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Effects of salinity on pre- and post-fertilization developmental events in the clam Anomalocardia flexuosa (Linnaeus, 1767)

Abstract

The knowledge about the effect of salinity on the physiological mechanism of bivalve reproduction is fundamental to improve production strategies in hatcheries. The present work evaluated the influence of different salinity concentrations (15, 20, 25, 30, 35 and 40 g⋅L−1) on pre- and post-fertilization development processes in the clam, Anomalocardia flexuosa, oocytes obtained by stripping. Salinity directly interfered with the germinal vesicle breakdown (GVBD) rate and in the cellular stability of unfertilized oocytes. Salinity concentrations between 30 and 35 g⋅L−1 provided better percentages of stable GVBD within 120 min, and incubation of oocytes in the salinity range of 30-35 g⋅L−1 for a time interval of 80-120 min provided > 80% GVBD. In the post-fertilization analysis, salinity affected the rate of the extrusion of the first and second polar bodies (PB1 and PB2). The release of 50% of the PBs was faster at a salinity of 35 g⋅L−1, with an estimated time of 10 min for PB1 and 30 min for PB2. Thus, chromosome manipulation methodologies aiming triploids should be applied at 35 g⋅L−1 salinity, with application of post-fertilization shock before 10 min for PB1 retention or before 30 min for PB2 retention.

Keywords:
tropical bivalve reproduction; stripping; germinal vesicle breakdown; polar body

Introduction

The production of bivalves in tropical regions has been consolidated as an important market for aquaculture (Nowland et al., 2020Nowland SJ, O’Connor WA, Osborne MWJ, Southgate PC. Current status and potential of tropical rock oyster aquaculture. Rev Fish Sci Aquacult. 2020;28(1):57-70. http://dx.doi.org/10.1080/23308249.2019.1670134.
http://dx.doi.org/10.1080/23308249.2019....
; Willer and Aldridge, 2020Willer DF, Aldridge DC. Sustainable bivalve farming can deliver food security in the tropics. Nat Food. 2020;1(7):384-8. http://dx.doi.org/10.1038/s43016-020-0116-8.
http://dx.doi.org/10.1038/s43016-020-011...
). However, tropical bivalve larviculture presents a series of obstacles that limits the continuous supply of seeds, essential to aquaculture and restocking (Nowland et al., 2021Nowland SJ, O’Connor WA, Elizur A, Southgate PC. Evaluating spawning induction methods for the tropical black-lip rock oyster, Saccostrea echinata. Aquacult Rep. 2021;20:100676. http://dx.doi.org/10.1016/j.aqrep.2021.100676.
http://dx.doi.org/10.1016/j.aqrep.2021.1...
). One promising species for tropical aquaculture (Lagreze-Squella et al., 2018Lagreze-Squella FJ, Sühnel S, Vieira G, Langdon C, Melo CMR. Optimizing broodstock conditioning for the tropical clam Anomalocardia brasiliana. J Shellfish Res. 2018;37:979-87. http://dx.doi.org/10.2983/035.037.0508.
http://dx.doi.org/10.2983/035.037.0508...
) is the clam Anomalocardia flexuosa, which is distributed from the Caribbean to Brazil (Abbott, 2011Abbott RT. American seashells. 2nd ed. New York: Van Nostrand Reinhold Company; 2011. https://doi.org/10.5962/bhl.title.7471.
https://doi.org/10.5962/bhl.title.7471...
), and plays an important role in feeding and in the income of traditional communities (Rios, 2009Rios EC. Compendium of Brazilian sea shells. Rio Grande: Evangraf; 2009.). Hatchery production methodologies based on feeding, maturation, reproduction, larviculture and settlement events under controlled laboratory conditions have been developed for this species (Lavander et al., 2014Lavander HD, Silva SR No, Sobral SC, Lima PCM, Rêgo MG, Gálvez AO. Manutenção e reprodução de Anomalocardia brasiliana em condições laboratoriais. Rev Bras Ciênc Agrár. 2014;9(2):269-76. http://dx.doi.org/10.5039/agraria.v9i2a3237.
http://dx.doi.org/10.5039/agraria.v9i2a3...
; Lagreze-Squella et al., 2018Lagreze-Squella FJ, Sühnel S, Vieira G, Langdon C, Melo CMR. Optimizing broodstock conditioning for the tropical clam Anomalocardia brasiliana. J Shellfish Res. 2018;37:979-87. http://dx.doi.org/10.2983/035.037.0508.
http://dx.doi.org/10.2983/035.037.0508...
; Lima et al., 2018Lima PCM, Lavander HD, Silva LOB, Gálvez AO. Larviculture of the sand clam cultivated in different densities. Bol Inst Pesca. 2018;44(2):1-7.).

The clam A. flexuosa, like most bivalve species, presents external fertilization. Stripping of the gonadal tissue gives access to undeveloped oocytes in maturation stages with intact germinal vesicle structure. This occurs because the oocytes of some species undergo a maturation process during the passage through the oviducts before their release (Colas and Dubé, 1998Colas P, Dubé F. Meiotic maturation in mollusc oocytes. Semin Cell Dev Biol. 1998;9(5):539-48. http://dx.doi.org/10.1006/scdb.1998.0248. PMid:9835642.
http://dx.doi.org/10.1006/scdb.1998.0248...
).

Germinal vesicle breakdown (GVBD) is a natural process occurring in the external environment when metaphase I oocytes are released (Guo et al., 1996Guo X, DeBrosse GA, Allen SK Jr. All-triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids. Aquaculture. 1996;142(3-4):149-61. http://dx.doi.org/10.1016/0044-8486(95)01243-5.
http://dx.doi.org/10.1016/0044-8486(95)0...
). This process is considered as a sign of oocyte maturation. To continue the GVBD stage, the oocytes may go through a period of hydration in the external environment (Melo et al., 2015Melo EMC, Gomes CHA M, Silva FC, Sühnel S, Melo CMR. Métodos químico e físico de indução à triploidia em Crassostrea gigas (Thunberg, 1793). Bol Inst Pesca. 2015;41:889-98.). The breakdown of the vesicle improves fertilization, decreases polyspermia, and increases efficiency in polyploidy induction processes (Qin et al., 2018Qin Y, Xiao S, Ma H, Mo R, Zhou Z, Wu X, Zhang Y, Yu Z. Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters, Crassostrea hongkongensis, in relation to tetraploid induction. Aquacult Res. 2018;49(11):3647-57. http://dx.doi.org/10.1111/are.13833.
http://dx.doi.org/10.1111/are.13833...
), and has been exploited in the hatchery of bivalves (Dégremont et al., 2012Dégremont L, Garcia C, Frank-Lawale A, Allen SK. Triploid oysters in the Chesapeake Bay: comparison of diploid and triploid Crassostrea virginica. J Shellfish Res. 2012;31(1):21-31. http://dx.doi.org/10.2983/035.031.0103.
http://dx.doi.org/10.2983/035.031.0103...
).

The process of embryogenesis begins from the time the sperm enters the oocyte and is characterized by the release of two polar bodies, PB1 and PB2 (Colas and Dubé, 1998Colas P, Dubé F. Meiotic maturation in mollusc oocytes. Semin Cell Dev Biol. 1998;9(5):539-48. http://dx.doi.org/10.1006/scdb.1998.0248. PMid:9835642.
http://dx.doi.org/10.1006/scdb.1998.0248...
). Polyploidy can be achieved by the physical or chemical treatment of fertilized eggs to inhibit the exit of PB1 or PB2 (Piferrer et al., 2009Piferrer F, Beaumont A, Falguière JC, Flajšhans M, Haffray P, Colombo L. Polyploid fish and shellfish: production, biology and applications to aquaculture for performance improvement and genetic containment. Aquaculture. 2009;293(3-4):125-56. http://dx.doi.org/10.1016/j.aquaculture.2009.04.036.
http://dx.doi.org/10.1016/j.aquaculture....
).

Environmental factors such as salinity affect the incubation time and the extrusion of polar bodies (Lavander et al., 2014Lavander HD, Silva SR No, Sobral SC, Lima PCM, Rêgo MG, Gálvez AO. Manutenção e reprodução de Anomalocardia brasiliana em condições laboratoriais. Rev Bras Ciênc Agrár. 2014;9(2):269-76. http://dx.doi.org/10.5039/agraria.v9i2a3237.
http://dx.doi.org/10.5039/agraria.v9i2a3...
). The knowledge of the effect of environmental variables on oocyte GVBD and the timing of polar bodies release is critical to increase fertilization success in hatcheries, achieve greater control in subsequent embryonic developmental stages, and for adjusting protocols for the use and control of polyploidy (Qin et al., 2018Qin Y, Xiao S, Ma H, Mo R, Zhou Z, Wu X, Zhang Y, Yu Z. Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters, Crassostrea hongkongensis, in relation to tetraploid induction. Aquacult Res. 2018;49(11):3647-57. http://dx.doi.org/10.1111/are.13833.
http://dx.doi.org/10.1111/are.13833...
). This study evaluated the influence of salinity on the time of oocyte GVBD and on the extrusion of polar bodies in A. flexuosa.

Methods

Broodstock

A total of 150 adults were collected in estuarine zone of the state of Maranhão (2º30’03”S, 44º03’40”W) and stored at the State University of Maranhão for the experiments. Approval by the Ethics Council for the Use of Experimental Animals does not apply to the invertebrate group, in accordance with the Brazilian law 11.794/08 (Brasil, 2008Brasil. Lei nº 11.794, de 8 de outubro de 2008. Regulamenta o inciso VII do § 1º do art. 225 da Constituição Federal, estabelecendo procedimentos para o uso científico de animais; revoga a Lei nº 6.638, de 8 de maio de 1979; e dá outras providências. Diário Oficial da União; Brasil; 9 October 2008.).

Clams were cleaned with a sodium hypochlorite solution (2%) and then stored in a 100 L tank in water treated by cartridge filters (50, 25, and 5 μm), biological filter (containing different media and nitrifying bacteria), and UV. The organisms were maintained at a salinity of 30 g⋅L−1, temperature of 24 °C, and constant aeration for 4 h of depuration in a Recirculating Aquaculture System (RAS). After this period, the animals were left out of the water overnight at 24 °C to prevent the release of gametes into the water.

Gametes

For sex identification, each individual was carefully opened using a knife and the gonadal tissue was observed under an optical microscope. Gametes from 30 females were obtained by stripping using a surgical blade. Subsequently, they were mixed and filtered in a 100 µm mesh and rinsed in 25 μm. A dilution of the gametes was carried out to reach a final rate of 50 oocytes/mL in each experimental unit.

GVBD at different salinities

The effect of salinity on GVBD was evaluated at salinities of 15, 20, 25, 30, 35, and 40 g⋅L−1, with five replicates per treatment. The marine water used in the experiment was collected at sea (original concentration of 42 g.L−1), filtered and sterilized in a UV filter in the laboratory. The dilution for the different saline concentrations was carried out by adding autoclaved freshwater.

The experimental units were thermostat regulated at 26 °C, which was considered as the ideal temperature for meiosis of the species (Lavander et al., 2017Lavander H, Santos G, Olivera A, Carvalho R, Guerra M, Coimbra MRM. Meiosis maturation in the marine clam Anomalocardia brasiliana (Veneridae). J Shellfish Res. 2017;36(3):601-5. http://dx.doi.org/10.2983/035.036.0308.
http://dx.doi.org/10.2983/035.036.0308...
). Diluted oocytes were distributed in 100 mL beakers, from which 1 mL aliquot was removed with the aid of a pipette for observation of the progression of GVBD (Figure 1). The germinal vesicle breakdown process was observed over a period of 120 min, from the observation of 1ml samples (approximately 50 oocytes) taken every 10 minutes for counting and recording.

Figure 1
Images registered in the program Mosaic V.2.2.1 with a camera attached to the microscope showing the appearance of Anomalocardia flexuosa oocytes before and after germinal vesicle breakdown (GVBD); A: Image of a set of oocytes without uniformity soon after undergoing the stripping process observed under 4X lens; B: Set of oocytes after hydration showing the GVBD under 4X lens; C: Image of a magnified oocyte before hydration with an arrow in yellow highlighting the structure of the intact germinal vesicle observed under 10X lens; D: Image of an oocyte magnified after hydration and with a yellow arrow indicating the uniformity of the nucleus as a function of the GVBD observed at 10X lens.

Extrusion of PB1 and PB2 in different salinities

The spermatozoa from 30 males were obtained by stripping, mixed, filtered using 80 µm mesh and quantified by optical microscopy. After 2 h of oocyte hydration for GVBD in different salinities (15, 20, 25, 30, 35, and 40 g⋅L−1) and at a constant temperature of 26 °C, the spermatozoa were added to each experimental unit at a ratio of 7 spermatozoa: 1 oocyte, according to the previously described methodology (Melo et al., 2015Melo EMC, Gomes CHA M, Silva FC, Sühnel S, Melo CMR. Métodos químico e físico de indução à triploidia em Crassostrea gigas (Thunberg, 1793). Bol Inst Pesca. 2015;41:889-98.). The first and second polar bodies (PB1 and PB2) extrusion rates were monitored at 5 min intervals over a period of 60 min using 1 ml samples (approximately 50 oocytes).

The average time to obtain the 50% of PB1 and PB2 extrusions for each treatment were determined as previously recommended (Melo et al., 2015Melo EMC, Gomes CHA M, Silva FC, Sühnel S, Melo CMR. Métodos químico e físico de indução à triploidia em Crassostrea gigas (Thunberg, 1793). Bol Inst Pesca. 2015;41:889-98.; Lavander et al., 2017Lavander H, Santos G, Olivera A, Carvalho R, Guerra M, Coimbra MRM. Meiosis maturation in the marine clam Anomalocardia brasiliana (Veneridae). J Shellfish Res. 2017;36(3):601-5. http://dx.doi.org/10.2983/035.036.0308.
http://dx.doi.org/10.2983/035.036.0308...
).

Statistical analyses

The averages of the five replicates of each salinity treatment were used to estimate the GVBD and the extrusion of PB1 and PB2 at the different time interval. The trends of the percentages of GVBD and PB released with time at each salinity concentration were estimated by fitting the best likelihood model with a generalized additive model (GAM) and considering a binomial distribution. A two-way analysis of variance (ANOVA) was used to measure treatment effects, and averages were compared using Tukey's post-hoc test at a p ≤ 0.05 significance level.

Statistical analyses were performed in R studio version 4.1.0, and the package “MGCV” (Wood, 2011Wood SN. Fast stable restricted maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models. J R Stat Soc Series B Stat Methodol. 2011;73(1):3-36. http://dx.doi.org/10.1111/j.1467-9868.2010.00749.x.
http://dx.doi.org/10.1111/j.1467-9868.20...
) was used to run the models. The steps for model selection were performed as previously described (Zuur, 2012Zuur AF. A beginner’s guide to generalized additive models with R. Newburgh: Highland Statistics Ltd; 2012.; Zuur et al., 2009Zuur AF, Ieno EN, Walker NJ, Saveliev AA, Smith GM. Mixed effects models and extensions in ecology with R. J Stat Softw. 2009;32:1-3. http://dx.doi.org/10.1007/978-0-387-87458-6.
http://dx.doi.org/10.1007/978-0-387-8745...
).

Results

Effect of salinity on GVBD

None of the salinity treatments resulted in 100% GVBD, however, high values were found at 30 and 35 g⋅L−1 salt concentrations (93.26 ± 1.04% and 93.76 ± 4.98%, respectively) at 120 min. Also, at 40 g⋅L−1 salt concentration 93.99% ± 7 were detected at 50 min, followed by a decrease in the breakdown in subsequent intervals. Salinity treatments at 15, 20 and 25 g⋅L−1 presented inferior values <85% after 120 min (Table 1 and Figure 2).

Table 1
Germinal vesicle breakdown of Anomalocardia flexuosa oocytes under salinity influence.
Figure 2
Mean of the percentages of germinal vesicle breakdown of oocytes obtained by stripping from Anomalocardia flexuosa.

Most of the treatments showed an increase in the percentage of GVBD as salinity raised, but in the 40 g⋅L−1 salinity treatment, the oocytes started to break up after 60 min to generate a significant loss until 120 min.

Effect of salinity on the extrusion of PB1 and PB2

Fertilization was conducted right after 120 min to guarantee maximum level of GVBD considering the previous section results. The extrusion of 50% PB in 20, 25, 30, and 40 g⋅L−1 treatment groups occurred nearly within the first 15 min. For the 15 g⋅L−1, the average time was 25 min, while for 35 g⋅L treatment group it happened in the first 10 min (Table 2 and Figure 3).

Table 2
Percentages of polar body 1 (PB1) extrusion of Anomalocardia flexuosa at each 5 min interval.
Figure 3
Mean of the percentages of polar body 1 release from fertilized eggs of Anomalocardia flexuosa.

For the extrusion of PB2, in treatments at 20, 25, and 30 g⋅L−1, nearly 50% was achieved in the first 35 min, while that rate was accomplished in the first 30 min for 35 and 40 g⋅L−1 s (Table 3 and Figure 4). Yet for 15 g⋅L−1, extrusion was accomplished later, in 45 min.

Table 3
Percentages of polar body 2 (PB2) extrusion of Anomalocardia flexuosa at each 5 min interval.
Figure 4
Mean percentage of polar body 2 release from fertilized eggs of Anomalocardia flexuosa.

Discussion

Effect of salinity on GVBD

Sodium-potassium antagonism is largely responsible for the oocyte germinal vesicle maintenance (Allen, 1953Allen RD. Fertilization and artificial activation in the egg of the surf-clam, Spisula solidissima. Biol Bull. 1953;105(2):213-39. http://dx.doi.org/10.2307/1538639.
http://dx.doi.org/10.2307/1538639...
). The presence of Ca2+ ions, on the other hand, is essential for triggering vesicle degradation (Colas and Dubé, 1998Colas P, Dubé F. Meiotic maturation in mollusc oocytes. Semin Cell Dev Biol. 1998;9(5):539-48. http://dx.doi.org/10.1006/scdb.1998.0248. PMid:9835642.
http://dx.doi.org/10.1006/scdb.1998.0248...
). Thus, the salt concentration during hydration of oocytes obtained by stripping directly influences the GVBD (Qin et al., 2018Qin Y, Xiao S, Ma H, Mo R, Zhou Z, Wu X, Zhang Y, Yu Z. Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters, Crassostrea hongkongensis, in relation to tetraploid induction. Aquacult Res. 2018;49(11):3647-57. http://dx.doi.org/10.1111/are.13833.
http://dx.doi.org/10.1111/are.13833...
). According to Allen and Bushek (1992)Allen SK Jr, Bushek D. Large-scale production of triploid oysters, Crassostrea virginica (Gmelin), using “stripped” gametes. Aquaculture. 1992;103(3-4):241-51. http://dx.doi.org/10.1016/0044-8486(92)90170-P.
http://dx.doi.org/10.1016/0044-8486(92)9...
, the “immersion time” in seawater is important for synchronization of oocytes that are in prophase I. Thus, GVBD is a signal that meiosis is preparing to advance to the metaphase I stage until fertilization (Eudeline et al., 2000Eudeline B, Allen SK Jr, Guo X. Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production. J Exp Mar Biol Ecol. 2000;248(2):151-61. http://dx.doi.org/10.1016/S0022-0981(00)00158-1. PMid:10771299.
http://dx.doi.org/10.1016/S0022-0981(00)...
).

The present study suggests the existence of an association between increasing salinity concentrations and GVBD rates. The obtained results suggested that there are important aspects of biochemical and biophysical characteristics of A. flexuosa oocytes, such as plasma membrane permeability and osmotic tolerance that interfere on GVBD rates. In hypertonic medium, the cells first react by shrinking owing to the exit of water from the cellular structure. This phenomenon is followed by cell swelling following the entry of water to maintain the osmotic balance (Salinas-Flores et al., 2008Salinas-Flores L, Adams SL, Lim MH. Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol. Cryobiology. 2008;56(1):43-52. http://dx.doi.org/10.1016/j.cryobiol.2007.10.175. PMid:18155687.
http://dx.doi.org/10.1016/j.cryobiol.200...
).

A. flexuosa is characterized as a species tolerant to variation in high salinities (Lagreze-Squella et al., 2018Lagreze-Squella FJ, Sühnel S, Vieira G, Langdon C, Melo CMR. Optimizing broodstock conditioning for the tropical clam Anomalocardia brasiliana. J Shellfish Res. 2018;37:979-87. http://dx.doi.org/10.2983/035.037.0508.
http://dx.doi.org/10.2983/035.037.0508...
) and, therefore, can adapt its reproduction to the environmental conditions. Based on our results, however, the oocyte stability seems to be associated with a tolerance limit for high saline concentrations. The osmotic tolerance limit may be exceeded if cells undergo excessive shrinkage or swelling that may lead to irreversible injury (Salinas-Flores et al., 2008Salinas-Flores L, Adams SL, Lim MH. Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol. Cryobiology. 2008;56(1):43-52. http://dx.doi.org/10.1016/j.cryobiol.2007.10.175. PMid:18155687.
http://dx.doi.org/10.1016/j.cryobiol.200...
). Although the salinity of 40 g⋅L−1 had a higher catalytic ability for GVBD in our study, the effort to maintain the osmotic balance seemed to be critical for oocytes, leading to the rupture of the cell structure after 60 min.

Different studies have already pointed out that there are factors that can disturb embryogenesis and larval development such as gonadal maturation, oocyte maturation, and polyspermia in bivalves (Lavander et al., 2011Lavander HD, Cardoso LO Jr, Oliveira RL, Silva SR No, Galvez AO, Peixoto SRM. Biologia reprodutiva da Anomalocardia brasiliana (Gmelin, 1791) no litoral norte de Pernambuco, Brasil. Rev Bras Ciênc Agrár. 2011;6(2):344-50. http://dx.doi.org/10.5039/agraria.v6i2a1139.
http://dx.doi.org/10.5039/agraria.v6i2a1...
; Qin et al., 2018Qin Y, Xiao S, Ma H, Mo R, Zhou Z, Wu X, Zhang Y, Yu Z. Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters, Crassostrea hongkongensis, in relation to tetraploid induction. Aquacult Res. 2018;49(11):3647-57. http://dx.doi.org/10.1111/are.13833.
http://dx.doi.org/10.1111/are.13833...
). The larviculture of A. flexuosa is characterized as a stage strongly affected by factors such as temperature and salinity, water quality, feeding, and management (Oliveira et al., 2016Oliveira RLM, Lavander HD, Santos LBG, Calazans NKF, Gálvez AO, Peixoto SRM. Hatchery rearing of the venerid clam Anomalocardia brasiliana (Gmelin, 1791). J Shellfish Res. 2016;35(1):27-30. http://dx.doi.org/10.2983/035.035.0104.
http://dx.doi.org/10.2983/035.035.0104...
). In this sense, identifying the best conditions for fertilization can be a tool to help seed production in hatchery. For A. flexuosa, 30 and 35 g⋅L−1 salinity in the time between 80 and 120 min were the optimum range of hydration with higher percentages of GVBD.

Effect of salinity on the 50% release of PB1 and PB2

The use of polyploidy has become popular in bivalve aquaculture (Ma et al., 2019Ma P, Wang Z, Yu R. Triploid induction by hyperosmotic shock in the Yesso scallop, Patinopecten yessoensis. J World Aquacult Soc. 2019;50(5):922-33. http://dx.doi.org/10.1111/jwas.12583.
http://dx.doi.org/10.1111/jwas.12583...
), either due to gonadal sterility (Piferrer et al., 2009Piferrer F, Beaumont A, Falguière JC, Flajšhans M, Haffray P, Colombo L. Polyploid fish and shellfish: production, biology and applications to aquaculture for performance improvement and genetic containment. Aquaculture. 2009;293(3-4):125-56. http://dx.doi.org/10.1016/j.aquaculture.2009.04.036.
http://dx.doi.org/10.1016/j.aquaculture....
; Dheilly et al., 2014Dheilly NM, Jouaux A, Boudry P, Favrel P, Lelong C. Transcriptomic profiling of gametogenesis in triploid pacific oysters Crassostrea gigas: towards an understanding of partial sterility associated with triploidy. PLoS One. 2014;9(11):e112094. http://dx.doi.org/10.1371/journal.pone.0112094. PMid:25375782.
http://dx.doi.org/10.1371/journal.pone.0...
; Zhang et al., 2017Zhang Y, Li J, Qin Y, Zhou Y, Zhang Y, Yu Z. A comparative study of the survival, growth and gonad development of the diploid and triploid Hong Kong oyster, Crassostrea hongkongensis (Lam & Morton 2003). Aquacult Res. 2017;48(5):2453-62. http://dx.doi.org/10.1111/are.13081.
http://dx.doi.org/10.1111/are.13081...
), to the increase in the cell volume and lack of compensation of cell number (Guo and Allen, 1994Guo X, Allen SK Jr. Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis say). Genetics. 1994;138(4):1199-206. http://dx.doi.org/10.1093/genetics/138.4.1199. PMid:7896101.
http://dx.doi.org/10.1093/genetics/138.4...
), or even to the increased heterozygosity that promote larger and faster growth in bivalves (Guo et al., 2009Guo X, Wang Y, Xu Z, Yang H. Chromosome set manipulation in shellfish. In: Burnell G, Allan G, editors. New technologies in aquaculture: improving production efficiency, quality and environmental management. Cambridge: Woodhead Publishing; 2009. p. 165–94. http://dx.doi.org/10.1533/9781845696474.1.165.
http://dx.doi.org/10.1533/9781845696474....
; Yang and Guo, 2018Yang H, Guo X. Triploid hard clams Mercenaria mercenaria produced by inhibiting polar body I or polar body II. Aquacult Res. 2018;49(1):449-61. http://dx.doi.org/10.1111/are.13476.
http://dx.doi.org/10.1111/are.13476...
).

There are different alternatives to obtain triploid bivalve individuals. One of them refers to the possibility of interrupting the meiotic process by inhibiting the extrusion of PB1 or PB2 (Piferrer et al., 2009Piferrer F, Beaumont A, Falguière JC, Flajšhans M, Haffray P, Colombo L. Polyploid fish and shellfish: production, biology and applications to aquaculture for performance improvement and genetic containment. Aquaculture. 2009;293(3-4):125-56. http://dx.doi.org/10.1016/j.aquaculture.2009.04.036.
http://dx.doi.org/10.1016/j.aquaculture....
). For this purpose, the determination of the moment of the exit of 50% of PB1 or PB2 after fertilization is important as a reference for the application of inducers (Guo et al., 1996Guo X, DeBrosse GA, Allen SK Jr. All-triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids. Aquaculture. 1996;142(3-4):149-61. http://dx.doi.org/10.1016/0044-8486(95)01243-5.
http://dx.doi.org/10.1016/0044-8486(95)0...
; Lavander et al., 2017Lavander H, Santos G, Olivera A, Carvalho R, Guerra M, Coimbra MRM. Meiosis maturation in the marine clam Anomalocardia brasiliana (Veneridae). J Shellfish Res. 2017;36(3):601-5. http://dx.doi.org/10.2983/035.036.0308.
http://dx.doi.org/10.2983/035.036.0308...
; Melo et al., 2015Melo EMC, Gomes CHA M, Silva FC, Sühnel S, Melo CMR. Métodos químico e físico de indução à triploidia em Crassostrea gigas (Thunberg, 1793). Bol Inst Pesca. 2015;41:889-98.). As in GVBD, salinity can also impact post-fertilization processes such as the timing of release or inhibition of polar bodies in bivalve mollusks (Ma et al., 2019Ma P, Wang Z, Yu R. Triploid induction by hyperosmotic shock in the Yesso scallop, Patinopecten yessoensis. J World Aquacult Soc. 2019;50(5):922-33. http://dx.doi.org/10.1111/jwas.12583.
http://dx.doi.org/10.1111/jwas.12583...
).

In the present study, we were unable to detect an explicit association between increasing salinity and the speed of events, as observed in GVBD evaluation. However, there was an optimal range for the extrusion of both polar bodies that occurred from 20 to 35 g⋅L−1 salinity at an interval of 10 to 20 min for PB1 and 30 to 40 min for PB2. The time to initiation of post-fertilization treatment and its duration are the main aspects influencing the success rate of polyploidy and embryo survival (Allen and Bushek, 1992Allen SK Jr, Bushek D. Large-scale production of triploid oysters, Crassostrea virginica (Gmelin), using “stripped” gametes. Aquaculture. 1992;103(3-4):241-51. http://dx.doi.org/10.1016/0044-8486(92)90170-P.
http://dx.doi.org/10.1016/0044-8486(92)9...
; Eudeline et al., 2000Eudeline B, Allen SK Jr, Guo X. Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production. J Exp Mar Biol Ecol. 2000;248(2):151-61. http://dx.doi.org/10.1016/S0022-0981(00)00158-1. PMid:10771299.
http://dx.doi.org/10.1016/S0022-0981(00)...
).

Salinities of 15 g⋅L−1 may hold back the induction to triploidy because the longer the process time for the release of PB1 or PB2 during chromosome manipulation, the higher are the chances of anomalies occurrence (Qin et al., 2018Qin Y, Xiao S, Ma H, Mo R, Zhou Z, Wu X, Zhang Y, Yu Z. Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters, Crassostrea hongkongensis, in relation to tetraploid induction. Aquacult Res. 2018;49(11):3647-57. http://dx.doi.org/10.1111/are.13833.
http://dx.doi.org/10.1111/are.13833...
). Although 40 g⋅L−1 concentration showed high percentages of extrusion of PB1 and PB2, the anticipated cell lysis process before fertilization made this treatment unfeasible. A previous report (Guimarães et al., 2008Guimarães IM, Antonio ÍG, Peixoto S, Olivera A. Salinity influence on the survival of mangrove oyster, Crassostrea rhizophorae. Arq Ciênc Mar. 2008;41(1):118-22.) showed that salinity above 40 g⋅L−1 can be lethal in the early life of estuarine bivalves.

Another study with A. flexuosa (Lavander et al., 2017Lavander H, Santos G, Olivera A, Carvalho R, Guerra M, Coimbra MRM. Meiosis maturation in the marine clam Anomalocardia brasiliana (Veneridae). J Shellfish Res. 2017;36(3):601-5. http://dx.doi.org/10.2983/035.036.0308.
http://dx.doi.org/10.2983/035.036.0308...
) also observed that the highest salinity tested (35 g⋅L−1) resulted in better extrusion rates of PB1 and PB2 as compared to 15 and 25 g⋅L−1, corroborating the hypothesis that salinity interferes with the exit time of polar bodies. On the other hand, under the same conditions of temperature and salinity (26°C and 35 g⋅L−1), these authors found higher extrusion rates, such as of 70% for PB1 until the first 10 min and 62.67% for PB2 after 16 min of fertilization. The discrepancy in the results may be attributed to the methodological differences of observation, prediction analysis, and mainly the methodology of obtaining gametes. Using stripping instead of natural spawning to obtain gametes configures a more controlled strategy for the various treatments applied. However, oocytes obtained by stripping will be in different stages of maturation (Downing and Allen, 1987Downing SL, Allen SK Jr. Induced triploidy in the Pacific oyster, Crassostrea gigas: optimal treatments with cytochalasin B depend on temperature. Aquaculture. 1987;61(1):1-15. http://dx.doi.org/10.1016/0044-8486(87)90332-2.
http://dx.doi.org/10.1016/0044-8486(87)9...
), contrasting to those obtained by natural or induced release (Lavander et al., 2017Lavander H, Santos G, Olivera A, Carvalho R, Guerra M, Coimbra MRM. Meiosis maturation in the marine clam Anomalocardia brasiliana (Veneridae). J Shellfish Res. 2017;36(3):601-5. http://dx.doi.org/10.2983/035.036.0308.
http://dx.doi.org/10.2983/035.036.0308...
).

Regardless of the method applied for spawning, either natural or stripping, the induction of triploidy is most suitable at 35 g⋅L−1 salinity, with the possibility of carrying post-fertilization shocks before 10 min for PB1 retention or before 30 min for PB2 retention. According to a previous study (Yang and Guo, 2018Yang H, Guo X. Triploid hard clams Mercenaria mercenaria produced by inhibiting polar body I or polar body II. Aquacult Res. 2018;49(1):449-61. http://dx.doi.org/10.1111/are.13476.
http://dx.doi.org/10.1111/are.13476...
), the formation of triploid clams by retention of both PB1 and PB2 is feasible.

Unlike bivalves from temperate climate where temperature largely influences spawning (Camacho et al., 2011Camacho EG, Dominguez GR, Armenta OOZ, Haws M, Supan J, Bajo LJA, Llamas GH, Valenzuela JEV, Juárez RNP. Developing hatchery methods for the mangrove oyster, Crassostrea corteziensis for the Pacific Coast of Mexico. Culiacán: Autonomous University of Sinaloa; 2011. Final reports: investigations 2009–2011.; O’Connor et al., 2008O’Connor W, Dove M, Finn B, O’Connor S. Manual for hatchery production of Sydney rock oysters (Saccostrea glomerata). Deakin: NSW Department of Primary Industries; 2008. Fisheries research report series nº 20.), those from tropical regions are more influenced by salinity in the reproductive cycle (Nowland et al., 2021Nowland SJ, O’Connor WA, Elizur A, Southgate PC. Evaluating spawning induction methods for the tropical black-lip rock oyster, Saccostrea echinata. Aquacult Rep. 2021;20:100676. http://dx.doi.org/10.1016/j.aqrep.2021.100676.
http://dx.doi.org/10.1016/j.aqrep.2021.1...
) because temperature does not change much throughout the year (Paixão et al., 2013Paixão L, Ferreira MA, Nunes Z, Fonseca-Sizo F, Rocha R. Effects of salinity and rainfall on the reproductive biology of the mangrove oyster (Crassostrea gasar): implications for the collection of broodstock oysters. Aquaculture. 2013;380-383:6-12. http://dx.doi.org/10.1016/j.aquaculture.2012.11.019.
http://dx.doi.org/10.1016/j.aquaculture....
). A previous study (Lavander et al., 2011Lavander HD, Cardoso LO Jr, Oliveira RL, Silva SR No, Galvez AO, Peixoto SRM. Biologia reprodutiva da Anomalocardia brasiliana (Gmelin, 1791) no litoral norte de Pernambuco, Brasil. Rev Bras Ciênc Agrár. 2011;6(2):344-50. http://dx.doi.org/10.5039/agraria.v6i2a1139.
http://dx.doi.org/10.5039/agraria.v6i2a1...
) identified that A. flexuosa species presented the fullest gonads during the dry period with salinity > 35 g⋅L−1 and released gametes during the rainy period with salinities between 25 and 35 g⋅L−1. Thus, the species may present a synchronous behavior with the rainy period.

The information that spawning occurs more frequently in monsoon and at intermediate salinities (Lavander et al., 2011Lavander HD, Cardoso LO Jr, Oliveira RL, Silva SR No, Galvez AO, Peixoto SRM. Biologia reprodutiva da Anomalocardia brasiliana (Gmelin, 1791) no litoral norte de Pernambuco, Brasil. Rev Bras Ciênc Agrár. 2011;6(2):344-50. http://dx.doi.org/10.5039/agraria.v6i2a1139.
http://dx.doi.org/10.5039/agraria.v6i2a1...
) along with the results of the present work that there is an optimal range for GVBD and post-fertilization processes corroborate the idea that there is a reproductive strategy of this animal that is intrinsically related to salinity.

It is also necessary to consider the influence of genetic factors of the population under analysis. Different geographic populations of the same species accumulate alleles that reflect environmental adaptability (Kim et al., 2014Kim WJ, Dammannagoda ST, Jung H, Baek IS, Yoon HS, Choi SD. Mitochondrial DNA sequence analysis from multiple gene fragments reveals genetic heterogeneity of Crassostrea ariakensis in East Asia. Genes Genomics. 2014;36(5):611-24. http://dx.doi.org/10.1007/s13258-014-0198-5.
http://dx.doi.org/10.1007/s13258-014-019...
; Ren et al., 2016Ren J, Hou Z, Wang H, Sun MA, Liu X, Liu B, Guo X. Intraspecific variation in mitogenomes of five Crassostrea species provides insight into oyster diversification and speciation. Mar Biotechnol. 2016;18(2):242-54. http://dx.doi.org/10.1007/s10126-016-9686-8. PMid:26846524.
http://dx.doi.org/10.1007/s10126-016-968...
). Organisms with external fertilization can develop their gametic plasticity to adapt to environmental conditions (Lettieri et al., 2019Lettieri G, Mollo V, Ambrosino A, Caccavale F, Troisi J, Febbraio F, Piscopo M. Molecular effects of copper on the reproductive system of Mytilus galloprovincialis. Mol Reprod Dev. 2019;86(10):1357-68. http://dx.doi.org/10.1002/mrd.23114. PMid:30648312.
http://dx.doi.org/10.1002/mrd.23114...
). However, the results presented herein provide important information for the control of the reproductive process and on chromosome manipulation, suggesting that for achieving triploidy in animals maintained at 35 g⋅L−1 salinity, the application of post-fertilization shocks should be carried out before 10 min for PB1 or before 30 min for PB2 retentions, respectively.

Conclusion

We concluded that in the tropical clam (Anomalocardia flexuosa) salinity plays a key role on the pre- and post-fertilization stages with best ranges between 30 and 35 g⋅L−1 for GVBD and between 20 to 35 g⋅L−1 for PB1 and PB2 extrusions. These findings have implications not only for chromosome manipulation approaches aiming triploidy as well as for the reproductive management in hatcheries.

Acknowledgements

Financial support for this study was provided by Fundação de Amparo à Pesquisa e Desenvolvimento Científico e Tecnológico do Maranhão – FAPEMA (Project UNIVERSAL -01084/18).

  • Financial support: IG received funding from Fundação de Amparo à Pesquisa e Desenvolvimento Científico e Tecnológico do Maranhão – FAPEMA (Project UNIVERSAL -01084/18).
  • How to cite: Lopes RGPS, Rego AP, Gomes SMJ, Ramos T, Antonio ÍG, Coimbra MRM. Effects of salinity on pre- and post-fertilization developmental events in the clam Anomalocardia flexuosa (Linnaeus, 1767). Anim Reprod. 2023;20(2):e20230005. https://doi.org/10.1590/1984-3143-AR2023-0005

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Publication Dates

  • Publication in this collection
    26 May 2023
  • Date of issue
    2023

History

  • Received
    05 Jan 2023
  • Accepted
    13 Apr 2023
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