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Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies
Cryobiology 59 (2009) 344–350
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Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies q Estefanía Paredes *, Juan Bellas Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain
a r t i c l e
i n f o
Article history: Received 28 July 2009 Accepted 22 September 2009 Available online 26 September 2009 Keywords: Cryoprotectant Cryopreservation Sea urchin Gametes Embryos Paracentrotus lividus
a b s t r a c t Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me2SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to 12 °C at 1 °C/min, holding for 2 min for seeding, cooling to 20 °C at 0.5 °C/min, and then cooling to 35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Marine pollution is one of the main environmental problems affecting developed countries during last decades, and its prevention and control has become a main task of Society at a global level. Current strategies of marine pollution monitoring integrate traditional methods of chemical analysis with biological parameters to evaluate the effects of pollution on living resources [7,9]. Among the most commonly used biological tools, the embryo-larval bioassays with marine invertebrates, in particular with bivalves and sea urchins, are in an advanced developmental stage, and have been routinely used for decades for the evaluation and monitoring of marine pollution worldwide (e.g. [18,21]). The application of these biological techniques in routine monitoring has allowed the identification of the major drawbacks that are limiting a more extensive use of embryo-larval bioassays. Thus, the availability of high quality biological material all year round, regardless of the seasonal reproductive cycle of the species, is one of the main limitations [18]. Although off-season broodstock conditioning may extend the natural spawning period of the test species, it is not q Support for this research was provided by the Galician Government (Consellería de Innovación e Industria) through the Research Project PGIDIT07MMA018312PR. * Corresponding author. Fax: +34 986 812556. E-mail address: [email protected] (E. Paredes).
0011-2240/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2009.09.010
possible to obtain fertile stocks all year round, and the quality of the spawn off-season is frequently low [8]. The cryopreservation of gametes, embryos, and larvae of marine invertebrates such as bivalves and sea urchins would provide a way to overcome this main constraint and provide biological material available throughout the year, enhancing the potential of such bioassays. The cryopreservation of sperm and larvae of marine invertebrates have been investigated in previous studies (e.g. [13,15,19,31,33,38,41]). Regarding sea urchins, positive results have been mainly achieved with larval stages from gastrula to pluteus [1,3–6]. However, scarce information is available about cryopreservation of earlier developmental stages (from the egg to the blastula stage) [2], which are the most sensitive stages to chemical pollution in the life cycle of marine invertebrates (e.g. [11,18,26,34]) and hence, the most suitable for ecotoxicological studies. The aims of the present study are: (i) to test different permeant and non-permeant cryoprotectant agents (CPAs), individually and in combination, in order to find suitable concentrations and CPAs combinations (on a toxicity basis) for cryopreservation of sea urchin embryos intended to be used in ecotoxicological bioassays; and (ii) to use these CPAs combinations to carry out preliminary cryopreservation protocols with those organisms. The test species chosen is the edible sea urchin Paracentrotus lividus (Lamarck 1816), a large regular sea urchin widely distributed throughout the Medi-
E. Paredes, J. Bellas / Cryobiology 59 (2009) 344–350
terranean Sea and European Atlantic coast with important ecological roles in the functioning, dynamics and structure of benthic assemblages [10,17]. Several studies have also shown the importance of sea urchin pluteus larvae in the composition and biomass of zooplankton communities, playing a significant role in the pelagic foodweb [25]. Moreover, in some European countries P. lividus is exploited for its highly valued gonads [10]. Materials and methods Biological material Mature sea urchins, P. lividus, were collected by scuba divers in outer part of the Ría de Vigo (Galicia, NW Iberian Peninsula) during the natural spawning season. Animals were transported to the laboratory in a portable icebox and maintained in aquaria with running natural seawater for at least 1 week until the experiments. Sea urchins were fed with the green algae Ulva lactuca. Gametes were obtained directly from the gonads with a Pasteur pipette after dissection of a single pair of adults according to Beiras and Saco-Álvarez [8]. Mature oocytes were transferred to a 100-mL measuring cylinder and their quality was checked under microscope. Only batches of mature eggs that were spherical and undamaged were used for the experiments. Sperm mobility was checked under microscope and the sperm solution was stored at 4 °C until use. Experimental solutions There is a wide range of CPAs used in invertebrate cryopreservation but we have focussed into the five most commonly used. Between the range of permeant CPAs we have chosen dimethyl sulfoxide (Me2SO), propylene glycol (PG), and ethylene glycol (EG), and among non-permeant CPAs we used trehalose (TRE) and polyvinylpyrrolidone (PVP). The reagents were obtained from Sigma–Aldrich chemicals. Table 1 shows the CPAs concentrations tested in individual and mixture experiments. Those concentrations were selected on the basis of previous studies carried with marine invertebrates (e.g. [1–4,12,15,24,28]). Toxicity tests A few microliter of motile sperm were added to the egg suspension and carefully stirred to allow fertilization. Fertilized eggs from one female and one male were used to minimize genetic variability [20,40]. The toxicity of the individual CPAs and their combinations, to the unfertilized egg, fertilized egg, and blastula stage, was tested. Approximately 500 eggs or embryos were delivered into 2 mL cryovials with 1 mL of artificial sea water (ASW). Then, the eggs or embryos were impregnated with the CPAs solutions. The expo-
Table 1 CPAs concentrations tested. Selected concentrations for mixture experiments are shown in boldface. CPA
Single use Concentration (M)
Blended Concentration (M)
Me2SO
0.1, 0.5, 1, 1.5, 2
EG
1, 1.5, 2
PG
0.68, 1.36, 2.04, 2.72, 3.4
+TRE 0.03, 0.04 +PVP 0.085, 0.425, 0.75 +TRE 0.03, 0.04 +PVP 0.085, 0.425, 0.75 +TRE 0.03, 0.04 +PVP 0.085, 0.425, 0.75
TRE PVP
0.01, 0.02, 0.03, 0.04, 0.05 0.085, 0.425, 0.75
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sure of cells to a high concentration of CPA causes the release of water from the cells as the concentration equilibrium is maintained, and the resulting dehydration can be lethal if it occurs too rapidly [23]. Thus, it is advised to add CPAs following a gradual stepwise protocol in order to reduce toxicity and osmotic stress to embryos that could lead to morphological damage (e.g. [1,32]). The CPAs were added in 15 fixed molar steps of 1 min, to a final volume of 2 mL. Those molar steps ranged from 0.03 M (for a final concentration of the CPA of 0.5 M) to 0.13 M (for a final concentration of 2 M). Furthermore, this impregnation protocol was compared with the addition of CPAs in 15 volume steps. The eggs or embryos were exposed to the CPAs for 30 min at 4 °C and the CPAs were then diluted with ASW in 12 fixed molar steps of 1 min ranging from 0.03 to 0.13 M. After dilution, the eggs or embryos were rinsed carefully with ASW to remove the excess of CPA by using a 50-lm mesh, transferred to experimental vials with 20 mL of ASW, and incubated for 48 h until 4-arm-pluteus stage was reached in controls. After the incubation period sea urchin larvae were preserved by adding a few drops of 40% buffered formalin, and the percentage of 4-arm pluteus larvae (n = 100), and the mean larval growth (n = 35) were recorded. Larval growth was defined as the maximum dimension in the first 35 individuals per vial (including embryos), subtracting the average of the diameter of the fertilized eggs (90 lm). Length of individuals was recorded in the inverted microscope using the Leica QWin image analysis software. Four replicates per treatment and four ASW controls were assayed for each experiment. Control embryogenesis success was always above 90%. Physicochemical conditions of the experiments were 34.8 ± 0.45 ppt salinity, 6.23 ± 0.87 mg/l O2, and 8.01 ± 0.14 pH (mean ± SD). Cryopreservation trials Following the toxicity assessment of the CPAs, cryopreservation experiments were undertaken on the three developmental stages. Approximately 500 eggs or embryos were delivered into 2 mL cryovials with 1 mL of artificial sea water (ASW). CPA impregnation protocol was performed as described above. Cryovials were then placed in a Freeze Control System CL-8800 (Cryologic Pty Ltd.) and different protocols were assayed. Cryovials were thawed by immersion in a water bath at 16 °C for 1 min. Dilution, rinse out, and incubation protocols were performed as described above. Statistical analyses Statistical analyses were conducted using the SPSSÒ version 15.0 statistical software. Differences in the percentage of normal larvae and larval growth among treatments were analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s test for calculation of the no observed effect concentrations (NOEC) and lowest observed effect concentrations (LOEC). For analysis, the percentage of normal larvae data were first arcsine-transformed to achieve normality [16]. Statistical tests were performed according to Newman [29] and Sokal and Rohlf [39]. Results Individual cryoprotectants Fig. 1 shows the comparison of two methods for the impregnation of P. lividus embryos with CPAs. These results indicate that impregnation of embryos with Me2SO in 15 fixed molar steps allowed considerably higher growth and survival of sea urchin larvae than impregnation in 15 fixed volume steps. Statistically significant differences were observed for fertilized eggs at all tested con-
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% 4-arm pluteus larvae
A
100 80 60 40 20 0 Fertilized egg Me2SO:
0.1 M
Blastula 0.5 M
1M
1.5 M
2M
% 4-arm pluteus larvae
B 100 80 60 40 20 0 Fertilized egg
Blastula
Fig. 1. Comparison of CPAs impregnation protocols in fertilized eggs and blastulae of P. lividus. (A) Percentage of larval growth after 30 min exposure of embryos to different concentrations of Me2SO added in 15 fixed volume steps. (B) Percentage of larval growth after 30 min exposure of embryos to different concentrations of Me2SO added in 15 fixed molar steps. Error bars represent standard deviations.
centrations, except 0.1 M Me2SO, and for blastulae, significant differences were obtained above 0.5 M (p < 0.01). From there on, CPAs were added in 15 fixed molar steps of 1 min. As expected, permeant CPAs were more toxic than non-permeant CPAs. The observed toxicity varied among different developmental stages as well as with the concentration and the type of CPA. Despite the CPA tested, the most sensitive developmental stage was, in general, the unfertilized egg, whereas the blastula stage was the most tolerant one (Tables 2 and 3). The three permeant CPAs significantly inhibited the embryonic development, assessed as the percentage of 4-arm fully developed pluteus larvae, and the larval growth of P. lividus within the experimental concentration range (Table 3). The NOEC and LOEC values of Me2SO were 0.5 and 1 M for the unfertilized egg, 1 and 1.5 M for the fertilized egg, and 1.5 and 2 M for the blastula stage. EG showed lower toxicity to the unfertilized egg and higher toxicity to the blastula, with NOEC and LOEC values of 1 and 1.5 M for the three developmental stages. The NOEC and LOEC of PG were 0.68 and 1.36 M for the egg and the fertilized egg stages, whilst for the blastula stage the values obtained were 1.36 and 2.04 M (Table 2). Non-permeant CPAs showed no toxicity at the tested concentrations (Tables 2 and 3).
Cryoprotectants combination Me2SO, EG, and PG were tested in combination with TRE and PVP. The toxicity observed for Me2SO in combination with non-permeant CPAs differed from the toxicity observed for individual Me2SO. Both TRE and PVP consistently reduced Me2SO toxicity to the unfertilized egg and TRE also reduced slightly Me2SO toxicity to the fertilized egg. On the other hand, both TRE and PVP increased Me2SO toxicity to the blastulae, whilst PVP increased Me2SO toxicity to the fertilized egg (Fig. 2 and Table 2). Moreover, in disagreement with the results observed for individual Me2SO, no significant differences in larval growth were detected among the three developmental stages for the Me2SO (1 M) + TRE combination (Fig. 2). The Me2SO + PVP combination yielded different results, though. In particular, larvae resulting from exposed fertilized eggs, were smaller than those resulting from exposed unfertilized eggs and blastulae at 1 M Me2SO, except for the combination with 0.75 M PVP, with significantly higher growth in larvae resulting from fertilized eggs than in those from exposed blastulae. PVP highly reduced EG toxicity. Thus, no significant toxicity was observed in the EG + PVP combination at 1 and 1.5 M EG, with similar results for the three exposed developmental stages. On the
Table 2 NOEC and LOEC for the tested CPAs alone and in combination. CPA
Me2SO EG PG TRE PVP Me2SO + TRE Me2SO + PVP EG + TRE EG + PVP PG + TRE PG + PVP
NOEC (M)
LOEC (M)
Egg
Fertilized egg
Blastula
Egg
Fertilized egg
Blastula
0.5 1 0.68 0.05 0.75 1 – 1 2 2.04 2.04
1 1 0.68 0.05 0.75 1 – 1 2 2.04 2.04
1.5 1 1.36 0.05 0.75 1 – 2 2 2.04 2.04
1 1.5 1.36 – – 1.5 1 1.5 – – –
1.5 1.5 1.36 – – 1.5 1 1.5 – – –
2 1.5 2.04 – – 1.5 1 – – – –
E. Paredes, J. Bellas / Cryobiology 59 (2009) 344–350 Table 3 Percentage of larval growth and normal larvae in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs and blastulae to different concentrations of Me2SO, EG, PG, PVP, and TRE (standard deviations are shown in brackets). Developmental stage
CPA
Concentration (M)
% Larval growth
% 4-arm pluteus normal larvae
Egg
Me2SO
0.1 0.5 1 1.5 2 1 1.5 2 0.68 1.36 2.04 0.01 0.02 0.03 0.04 0.05 0.085 0.425 0.75
100 ± 14.7 91.9 ± 0.4 24.8 ± 3.1 0.1 ± 3.9 0 85.9 ± 3.7 46.6 ± 5.9 1.6 ± 2.4 100 ± 12.4 52.8 ± 15.1 16.51 ± 14.6 100 ± 5.9 100 ± 27.1 97.2 ± 10.8 89.7 ± 10.8 94.8 ± 17.1 78.1 ± 49.1 100 ± 16.0 100 ± 3.08
99.9 ± 2.0 98.4 ± 1.5 29.8 ± 2.8 6.4 ± 10.5 0 87.9 ± 1.1 46.1 ± 3.0 0 99.8 ± 0.4 60.0 ± 1.8 21.0 ± 5.2 97.4 ± 1.8 90.7 ± 8.7 96.3 ± 2.8 79.9 ± 8.9 76.9 ± 15.6 73.9 ± 36.9 94.4 ± 6.1 97.0 ± 5.1
0.1 0.5 1 1.5 2 1 1.5 2 0.68 1.36 2.04 2.72 3.4 0.01 0.02 0.03 0.04 0.05 0.085 0.425 0.75
86.1 ± 4.0 96.5 ± 6.5 84.6 ± 7.3 34.5 ± 10.9 26.7 ± 17.4 95.2 ± 4.5 46.8 ± 21.4 5.8 ± 1.9 85.0 ± 8.6 30.8 ± 2.0 48.5 ± 6.2 53.8 ± 5.5 15.5 ± 6.2 100 ± 1.2 100 ± 2.6 100 ± 2.9 99.8 ± 4.3 100 ± 5.0 99.9 ± 2.1 100 ± 2.3 100 ± 1.7
98.1 ± 1.6 100 ± 1.4 81.4 ± 3.5 34.0 ± 10.9 26.4 ± 13.9 98.7 ± 1.2 33.2 ± 5.1 0 90.3 ± 1.2 46.9 ± 8.9 57.7 ± 13.1 64.2 ± 18.0 29.4 ± 5.1 97.9 ± 0.8 99.5 ± 1.0 98.9 ± 1.2 97.3 ± 1.5 99.5 ± 0.6 98.5 ± 2.6 99.2 ± 1.4 98.9 ± 2.0
0.1 0.5 1 1.5 2 1 1.5 2 0.68 1.36 2.04 2.72 3.4 0.01 0.02 0.03 0.04 0.05 0.085 0.425 0.75
96.9 ± 0.9 96.8 ± 4.5 94.1 ± 5.4 89.6 ± 7.2 73.0 ± 14.7 99.2 ± 2.3 89.0 ± 1.5 77.4 ± 4.7 97.5 ± 7.0 89.9 ± 5.0 79.3 ± 4.2 59.2 ± 7.1 39.8 ± 6.9 99.5 ± 4.8 100 ± 6.0 100 ± 4.6 100 ± 8.3 100 ± 3.5 99.8 ± 4.6 99.0 ± 6.0 100 ± 3.0
96.9 ± 3.3 97.6 ± 2.4 96.2 ± 2.6 97.9 ± 1.6 75.1 ± 3.6 97.6 ± 1.2 94.5 ± 2.1 90.1 ± 6.3 100 ± 0.1 94.5 ± 2.7 80.6 ± 3.0 56.6 ± 3.0 46.9 ± 0.1 93.6 ± 3.5 99.5 ± 1.3 98.7 ± 2.9 97.6 ± 0.1 98.1 ± 1.8 96.6 ± 3.2 96.9 ± 2.7 95.2 ± 6.6
EG
PG
TRE
PVP
Fertilized egg
Me2SO
EG
PG
TRE
PVP
Blastula
Me2SO
EG
PG
TRE
PVP
other hand, significant toxic effects were detected in the EG + TRE combination for the egg and fertilized egg stages, but not for the blastula stage. Although TRE increased EG toxicity to the unfertilized egg, toxicity to the fertilized egg and blastula was significantly reduced (Fig. 3 and Table 2). The PG + PVP and the PG + TRE combinations tested were not toxic to the unfertilized egg, fertilized egg, or blastula stages at
347
the tested concentrations (Fig. 4 and Table 2). Therefore, both PVP and TRE seem to have a protective role reducing consistently PG toxicity regardless of the developmental stage tested. Cryopreservation trials Among the experiments conducted with different CPAs combinations, the most successful cryopreservation involved impregnation of fertilized eggs and blastulae with PG (2 M) + PVP (0.75 M). The following protocols, based on successful methods used for sea urchin and bivalve species [1,15,24], were assayed: (a) cool from 4 to 12 °C at 1 °C/min, hold for 2 min for seeding, and then cool to 35 °C at 1 °C/min, and (b) cool from 4 to 12 °C at 1 °C/ min, hold 2 min for seeding, cool to 20 °C at 0.5 °C/min and then cool to 35 °C at 1 °C/min. Larvae developed from fertilized eggs frozen by this method achieved a 30.1% (protocol a) and 42.9% (protocol b) growth with respect to control unfrozen eggs, whereas the percentage of pluteus larvae was 41.5% (13% 4-arm pluteus larvae) and 40.5% (27% 4-arm pluteus larvae). On the other hand, larvae developed from frozen blastulae achieved a 30.3% (protocol a) and 60.5% (protocol b) growth, whereas the percentage of pluteus larvae was 40.5% (27% 4-arm pluteus larvae) and 68.5% (25.9% 4arm pluteus larvae). Discussion The successful cryopreservation of early developmental stages of marine organisms depends on the selection of suitable CPA treatments, taking into account both its toxicity and its ability to avoid or minimize the formation of internal and external ice crystals, i.e. its efficiency [32,35]. Cryoprotectants can suppress most cryoinjuries but, when used at higher (more effective) concentrations, most of them become toxic. For instance, Me2SO has diverse effects on ion transporters and pumps and inhibits catalase and peroxide activity [22,30,37], whereas EG and PG cause membrane damage by decreasing the polarity of the aqueous phase and changing the partition of hydrophobic molecules between the cell membrane and the external phase [14,22]. Taking this into account, it must be ensured that a right balance between their toxicity, if any, and their protective ability, is achieved, in order to offer protection rather than damage to the cells. There is a wide range of CPAs that have been used for the cryopreservation of marine invertebrate larvae. Among those, we have chosen the permeant CPAs Me2SO, PG, and EG, and the non-permeant TRE and PVP, to study their suitability for the cryopreservation of early embryos of the sea urchin P. lividus. In general, Me2SO showed the highest toxicity to the three tested developmental stages after 30 min exposure, followed by EG and PG, whilst the non-permeant CPAs were no toxic at the experimental concentrations. From these results it can be concluded that PG and EG are, in terms of its toxicity, the most suitable CPAs for the cryopreservation of P. lividus unfertilized eggs and blastulae, at concentrations of no more than 1 M, and EG seem to be also the best CPA to be used with fertilized eggs at the same concentrations. In this line, Asahina and Takahashi [3] and Adams et al. [1] found that EG was less toxic than Me2SO to eggs and 4-arm pluteus larvae of the sea urchins Hemicentrotus pulcherrimus and Evechinus chloroticus, following CPA exposure and removal. However, when the gastrula stage was evaluated, EG was more toxic than Me2SO at a concentration of 2 M [1]. Despite methodological differences, previous studies involving gametes and embryos of bivalves report, usually, higher toxicity of PG, followed by EG and Me2SO, which showed the lowest toxicity [12,28,36]. However, some studies carried out with embryos and larvae of the oyster Crassostrea gigas and the abalone Haliotis midae, report higher toxicity of Me2SO than EG and PG [15,35];
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E. Paredes, J. Bellas / Cryobiology 59 (2009) 344–350
% Larval growth
A
B
Me2SO 1 M
Me2SO 1.5 M
100 80 60 40 20 0 Egg
Fert egg
PVP 0.085 M
C
Egg
Blastula
PVP 0.425 M
Fert egg PVP 0.75 M
D
Me2SO 1 M
Blastula
E
Me2SO 1.5 M
Me2SO 2 M
% Larval growth
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg TRE 0.03 M
Blastula
Egg
Fert egg
Blastula
TRE 0.04 M
Fig. 2. Percentage of larval growth in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs, and blastulae to different combinations of Me2SO with PVP and TRE. (A) Me2SO 1 M + PVP; (B) Me2SO 1.5 M + PVP; (C) Me2SO 1 M + TRE; (D) Me2SO 1.5 M + TRE; (E) Me2SO 2 M + TRE. Error bars represent standard deviations.
A
B
EG 1 M
C
EG 1.5 M
EG 2 M
% Larval growt
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg PVP 0.425 M
D
E
EG 1 M
Blastula
Egg
Fert egg
Blastula
PVP 0.75 M
F
EG 1.5 M
EG 2 M
% Larval growt
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg TRE 0.03 M
Blastula
Egg
Fert egg
Blastula
TRE 0.04 M
Fig. 3. Percentage of larval growth in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs, and blastulae to different combinations of EG with PVP and TRE. (A) EG 1 M + PVP; (B) EG 1.5 M + PVP; (C) EG 2 M + PVP; (D) EG 1 M + TRE; (E) EG 1.5 M + TRE; (F) EG 2 M + TRE. Error bars represent standard deviations.
whereas similar toxicity of the three cryoprotectants for oyster embryos and larvae has also been found [24]. The results obtained in the present study also indicate that the blastula stage was more resistant to the CPAs than the unfertilized and fertilized eggs. This finding supports data from previous studies conducted with bivalves, reporting higher resistance of the trochophore stage to cryoprotectants than earlier embryos or gametes [15,28,36]. Therefore,
it is evident from this and previous studies that CPAs tolerance in marine invertebrates is species and stage specific. Thus, the studies of suitability of CPAs for the cryopreservation of embryos and larvae of marine invertebrates should be carried out with the species and developmental stages intended to be used. Since the efficiency of CPAs in their protective role against freezing injuries increases when used at higher concentrations,
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% Larval growth
A
B
PG 0.68 M
C
PG 1.36 M
PG 2.04 M
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg
PVP 0.425 M
D
E
PG 0.68 M
Blastula
Egg
Fert egg
Blastula
PVP 0.75 M
F
PG 1.36 M
PG 2.04 M
% Larval growth
100 80 60 40 20 0 Fert egg
Blastula
Fert egg
Blastula
TRE 0.03 M
Fert egg
Blastula
TRE 0.04 M
Fig. 4. Percentage of larval growth in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs and blastulae to different combinations of PG with PVP and TRE. (A) PG 0.68 M + PVP; (B) PG 1.36 M + PVP; (C) PG 2.04 M + PVP; (D) PG 0.68 M + TRE; (E) PG 1.36 M + TRE; (F) PG 2.04 M + TRE. Error bars represent standard deviations.
several methods need to be applied in order to mitigate the increase of toxicity caused by those high concentrations. One of the approaches used is to add and remove the CPAs in a gradual stepwise protocol of several fixed molar steps, in order to reduce the osmotic stress to eggs and embryos [1,32]. In the present work, the addition of CPAs in 15 fixed molar steps instead of 15 fixed volume steps, reduced significantly the larval abnormalities and increased larval growth. Furthermore, the addition of high molecular weight non-permeant CPAs has been reported as a complementary approach to reduce the toxicity of permeant CPAs [12]. The beneficial effect of combining TRE and PVP with permeant CPAs, which was demonstrated in the present study for P. lividus, would allow loading eggs and embryos with higher, and therefore more effective, concentrations of permeant CPAs. It must be noted, though, that the protective ability of TRE and PVP was dependant on the permeant CPA and on the developmental stage tested. PVP consistently reduced EG and PG toxicity to the eggs and embryos of P. lividus and reduced Me2SO toxicity to the unfertilized egg. On the other hand, TRE reduced PG toxicity to the eggs and embryos, the toxicity of EG to the blastula stage and the toxicity of Me2SO to the unfertilized egg. Although previous studies with sea urchin embryos and larvae found that Me2SO was better as a cryoprotectant than EG [1,4– 6,27], on the basis of the toxicity results obtained here, it would be recommended to use solutions with 2 M PG or EG in combination with 0.75 M PVP, for the cryopreservation of P. lividus embryos. Following the evaluation of CPAs toxicity, cryopreservation trials were done. Freezing and thawing experiments were conducted using PG and PVP combinations and different cryopreservation protocols were tested. Different cooling rates and holding temperatures were examined, as well as the effects of incorporating a seeding step during cooling. Since unfertilized eggs showed very poor post-thaw normal development, experimental efforts focused mainly on fertilized eggs and blastulae. Solutions containing 2 M PG and 0.75 M PVP were the most effective, and the incorporation
of a seeding step at 12 °C into the freezing protocol improved the normal embryonic development post-thawing. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively. In conclusion, this study provides results on the toxicity of five CPAs to eggs and blastulae of the sea urchin P. lividus. Our results indicate that, on the basis of their toxicity, PG and EG in combination with PVP might be the most suitable CPAs for this species. Promising results have been achieved using PG and PVP combinations in cryopreservation trials with fertilized eggs and blastulae. However, since Me2SO has been proven to be an efficient CPA for several marine invertebrates, including sea urchins, this CPA should not be discarded in future cryopreservation studies with P. lividus embryos. Future studies also should focus on a detailed evaluation of the cryopreservation protocol, including the impact of plunging into liquid nitrogen and the conservation of the biological material. Acknowledgments We thank Ricardo Beiras for valuable comments on the manuscript and the personnel from the Estación de Ciencias Mariñas de Toralla (Universidade de Vigo) for providing us with sea urchins. References [1] S.L. Adams, P.A. Hessian, P.V. Mladenov, The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus, Cryobiology 52 (2006) 139–145. [2] E. Asahina, T. Takahashi, Survival of sea urchin spermatozoa and embryos at very low temperatures, Cryobiology 14 (1977) 703. [3] E. Asahina, T. Takahashi, Freezing tolerance in embryos and spermatozoa of the sea urchin, Cryobiology 15 (1978) 122–127. [4] E. Asahina, T. Takahashi, Cryopreservation of sea urchins embryos and sperm, Development Growth and Differentiation 21 (1979) 423–430. [5] C. Barros, A. Muller, M.J. Wood, D.G. Whittingham, High survival of sea urchin semen (Tetrapigus niger) pluteus larvae (Loxechinus albus) frozen in 1.0 M Me2SO, Cryobiology 33 (1996) 646.
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Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies q Estefanía Paredes *, Juan Bellas Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain
a r t i c l e
i n f o
Article history: Received 28 July 2009 Accepted 22 September 2009 Available online 26 September 2009 Keywords: Cryoprotectant Cryopreservation Sea urchin Gametes Embryos Paracentrotus lividus
a b s t r a c t Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me2SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to 12 °C at 1 °C/min, holding for 2 min for seeding, cooling to 20 °C at 0.5 °C/min, and then cooling to 35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Marine pollution is one of the main environmental problems affecting developed countries during last decades, and its prevention and control has become a main task of Society at a global level. Current strategies of marine pollution monitoring integrate traditional methods of chemical analysis with biological parameters to evaluate the effects of pollution on living resources [7,9]. Among the most commonly used biological tools, the embryo-larval bioassays with marine invertebrates, in particular with bivalves and sea urchins, are in an advanced developmental stage, and have been routinely used for decades for the evaluation and monitoring of marine pollution worldwide (e.g. [18,21]). The application of these biological techniques in routine monitoring has allowed the identification of the major drawbacks that are limiting a more extensive use of embryo-larval bioassays. Thus, the availability of high quality biological material all year round, regardless of the seasonal reproductive cycle of the species, is one of the main limitations [18]. Although off-season broodstock conditioning may extend the natural spawning period of the test species, it is not q Support for this research was provided by the Galician Government (Consellería de Innovación e Industria) through the Research Project PGIDIT07MMA018312PR. * Corresponding author. Fax: +34 986 812556. E-mail address: [email protected] (E. Paredes).
0011-2240/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2009.09.010
possible to obtain fertile stocks all year round, and the quality of the spawn off-season is frequently low [8]. The cryopreservation of gametes, embryos, and larvae of marine invertebrates such as bivalves and sea urchins would provide a way to overcome this main constraint and provide biological material available throughout the year, enhancing the potential of such bioassays. The cryopreservation of sperm and larvae of marine invertebrates have been investigated in previous studies (e.g. [13,15,19,31,33,38,41]). Regarding sea urchins, positive results have been mainly achieved with larval stages from gastrula to pluteus [1,3–6]. However, scarce information is available about cryopreservation of earlier developmental stages (from the egg to the blastula stage) [2], which are the most sensitive stages to chemical pollution in the life cycle of marine invertebrates (e.g. [11,18,26,34]) and hence, the most suitable for ecotoxicological studies. The aims of the present study are: (i) to test different permeant and non-permeant cryoprotectant agents (CPAs), individually and in combination, in order to find suitable concentrations and CPAs combinations (on a toxicity basis) for cryopreservation of sea urchin embryos intended to be used in ecotoxicological bioassays; and (ii) to use these CPAs combinations to carry out preliminary cryopreservation protocols with those organisms. The test species chosen is the edible sea urchin Paracentrotus lividus (Lamarck 1816), a large regular sea urchin widely distributed throughout the Medi-
E. Paredes, J. Bellas / Cryobiology 59 (2009) 344–350
terranean Sea and European Atlantic coast with important ecological roles in the functioning, dynamics and structure of benthic assemblages [10,17]. Several studies have also shown the importance of sea urchin pluteus larvae in the composition and biomass of zooplankton communities, playing a significant role in the pelagic foodweb [25]. Moreover, in some European countries P. lividus is exploited for its highly valued gonads [10]. Materials and methods Biological material Mature sea urchins, P. lividus, were collected by scuba divers in outer part of the Ría de Vigo (Galicia, NW Iberian Peninsula) during the natural spawning season. Animals were transported to the laboratory in a portable icebox and maintained in aquaria with running natural seawater for at least 1 week until the experiments. Sea urchins were fed with the green algae Ulva lactuca. Gametes were obtained directly from the gonads with a Pasteur pipette after dissection of a single pair of adults according to Beiras and Saco-Álvarez [8]. Mature oocytes were transferred to a 100-mL measuring cylinder and their quality was checked under microscope. Only batches of mature eggs that were spherical and undamaged were used for the experiments. Sperm mobility was checked under microscope and the sperm solution was stored at 4 °C until use. Experimental solutions There is a wide range of CPAs used in invertebrate cryopreservation but we have focussed into the five most commonly used. Between the range of permeant CPAs we have chosen dimethyl sulfoxide (Me2SO), propylene glycol (PG), and ethylene glycol (EG), and among non-permeant CPAs we used trehalose (TRE) and polyvinylpyrrolidone (PVP). The reagents were obtained from Sigma–Aldrich chemicals. Table 1 shows the CPAs concentrations tested in individual and mixture experiments. Those concentrations were selected on the basis of previous studies carried with marine invertebrates (e.g. [1–4,12,15,24,28]). Toxicity tests A few microliter of motile sperm were added to the egg suspension and carefully stirred to allow fertilization. Fertilized eggs from one female and one male were used to minimize genetic variability [20,40]. The toxicity of the individual CPAs and their combinations, to the unfertilized egg, fertilized egg, and blastula stage, was tested. Approximately 500 eggs or embryos were delivered into 2 mL cryovials with 1 mL of artificial sea water (ASW). Then, the eggs or embryos were impregnated with the CPAs solutions. The expo-
Table 1 CPAs concentrations tested. Selected concentrations for mixture experiments are shown in boldface. CPA
Single use Concentration (M)
Blended Concentration (M)
Me2SO
0.1, 0.5, 1, 1.5, 2
EG
1, 1.5, 2
PG
0.68, 1.36, 2.04, 2.72, 3.4
+TRE 0.03, 0.04 +PVP 0.085, 0.425, 0.75 +TRE 0.03, 0.04 +PVP 0.085, 0.425, 0.75 +TRE 0.03, 0.04 +PVP 0.085, 0.425, 0.75
TRE PVP
0.01, 0.02, 0.03, 0.04, 0.05 0.085, 0.425, 0.75
345
sure of cells to a high concentration of CPA causes the release of water from the cells as the concentration equilibrium is maintained, and the resulting dehydration can be lethal if it occurs too rapidly [23]. Thus, it is advised to add CPAs following a gradual stepwise protocol in order to reduce toxicity and osmotic stress to embryos that could lead to morphological damage (e.g. [1,32]). The CPAs were added in 15 fixed molar steps of 1 min, to a final volume of 2 mL. Those molar steps ranged from 0.03 M (for a final concentration of the CPA of 0.5 M) to 0.13 M (for a final concentration of 2 M). Furthermore, this impregnation protocol was compared with the addition of CPAs in 15 volume steps. The eggs or embryos were exposed to the CPAs for 30 min at 4 °C and the CPAs were then diluted with ASW in 12 fixed molar steps of 1 min ranging from 0.03 to 0.13 M. After dilution, the eggs or embryos were rinsed carefully with ASW to remove the excess of CPA by using a 50-lm mesh, transferred to experimental vials with 20 mL of ASW, and incubated for 48 h until 4-arm-pluteus stage was reached in controls. After the incubation period sea urchin larvae were preserved by adding a few drops of 40% buffered formalin, and the percentage of 4-arm pluteus larvae (n = 100), and the mean larval growth (n = 35) were recorded. Larval growth was defined as the maximum dimension in the first 35 individuals per vial (including embryos), subtracting the average of the diameter of the fertilized eggs (90 lm). Length of individuals was recorded in the inverted microscope using the Leica QWin image analysis software. Four replicates per treatment and four ASW controls were assayed for each experiment. Control embryogenesis success was always above 90%. Physicochemical conditions of the experiments were 34.8 ± 0.45 ppt salinity, 6.23 ± 0.87 mg/l O2, and 8.01 ± 0.14 pH (mean ± SD). Cryopreservation trials Following the toxicity assessment of the CPAs, cryopreservation experiments were undertaken on the three developmental stages. Approximately 500 eggs or embryos were delivered into 2 mL cryovials with 1 mL of artificial sea water (ASW). CPA impregnation protocol was performed as described above. Cryovials were then placed in a Freeze Control System CL-8800 (Cryologic Pty Ltd.) and different protocols were assayed. Cryovials were thawed by immersion in a water bath at 16 °C for 1 min. Dilution, rinse out, and incubation protocols were performed as described above. Statistical analyses Statistical analyses were conducted using the SPSSÒ version 15.0 statistical software. Differences in the percentage of normal larvae and larval growth among treatments were analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s test for calculation of the no observed effect concentrations (NOEC) and lowest observed effect concentrations (LOEC). For analysis, the percentage of normal larvae data were first arcsine-transformed to achieve normality [16]. Statistical tests were performed according to Newman [29] and Sokal and Rohlf [39]. Results Individual cryoprotectants Fig. 1 shows the comparison of two methods for the impregnation of P. lividus embryos with CPAs. These results indicate that impregnation of embryos with Me2SO in 15 fixed molar steps allowed considerably higher growth and survival of sea urchin larvae than impregnation in 15 fixed volume steps. Statistically significant differences were observed for fertilized eggs at all tested con-
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% 4-arm pluteus larvae
A
100 80 60 40 20 0 Fertilized egg Me2SO:
0.1 M
Blastula 0.5 M
1M
1.5 M
2M
% 4-arm pluteus larvae
B 100 80 60 40 20 0 Fertilized egg
Blastula
Fig. 1. Comparison of CPAs impregnation protocols in fertilized eggs and blastulae of P. lividus. (A) Percentage of larval growth after 30 min exposure of embryos to different concentrations of Me2SO added in 15 fixed volume steps. (B) Percentage of larval growth after 30 min exposure of embryos to different concentrations of Me2SO added in 15 fixed molar steps. Error bars represent standard deviations.
centrations, except 0.1 M Me2SO, and for blastulae, significant differences were obtained above 0.5 M (p < 0.01). From there on, CPAs were added in 15 fixed molar steps of 1 min. As expected, permeant CPAs were more toxic than non-permeant CPAs. The observed toxicity varied among different developmental stages as well as with the concentration and the type of CPA. Despite the CPA tested, the most sensitive developmental stage was, in general, the unfertilized egg, whereas the blastula stage was the most tolerant one (Tables 2 and 3). The three permeant CPAs significantly inhibited the embryonic development, assessed as the percentage of 4-arm fully developed pluteus larvae, and the larval growth of P. lividus within the experimental concentration range (Table 3). The NOEC and LOEC values of Me2SO were 0.5 and 1 M for the unfertilized egg, 1 and 1.5 M for the fertilized egg, and 1.5 and 2 M for the blastula stage. EG showed lower toxicity to the unfertilized egg and higher toxicity to the blastula, with NOEC and LOEC values of 1 and 1.5 M for the three developmental stages. The NOEC and LOEC of PG were 0.68 and 1.36 M for the egg and the fertilized egg stages, whilst for the blastula stage the values obtained were 1.36 and 2.04 M (Table 2). Non-permeant CPAs showed no toxicity at the tested concentrations (Tables 2 and 3).
Cryoprotectants combination Me2SO, EG, and PG were tested in combination with TRE and PVP. The toxicity observed for Me2SO in combination with non-permeant CPAs differed from the toxicity observed for individual Me2SO. Both TRE and PVP consistently reduced Me2SO toxicity to the unfertilized egg and TRE also reduced slightly Me2SO toxicity to the fertilized egg. On the other hand, both TRE and PVP increased Me2SO toxicity to the blastulae, whilst PVP increased Me2SO toxicity to the fertilized egg (Fig. 2 and Table 2). Moreover, in disagreement with the results observed for individual Me2SO, no significant differences in larval growth were detected among the three developmental stages for the Me2SO (1 M) + TRE combination (Fig. 2). The Me2SO + PVP combination yielded different results, though. In particular, larvae resulting from exposed fertilized eggs, were smaller than those resulting from exposed unfertilized eggs and blastulae at 1 M Me2SO, except for the combination with 0.75 M PVP, with significantly higher growth in larvae resulting from fertilized eggs than in those from exposed blastulae. PVP highly reduced EG toxicity. Thus, no significant toxicity was observed in the EG + PVP combination at 1 and 1.5 M EG, with similar results for the three exposed developmental stages. On the
Table 2 NOEC and LOEC for the tested CPAs alone and in combination. CPA
Me2SO EG PG TRE PVP Me2SO + TRE Me2SO + PVP EG + TRE EG + PVP PG + TRE PG + PVP
NOEC (M)
LOEC (M)
Egg
Fertilized egg
Blastula
Egg
Fertilized egg
Blastula
0.5 1 0.68 0.05 0.75 1 – 1 2 2.04 2.04
1 1 0.68 0.05 0.75 1 – 1 2 2.04 2.04
1.5 1 1.36 0.05 0.75 1 – 2 2 2.04 2.04
1 1.5 1.36 – – 1.5 1 1.5 – – –
1.5 1.5 1.36 – – 1.5 1 1.5 – – –
2 1.5 2.04 – – 1.5 1 – – – –
E. Paredes, J. Bellas / Cryobiology 59 (2009) 344–350 Table 3 Percentage of larval growth and normal larvae in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs and blastulae to different concentrations of Me2SO, EG, PG, PVP, and TRE (standard deviations are shown in brackets). Developmental stage
CPA
Concentration (M)
% Larval growth
% 4-arm pluteus normal larvae
Egg
Me2SO
0.1 0.5 1 1.5 2 1 1.5 2 0.68 1.36 2.04 0.01 0.02 0.03 0.04 0.05 0.085 0.425 0.75
100 ± 14.7 91.9 ± 0.4 24.8 ± 3.1 0.1 ± 3.9 0 85.9 ± 3.7 46.6 ± 5.9 1.6 ± 2.4 100 ± 12.4 52.8 ± 15.1 16.51 ± 14.6 100 ± 5.9 100 ± 27.1 97.2 ± 10.8 89.7 ± 10.8 94.8 ± 17.1 78.1 ± 49.1 100 ± 16.0 100 ± 3.08
99.9 ± 2.0 98.4 ± 1.5 29.8 ± 2.8 6.4 ± 10.5 0 87.9 ± 1.1 46.1 ± 3.0 0 99.8 ± 0.4 60.0 ± 1.8 21.0 ± 5.2 97.4 ± 1.8 90.7 ± 8.7 96.3 ± 2.8 79.9 ± 8.9 76.9 ± 15.6 73.9 ± 36.9 94.4 ± 6.1 97.0 ± 5.1
0.1 0.5 1 1.5 2 1 1.5 2 0.68 1.36 2.04 2.72 3.4 0.01 0.02 0.03 0.04 0.05 0.085 0.425 0.75
86.1 ± 4.0 96.5 ± 6.5 84.6 ± 7.3 34.5 ± 10.9 26.7 ± 17.4 95.2 ± 4.5 46.8 ± 21.4 5.8 ± 1.9 85.0 ± 8.6 30.8 ± 2.0 48.5 ± 6.2 53.8 ± 5.5 15.5 ± 6.2 100 ± 1.2 100 ± 2.6 100 ± 2.9 99.8 ± 4.3 100 ± 5.0 99.9 ± 2.1 100 ± 2.3 100 ± 1.7
98.1 ± 1.6 100 ± 1.4 81.4 ± 3.5 34.0 ± 10.9 26.4 ± 13.9 98.7 ± 1.2 33.2 ± 5.1 0 90.3 ± 1.2 46.9 ± 8.9 57.7 ± 13.1 64.2 ± 18.0 29.4 ± 5.1 97.9 ± 0.8 99.5 ± 1.0 98.9 ± 1.2 97.3 ± 1.5 99.5 ± 0.6 98.5 ± 2.6 99.2 ± 1.4 98.9 ± 2.0
0.1 0.5 1 1.5 2 1 1.5 2 0.68 1.36 2.04 2.72 3.4 0.01 0.02 0.03 0.04 0.05 0.085 0.425 0.75
96.9 ± 0.9 96.8 ± 4.5 94.1 ± 5.4 89.6 ± 7.2 73.0 ± 14.7 99.2 ± 2.3 89.0 ± 1.5 77.4 ± 4.7 97.5 ± 7.0 89.9 ± 5.0 79.3 ± 4.2 59.2 ± 7.1 39.8 ± 6.9 99.5 ± 4.8 100 ± 6.0 100 ± 4.6 100 ± 8.3 100 ± 3.5 99.8 ± 4.6 99.0 ± 6.0 100 ± 3.0
96.9 ± 3.3 97.6 ± 2.4 96.2 ± 2.6 97.9 ± 1.6 75.1 ± 3.6 97.6 ± 1.2 94.5 ± 2.1 90.1 ± 6.3 100 ± 0.1 94.5 ± 2.7 80.6 ± 3.0 56.6 ± 3.0 46.9 ± 0.1 93.6 ± 3.5 99.5 ± 1.3 98.7 ± 2.9 97.6 ± 0.1 98.1 ± 1.8 96.6 ± 3.2 96.9 ± 2.7 95.2 ± 6.6
EG
PG
TRE
PVP
Fertilized egg
Me2SO
EG
PG
TRE
PVP
Blastula
Me2SO
EG
PG
TRE
PVP
other hand, significant toxic effects were detected in the EG + TRE combination for the egg and fertilized egg stages, but not for the blastula stage. Although TRE increased EG toxicity to the unfertilized egg, toxicity to the fertilized egg and blastula was significantly reduced (Fig. 3 and Table 2). The PG + PVP and the PG + TRE combinations tested were not toxic to the unfertilized egg, fertilized egg, or blastula stages at
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the tested concentrations (Fig. 4 and Table 2). Therefore, both PVP and TRE seem to have a protective role reducing consistently PG toxicity regardless of the developmental stage tested. Cryopreservation trials Among the experiments conducted with different CPAs combinations, the most successful cryopreservation involved impregnation of fertilized eggs and blastulae with PG (2 M) + PVP (0.75 M). The following protocols, based on successful methods used for sea urchin and bivalve species [1,15,24], were assayed: (a) cool from 4 to 12 °C at 1 °C/min, hold for 2 min for seeding, and then cool to 35 °C at 1 °C/min, and (b) cool from 4 to 12 °C at 1 °C/ min, hold 2 min for seeding, cool to 20 °C at 0.5 °C/min and then cool to 35 °C at 1 °C/min. Larvae developed from fertilized eggs frozen by this method achieved a 30.1% (protocol a) and 42.9% (protocol b) growth with respect to control unfrozen eggs, whereas the percentage of pluteus larvae was 41.5% (13% 4-arm pluteus larvae) and 40.5% (27% 4-arm pluteus larvae). On the other hand, larvae developed from frozen blastulae achieved a 30.3% (protocol a) and 60.5% (protocol b) growth, whereas the percentage of pluteus larvae was 40.5% (27% 4-arm pluteus larvae) and 68.5% (25.9% 4arm pluteus larvae). Discussion The successful cryopreservation of early developmental stages of marine organisms depends on the selection of suitable CPA treatments, taking into account both its toxicity and its ability to avoid or minimize the formation of internal and external ice crystals, i.e. its efficiency [32,35]. Cryoprotectants can suppress most cryoinjuries but, when used at higher (more effective) concentrations, most of them become toxic. For instance, Me2SO has diverse effects on ion transporters and pumps and inhibits catalase and peroxide activity [22,30,37], whereas EG and PG cause membrane damage by decreasing the polarity of the aqueous phase and changing the partition of hydrophobic molecules between the cell membrane and the external phase [14,22]. Taking this into account, it must be ensured that a right balance between their toxicity, if any, and their protective ability, is achieved, in order to offer protection rather than damage to the cells. There is a wide range of CPAs that have been used for the cryopreservation of marine invertebrate larvae. Among those, we have chosen the permeant CPAs Me2SO, PG, and EG, and the non-permeant TRE and PVP, to study their suitability for the cryopreservation of early embryos of the sea urchin P. lividus. In general, Me2SO showed the highest toxicity to the three tested developmental stages after 30 min exposure, followed by EG and PG, whilst the non-permeant CPAs were no toxic at the experimental concentrations. From these results it can be concluded that PG and EG are, in terms of its toxicity, the most suitable CPAs for the cryopreservation of P. lividus unfertilized eggs and blastulae, at concentrations of no more than 1 M, and EG seem to be also the best CPA to be used with fertilized eggs at the same concentrations. In this line, Asahina and Takahashi [3] and Adams et al. [1] found that EG was less toxic than Me2SO to eggs and 4-arm pluteus larvae of the sea urchins Hemicentrotus pulcherrimus and Evechinus chloroticus, following CPA exposure and removal. However, when the gastrula stage was evaluated, EG was more toxic than Me2SO at a concentration of 2 M [1]. Despite methodological differences, previous studies involving gametes and embryos of bivalves report, usually, higher toxicity of PG, followed by EG and Me2SO, which showed the lowest toxicity [12,28,36]. However, some studies carried out with embryos and larvae of the oyster Crassostrea gigas and the abalone Haliotis midae, report higher toxicity of Me2SO than EG and PG [15,35];
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% Larval growth
A
B
Me2SO 1 M
Me2SO 1.5 M
100 80 60 40 20 0 Egg
Fert egg
PVP 0.085 M
C
Egg
Blastula
PVP 0.425 M
Fert egg PVP 0.75 M
D
Me2SO 1 M
Blastula
E
Me2SO 1.5 M
Me2SO 2 M
% Larval growth
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg TRE 0.03 M
Blastula
Egg
Fert egg
Blastula
TRE 0.04 M
Fig. 2. Percentage of larval growth in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs, and blastulae to different combinations of Me2SO with PVP and TRE. (A) Me2SO 1 M + PVP; (B) Me2SO 1.5 M + PVP; (C) Me2SO 1 M + TRE; (D) Me2SO 1.5 M + TRE; (E) Me2SO 2 M + TRE. Error bars represent standard deviations.
A
B
EG 1 M
C
EG 1.5 M
EG 2 M
% Larval growt
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg PVP 0.425 M
D
E
EG 1 M
Blastula
Egg
Fert egg
Blastula
PVP 0.75 M
F
EG 1.5 M
EG 2 M
% Larval growt
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg TRE 0.03 M
Blastula
Egg
Fert egg
Blastula
TRE 0.04 M
Fig. 3. Percentage of larval growth in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs, and blastulae to different combinations of EG with PVP and TRE. (A) EG 1 M + PVP; (B) EG 1.5 M + PVP; (C) EG 2 M + PVP; (D) EG 1 M + TRE; (E) EG 1.5 M + TRE; (F) EG 2 M + TRE. Error bars represent standard deviations.
whereas similar toxicity of the three cryoprotectants for oyster embryos and larvae has also been found [24]. The results obtained in the present study also indicate that the blastula stage was more resistant to the CPAs than the unfertilized and fertilized eggs. This finding supports data from previous studies conducted with bivalves, reporting higher resistance of the trochophore stage to cryoprotectants than earlier embryos or gametes [15,28,36]. Therefore,
it is evident from this and previous studies that CPAs tolerance in marine invertebrates is species and stage specific. Thus, the studies of suitability of CPAs for the cryopreservation of embryos and larvae of marine invertebrates should be carried out with the species and developmental stages intended to be used. Since the efficiency of CPAs in their protective role against freezing injuries increases when used at higher concentrations,
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% Larval growth
A
B
PG 0.68 M
C
PG 1.36 M
PG 2.04 M
100 80 60 40 20 0 Egg
Fert egg
Blastula
Egg
Fert egg
PVP 0.425 M
D
E
PG 0.68 M
Blastula
Egg
Fert egg
Blastula
PVP 0.75 M
F
PG 1.36 M
PG 2.04 M
% Larval growth
100 80 60 40 20 0 Fert egg
Blastula
Fert egg
Blastula
TRE 0.03 M
Fert egg
Blastula
TRE 0.04 M
Fig. 4. Percentage of larval growth in P. lividus after 30 min exposure of unfertilized eggs, fertilized eggs and blastulae to different combinations of PG with PVP and TRE. (A) PG 0.68 M + PVP; (B) PG 1.36 M + PVP; (C) PG 2.04 M + PVP; (D) PG 0.68 M + TRE; (E) PG 1.36 M + TRE; (F) PG 2.04 M + TRE. Error bars represent standard deviations.
several methods need to be applied in order to mitigate the increase of toxicity caused by those high concentrations. One of the approaches used is to add and remove the CPAs in a gradual stepwise protocol of several fixed molar steps, in order to reduce the osmotic stress to eggs and embryos [1,32]. In the present work, the addition of CPAs in 15 fixed molar steps instead of 15 fixed volume steps, reduced significantly the larval abnormalities and increased larval growth. Furthermore, the addition of high molecular weight non-permeant CPAs has been reported as a complementary approach to reduce the toxicity of permeant CPAs [12]. The beneficial effect of combining TRE and PVP with permeant CPAs, which was demonstrated in the present study for P. lividus, would allow loading eggs and embryos with higher, and therefore more effective, concentrations of permeant CPAs. It must be noted, though, that the protective ability of TRE and PVP was dependant on the permeant CPA and on the developmental stage tested. PVP consistently reduced EG and PG toxicity to the eggs and embryos of P. lividus and reduced Me2SO toxicity to the unfertilized egg. On the other hand, TRE reduced PG toxicity to the eggs and embryos, the toxicity of EG to the blastula stage and the toxicity of Me2SO to the unfertilized egg. Although previous studies with sea urchin embryos and larvae found that Me2SO was better as a cryoprotectant than EG [1,4– 6,27], on the basis of the toxicity results obtained here, it would be recommended to use solutions with 2 M PG or EG in combination with 0.75 M PVP, for the cryopreservation of P. lividus embryos. Following the evaluation of CPAs toxicity, cryopreservation trials were done. Freezing and thawing experiments were conducted using PG and PVP combinations and different cryopreservation protocols were tested. Different cooling rates and holding temperatures were examined, as well as the effects of incorporating a seeding step during cooling. Since unfertilized eggs showed very poor post-thaw normal development, experimental efforts focused mainly on fertilized eggs and blastulae. Solutions containing 2 M PG and 0.75 M PVP were the most effective, and the incorporation
of a seeding step at 12 °C into the freezing protocol improved the normal embryonic development post-thawing. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively. In conclusion, this study provides results on the toxicity of five CPAs to eggs and blastulae of the sea urchin P. lividus. Our results indicate that, on the basis of their toxicity, PG and EG in combination with PVP might be the most suitable CPAs for this species. Promising results have been achieved using PG and PVP combinations in cryopreservation trials with fertilized eggs and blastulae. However, since Me2SO has been proven to be an efficient CPA for several marine invertebrates, including sea urchins, this CPA should not be discarded in future cryopreservation studies with P. lividus embryos. Future studies also should focus on a detailed evaluation of the cryopreservation protocol, including the impact of plunging into liquid nitrogen and the conservation of the biological material. Acknowledgments We thank Ricardo Beiras for valuable comments on the manuscript and the personnel from the Estación de Ciencias Mariñas de Toralla (Universidade de Vigo) for providing us with sea urchins. References [1] S.L. Adams, P.A. Hessian, P.V. Mladenov, The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus, Cryobiology 52 (2006) 139–145. [2] E. Asahina, T. Takahashi, Survival of sea urchin spermatozoa and embryos at very low temperatures, Cryobiology 14 (1977) 703. [3] E. Asahina, T. Takahashi, Freezing tolerance in embryos and spermatozoa of the sea urchin, Cryobiology 15 (1978) 122–127. [4] E. Asahina, T. Takahashi, Cryopreservation of sea urchins embryos and sperm, Development Growth and Differentiation 21 (1979) 423–430. [5] C. Barros, A. Muller, M.J. Wood, D.G. Whittingham, High survival of sea urchin semen (Tetrapigus niger) pluteus larvae (Loxechinus albus) frozen in 1.0 M Me2SO, Cryobiology 33 (1996) 646.
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