ml); E is the
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extinction coefficient (1900); and G is the sample mass (g).
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2.5 Statistical analysis
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The results were subjected to one-way analysis of variance (ANOVA) and data was given as mean ± standard error (SE). Additionally,
188
correlations among the survival rate, TCC, and TAC were analyzed using
189
Pearson correlation. All statistical analyses were done on SPSS Software
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for Windows (SPSS, 21.0, IBM, USA) and significance for all analyses
191
was set to P < 0.05 unless noted otherwise.
192
3. Results
193
3.1 Effects of high temperature stress on the survival rate of selected P.
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fucata families with different carotenoids levels
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The survival rates of selected P. fucata families with different
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carotenoids contents are shown in Table 1. The survival rates of all
197
families declined with increasing temperature. The survival rates of F1-6
198
and the control group were significantly different (P < 0.05) at all
199
temperatures. Compared with the control group, the F1-6 families had a
200
much higher survival rate (P < 0.05). The families with higher
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carotenoids contents had higher survival rates. There were remarkable
202
differences among various families at different TCC levels (P < 0.05).
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Differences in survival rates among various families with the same TCC
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level, however, were not substantial (P > 0.05).
205
3.2 Effects of high temperature stress on TCC and TAC of selected P.
206
fucata families with different carotenoids levels
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Figure 1 shows the variation of TCC and TAC in selected P. fucata
208
families with stress time at temperatures of 30 °C. TCC in the control
209
group and all the 6 experimental groups (F1-6) declined significantly with
210
increasing stress time from 0–96 h (P < 0.05), as indicated by lowercase
211
letters in Fig. 1A, and reached a minimum as follows: 2.017, 8.469, 8.549,
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3.391, 3.259, 2.829, and 2.866 µg·g-1 after 96 h, respectively. TCC levels
213
in the experimental group were significantly higher than those in the
214
control group at the same corresponding points in time (P < 0.05). The
215
differences between families with different carotenoids levels were
216
significant at the same points in time as indicated by bars having different
217
capital letters in Fig. 1A, but the differences between two families with
218
the same carotenoids level (including high (F1-2), intermediate (F3-4)
219
and low (F5-6) levels) were not significant (P > 0.05) at some points in
220
time as indicated by bars having the same capital letters in Fig. 1A.
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Similar to TCC, TAC in the experimental group and the control
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group declined significantly with increasing stress time and reached a
223
minimum as follows: 0.225, 1.238, 1.238, 0.559, 0.559, 0.267, and 0.266
224
U·mgprot-1 after 96 h (Fig. 1B), respectively. TAC was much higher in 10
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the experimental group than in the control group after the same stress
226
time (P < 0.05). There were remarkable differences in TAC between the
227
selected P. fucata families with different carotenoids levels (P < 0.05).
228
The difference in TAC between the two families with the same TCC level
229
was not significant (P > 0.05).
230
3.3 Effects of high temperature stress on the activity of antioxidant
231
enzymes in selected P. fucata families with different carotenoids levels
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Figure 2 shows the variation of SOD and CAT activity of the
233
selected P. fucata families with the stress time. As seen in Fig. 2A and 2B,
234
the SOD and CAT activity levels in the control group increased gradually
235
with increasing stress time, while the experimental group exhibited a
236
varying trend of increase-decrease-increase, reaching the first peak after 3
237
h, then decreasing to its lowest levels after 24 h, and then reaching its
238
highest after 96 h. Generally, the activities of SOD and CAT were lower
239
in selected families than in the control; when TCC was higher, the SOD
240
and CAT activity levels were generally lower, especially for CAT. The
241
enzyme activities were significantly different between families at the
242
same TCC level at the same point in time as indicated by the capital
243
letters in Fig. 2. However, the enzyme activities in some families with the
244
same carotenoids level were not significantly different at the same stress
245
times as indicated by lowercase letters in Fig. 2.
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3.4 Effect of high temperature stress on MDA in selected P. fucata
247
families with different carotenoids levels As shown in Fig. 3, the MDA in the control group and selected
249
families increased gradually with increasing stress time. Similar to CAT,
250
MDA content in selected families was significantly lower than in the
251
control (P < 0.05); when TCC was higher, MDA content was generally
252
lower. The MDA content was significantly different between families
253
with the same TCC level at the same point in time as indicated by the
254
capital letters in Fig. 3. However, the MDA content in some families with
255
the same carotenoids level was not significantly different at the same
256
stress times as indicated by lowercase letters in Fig. 3.
257
3.5 Correlations among survival rate, TAC, and TCC in selected P. fucata
258
with different carotenoids levels
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Pearson correlation coefficients among the survival rate (SR), TAC,
260
and TCC in P. fucata are listed in Table 2, indicating that they exhibited
261
positive correlations (P < 0.05). The survival rate exhibited S-like
262
relationships with both TCC and TAC (Fig. 4A and 4B). The inflection
263
points for SR-TCC and SR-TAC curves were at 56.05 µg·g-1 and 3.66
264
U·mgprot-1, respectively. The equation for survival rate plotted against
265
TCC was y = 24.34ln(x) + 2.00 and the equation for survival rate plotted
266
against TAC was y = 33.04ln(x) + 57.17. TAC exhibited a linear
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relationship with TCC, with its linear equation being y = 0.06x + 0.27, as
268
shown in Fig. 4C.
269
4 Discussion
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4.1 Resistance of selected P. fucata families with different carotenoids
271
levels to high temperature stress
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Shellfish are poikilotherms, which means that their physiological
273
activities are influenced by the surrounding temperature [24]. In our
274
research, the survival rate of carotenoids-enriched P. fucata decreased
275
slowly with temperatures rising from 26 °C to 34 °C. A lower carotenoids
276
content led to a substantially decreased survival rate (Table 1). When the
277
temperature was higher than 32 °C, the survival rate in the control group
278
was only 0.5%–1% while the survival rates in the experimental groups
279
ranged from 10% to 50% (Table 1). However, the survival rates in test
280
groups were over 30% when temperature was lower than 28 °C, with the
281
highest being 98.4% in the family of the highest carotenoids content. The
282
resistance to high temperature stress in the experimental group was much
283
higher than that in the control group (P < 0.05). The survival rate of the
284
selectively bred P. fucata families with high, intermediate, and low
285
carotenoids levels cultured in Xincun Port, Hainan Island were 80.25%,
286
67.25%, and 60.25%, respectively, while the survival rates of the previous
287
selectively bred high-growth lines were only 30%–40% in September and
288
October (the seawater temperature is usually high during these months)
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(data not published), indicating that carotenoids can enhance resistance of
290
P. fucata to high temperatures. Similar situations have also been found in Haida Golden Scallop
292
Patinopecten yessoensis [25-26]. The survival rate of “Haida Golden
293
Scallop” with higher carotenoids content was 86.7% while that of the
294
ordinary P. yessoensis is only 60%. In addition, the survival rates of
295
Haliotis discus hannai Ino [27] and Lutraria sieboldii [ 28] also decrease
296
substantially under high temperature stress. High temperature stress can
297
aggravate the oxidative stress, which severely damages the tissue or cells
298
of shellfish and significantly influences their survival. Carotenoids can
299
remove the ROS and free radicals produced by oxidative stress as a
300
means of reducing the damage to the organism [29]. This study found that
301
TCC exhibits a significant decreasing trend with increasing stress time (P
302
<0.05) (Fig. 1), indicating that carotenoids were consumed significantly
303
with increasing temperature. It also indicated that carotenoids play a
304
positive role for shellfish under high temperature stress. The correlation
305
analysis further indicates that TCC and the survival rate exhibit a
306
curvilinear correlation (Fig. 4), which means that the survival rate will
307
rapidly increase with the increasing TCC to a plateau of 56.05 µg·g-1, an
308
inflection point that can be used as a selective breeding index.
309
4.2 Immune responses of selected P. fucata with different carotenoids
310
levels to high temperature stress
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The antioxidant system is an important regulatory mechanism of the
312
oxidization/reduction balance in organisms. The antioxidant defensive
313
system automatically maintains the balance between oxidization and
314
reduction by utilizing antioxidant components including enzymes and
315
non-enzymes, which are able to eliminate the ROS and free radicals [7]. As
316
an overall measure of the antioxidant level in an organism, TAC
317
represents the antioxidant ability, including all enzymes and non-enzymes,
318
within the organism. In our research, TAC, both in the control group and
319
in the experimental group, decreases with increasing stress time (Fig. 2),
320
indicating that antioxidant ability is gradually consumed under high
321
temperature stress. The data from different points in stress time shows
322
that a higher TCC would lead to a higher TAC, displaying a linear
323
correlation. Research on Chlamys nobilis by Zheng [30] et al. and Zhang
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et al. has also shown that TAC and TCC exhibit a significant positive
correlation. TAC and the survival rate (SR) (at 30 °C) exhibit a
326
curvilinear positive correlation (Fig. 4), indicating that SR will reach an
327
inflection point with increasing TAC. Thus, the survival rate tends to be
328
stable with the increase in TAC. It can reach a limit value, which is
329
consistent with similar changes in TCC (Fig. 4). Therefore, the TAC at
330
the inflection point of SR (3.66 U·mgprot-1) can also serve as a threshold
331
index for selective breeding of stress-resistant lines.
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SOD and CAT are important antioxidant enzymes in organisms [32-33]. In our research, the SOD and CAT activities in the control group
334
constantly increased with increasing stress time (Fig. 2), indicating that
335
they responded positively and rapidly in normal individuals under high
336
temperature stress. A similar situation has also been observed in
337
Chamelea gallina [34] in which the SOD activity increased significantly
338
when the temperature rose from 20 °C to 30 °C. However, the SOD and
339
CAT activities in the selected families with higher TCC are significantly
340
lower than those in the control group (P < 0.05), and when TCC was
341
higher, the SOD and CAT activity levels were generally lower, indicating
342
that TCC is responsible in part for high temperature stress. Thus, SOD
343
and CAT showed low responses at the presence of carotenoids. It has also
344
been observed that the SOD activity (176.59 U·g-1) in the experimental
345
group is lower than that in the control group (328.37 U·g-1) after
346
Hyriopsis cumingii [35] were fed with β-carotenoid in different
347
concentrations. In addition, research on Anadara inaequivalvis by
348
Gostyukhina [19] et al. has also found that a higher carotenoids content in
349
the organism tissue would lead to a lower activity of the key antioxidant
350
enzymes such as SOD and CAT. The research indicates that carotenoids
351
can take the place of SOD and CAT in antioxidant system of shellfish to
352
some extent [36].
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Malondialdehyde (MDA) is the product of peroxidation of lipids in
354
organisms under stress, reflecting the influence of high temperature stress
355
on individuals. A higher MDA level indicates greater stress on the
356
organism. In this study, the MDA content in the control group increased
357
significantly and rapidly and tended to be stable with ongoing stress,
358
indicating that the members of the control group generated a stress
359
response, enhancing lipid peroxidation. The variation of MDA in the
360
experimental group was similar to that of the control group, but the MDA
361
levels in the experimental group were significantly lower than in the
362
control group (P < 0.05). Higher carotenoids content would lead to a
363
lower MDA. There were significant differences among the families with
364
different TCC levels (P < 0.05), but no significant differences among the
365
families with the same TCC level (P > 0.05). These findings indicate that
366
carotenoids are able to resist the peroxidation of lipids in response to high
367
temperature stress. It has been reported that the MDA content in
368
Adamussium colbecki and Pecten jacobaeus containing 813.76 µg·g-1 and
369
454.93 µg·g-1 carotenoids, respectively, increased significantly when the
370
temperature rose from 0 °C to 25 °C. The MDA content in P. jacobaeus
371
was nearly twice as high as in A. colbecki [37], indicating that shellfish
372
with higher carotenoids levels will have lower MDA levels in response to
373
high temperature stress. In other words, carotenoids can protect
374
organisms in adverse environments.
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4.3 Concluding remarks In summary, high temperature resistance, as an essential objective
377
for breeding shellfish like P. fucata, has a direct bearing on the survival of
378
shellfish in summer. The results obtained from our study suggest that the
379
antioxidant system, including enzymes and non-enzymes, participated in
380
maintaining the oxidization/reduction balance when P. fucata was being
381
exposed to high temperature stress. Furthermore, a more interesting and
382
significant result in the present study is that there were significantly
383
positive correlations among the survival rate, TAC, and TCC in P. fucata,
384
which demonstrated that carotenoids, as a kind of antioxidant active
385
substance, are an important part of the antioxidant system in P. fucata.
386
That may be the reason why carotenoids play a positive role in
387
strengthening the ability of the pearl oyster to resist adverse environments.
388
Thus, the P. fucata with high carotenoids content or high total antioxidant
389
capacity can be selectively bred for stress-resistant lines in the future.
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Competing interests
398
The authors declare that they have no competing interests.
399
Acknowledgments
401
This work was supported by the Earmarked Fund for China Agriculture
402
Research System (grant no. CARS-48), Special Fund for Marine
403
Fisheries Research and Extension of Guangdong Province (Z2014006,
404
Z2015006,
405
Agro-scientific Research in the Public Interest (2015TS08) and Special
406
Fund by Sanya Government (2014KS04).
and
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409
415 416 417
for
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Table list
525
Table 1
526
Resistance to high temperature stress in the pearl oyster Pinctada fucata
527
with different carotenoids contents
528
Note：Different capital letters indicate significant differences ( P＜0.05)
529
in different families with same temperature, the same capital letter
530
indicates no significant difference (P＞0.05); Different lowercase letters
531
indicate significant differences (P＜0.05) in different temperatures with
532
same family, the same lowercase letter indicates no significant difference
533
(P＞0.05)
M AN U
SC
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524
534
Table2
536
Correlations between survival rate, TAC and TCC in the pearl oyster
537
Pinctada fucata
538
Note: ** every significant at 0.01 level (P＜0.01)
540 541
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Figure legends
543
Figure.1. Effects of stress time on TCC and TAC in the pearl oyster
544
Pinctada fucata. (A), (B), represented the changes of TCC and TAC in
545
the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃.
546
The bars represented the mean±SD (n=3). Different capital letters
547
represented significant differences (P＜0.05) in different families with
548
same stress time, the same capital letter indicates no significant difference
549
(P ＞ 0.05) and different lowercase letters represented significant
550
differences (P＜0.05) in different stress times with same family ,the same
551
lowercase letter indicates no significant difference (P＞0.05).
M AN U
SC
RI PT
542
552
Figure.2. Effects of stress time on SOD and CAT in the pearl oyster
554
Pinctada fucata. (A), (B), represented the changes of SOD and CAT in
555
the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃.
556
The bars represented the mean±SD (n=3). Different capital letters
557
represented significant differences (P＜0.05) in different families with
558
same stress time, the same capital letter indicates no significant difference
559
(P＞0.05) and different lowercase letters represented significant
560
differences (P＜0.05) in different stress times with same family, the same
561
lowercase letter indicates no significant difference (P＞0.05).
AC C
EP
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553
562 563
24
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Figure.3. Effect of stress time on MDA in the pearl oyster Pinctada
565
fucata after 96 h stress at 30 ℃. The bars represented the mean±SD (n=3).
566
Different capital letters represented significant differences (P＜0.05) in
567
different families with same stress time, the same capital letter indicates
568
no significant difference (P ＞ 0.05) and different lowercase letters
569
represented significant differences (P＜0.05) in different stress times with
570
same family, the same lowercase letter indicates no significant difference
571
(P＞0.05).
572
M AN U
SC
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564
Figure. 4. Correlations between the survival rate, TCC and TAC in the
574
pearl oyster Pinctada fucata
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ACCEPTED MANUSCRIPT Table 1 Resistance to high temperature stress in the pearl oyster Pinctada fucata with different carotenoids contents Survival rate /%
Total carotenoids Family
Temperature /
content -1
(TCC)/µg·g F1
26 98.4±1.67A a
60.81
28
30
32
34
86.6±0.12A b
80.8±0.31A c
48.4±0.14A d
34.3±2.75A e
73.53
98.4±0.86A a
86.7±0.02A b
80.2±0.13A c
47.8±0.03A d
34.3±2.75A e
F3
35.58
90.2±0.24B a
59.9±0.01B b
55.4±.013B c
23.5±0.38B d
12.1±0.74B e
F4
27.01
90.1±0.01B a
59.9±0.02B b
55.3±0.14B c
22.9±0.94B d
12.1±0.17B e
F5
15.18
80.8±0.17C a
36.5±0.03C b
27.0±0.00C c
12.3±0.19C d
0.5±0.00C e
F6
21.29
80.6±0.04C a
36.5±0.01C b
27.0±0.03C c
11.9±0.62C d
0.5±0.00C e
13.53
Da
Db
Dc
Control/C
45.8±1.36
30.6±0.05
20.4±0.34
RI PT
F2
1.2±0.07
Dd
0.5±0.13C d
Note：Different capital letters indicate significant differences ( P＜0.05) in different families with same temperature, the same capital
SC
letter indicates no significant difference (P＞0.05); Different lowercase letters indicate significant differences (P＜0.05) in different temperatures with same family, the same lowercase letter indicates no significant difference (P＞0.05)
Table 2 Correlations among survival rate, TAC, and TCC in the pearl oyster Pinctada fucata Survival rate (SR)
TAC
M AN U
Survival rate (SR)
0.809
Total antioxidant capacity (TAC)
0.809**
Total carotenoids content (TCC)
0.739**
AC C
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Note: **. very significant at 0.01 level (P＜0.01)
**
0.951**
TCC 0.739** 0.951**
C F1
B a
B b
60
F2
A b
F3 F4
AA cc
50
F5 F6
C a
40
D a
30
F a
a
B c
D b E b F b G c
E a
20 G
AA dd
C b
G b
10
C c
AA e e BB dd
E Fc E c d
C Dd d D e
0 0
3
6
12
A a
4
A b A b
B a
A c A c
B a
BB bb
C Ca a
D a
B c
EP
2
1
A d
TE D
3
C Cab b
D b
AC C
Total antioxidant capacity (TAC)/nmol· mgprot
-1
A a
AA BB gg BBCC f f CC D f f gggg g
D f
48
96
M AN U
5
AA f f
24
Time/h
B
BB e e CC ee
RI PT
70
A a
SC
80
-1
A Total carotenoids content (TCC) /µg· g )
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F2 F3 F4 F5
A d
F6 A e
A e
A e
A f
B c
A f
B d
B C e Cb c D c
C F1
B dB e C Cc d D e
D d
C Cd e
BB ee C Ce D f f
D e
A g BB f f C Cf f
0
0
3
6
12
24
48
96
Time/h
Figure.1. Effects of stress time on TCC and TAC in the pearl oyster Pinctada fucata. (A), (B), represented the changes of TCC and TAC in the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃. The bars represented the mean±SD (n=3). Different capital letters
ACCEPTED MANUSCRIPT
represented significant differences (P＜0.05) in different families with same stress time, the same capital letter indicates no significant difference (P ＞ 0.05) and different lowercase letters represented
RI PT
significant differences (P＜0.05) in different stress times with same family, the same lowercase letter indicates no significant difference (P
AC C
EP
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M AN U
SC
＞0.05).
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A e
120 100
A f
AB A b CD b e bb
CC gf
F5
F6
A a A c
A d
DD cc BC DD e f ee
EE cd
BB DD f e f e
EE de
0 6
48
24
12
M AN U
3
EE aa
EE cc
EE de
20
0
CD aa
BB cc
CD dd
DD ef
BB aa
A b
EE bb
60 40
F4
BB dd
BB gf
80
F2
F3
SC
Superoxide dismutase(SOD)/nmol· mgprot
140
C F1
RI PT
160
-1
A
96
Time/h
B
90
70
40
f
F3 F4 F5 F6
BB aa
BB bb
30 A
BB de
DD bb
AC C
CC ef
BB bb
CC aa
BB cc
CC bb
DD f f
C F1 F2
TE D
50
10
A d
A e
60
20
A b
A c
EP
Catalase(CAT)/nmol· mgprot
-1
80
A a
A b
BB dd
CC dd DD dd
DD dd
CC ee
DD ee
CC ee
CC cc
BB de DD cc
DD aa
0
0
3
6
12
24
48
96
Time/h
Figure.2. Effect of stress time on SOD and CAT in the pearl oyster Pinctada fucata. (A), (B), represented the changes of SOD and CAT in the pearl oyster Pinctada fucata respectively after 96 h stress at 30 ℃. The bars represented the mean±SD (n=3). Different capital letters
ACCEPTED MANUSCRIPT
represented significant differences (P＜0.05) in different families with same stress time, the same capital letter indicates no significant difference (P＞0.05) and different lowercase l letters represented significant
RI PT
differences (P＜0.05) in different stress times with same family, the same
AC C
EP
TE D
M AN U
SC
lowercase letter indicates no significant difference (P＞0.05).
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25
F2
F3
F4
F5
F6
A b
A c
A c
A d
A e
A a
B Ba a
20
15
10 A f
5
CC cc
A Af Bf Be e
DD bb
B Bc c
B Bd d
B Bd Cd Cc c
DD dd
B Be e
C Ce f D deD d
C Ce f
0 3
6
12
DD ee
24
C Ca a
C Cb b
DD bb
48
DD aa
96
M AN U
0
C Cd d
RI PT
B Bb b
SC
Malonic dialdehyde(MDA)/nmol· mgprot
-1
30
C F1
Time/h
Figure.3. Effect of stress time on MDA in the pearl oyster Pinctada fucata after 96 h stress at 30 ℃. The bars represented the mean±SD
TE D
(n=3). Different capital letters represented significant differences (P＜ 0.05) in different families with same stress time, the same capital letter indicates no significant difference (P＞0.05) and different lowercase
EP
letters represented significant differences (P＜0.05) in different stress
AC C
times with same family, the same lowercase letter indicates no significant difference (P＞0.05).
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A
100
60
RI PT
Survival rate(SR)/%
80
y=24.34ln(x)+2.00 2 R =0.775
40
SC
20
0 15
30
45
60
75
M AN U
0
90
-1
Total carotenoids content(TCC)/µg· g
B
100
40
TE D EP
60
y= 33.04ln(x)+57.17 2 R =0.821
20
AC C
Survival rate(SR)/%
80
0
0
1
2
3
4 -1
Total antioxidant capacity(TAC)/U· mgprot
5
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5
4
RI PT
3
2
y=0.27+0.06x 2 R =0.905 1
0 15
30
45
60
M AN U
0
SC
Total antioxidant capacity(TAC)/U· mgprot
-1
C
75
90
-1
Total carotenoids content(TCC)/µg· g
Figure. 4. Correlations between the survival rate, TCC and TAC in the
AC C
EP
TE D
pearl oyster Pinctada fucata
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Highlights
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M AN U
SC
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Indexes to selectively breed high temperature resistance in shellfish are proposed. Antioxidant enzymes had low activity in the presence of carotenoids. High temperature stress in Pinctada fucata might be mitigated by carotenoids.
AC C
• • •