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EFFECTS OF THE TETRAPLOID CONDITION ON GAMETE SIZE AND ON TESTIS VERSUS OVARY ALLOCATION IN THE TRUE FUNCTIONAL HERMAPHRODITIC SCALLOP ARGOPECTEN VENTRICOSUS (SOWERBY II, 1842).

INTRODUCTION

The Catarina scallop Argopecten ventricosus (Sowerby II, 1842) is a true functional hermaphroditic (monoecious) organism, in which self-fertilization is possible (Ibarra et al. 1995) and in which the male and female gamete cells develop synchronously in one gonad organ spatially divided into a testis and an ovary. When in triploid condition, significant suppression of gamete development occurs (Ruiz-Verdugo et al. 2000), although the sterility induced is partial and varies among organisms (Ruiz-Verdugo et al. 2001). In partially sterile triploids, besides showing a reduced frequency and an increased size of all gamete cell types when compared with those in diploids, the testis part of the gonad organ is characterized by development of mixed-sex acini, in which some of the acini develop male gametes whereas others develop female gametes (Maldonado-Amparo & Ibarra 2002a, 2002b).

Induction to a larger ploidy other than a triploid, such as a tetraploid, is viable when eggs derived from triploids are fertilized with sperm from diploids, inhibiting the extrusion of the first polar body during meiosis I (Guo & Allen 1994b). Using this method, viable adult tetraploids have been obtained for the Pacific oyster Crassostrea gigas (Thunberg, 1793) (Guo & Allen 1994b), the pearl oyster Pinctada martensii (Dunker. 1872) (He et al. 2000), and the Catarina scallop Argopecten ventricosus (Maldonado-Amparo et al. 2003). The resulting sex and gonadal development characteristics in tetraploids are important because their reproductive fitness will determine their potential to be used in establishing a breeding line. For example, in the protandric dioecious and occasional hermaphrodite Pacific oyster C. gigas, generational maintenance of a full tetraploid complement of chromosomes in the Pacific oyster has proven to be difficult and is attributed to random chromosome loss resulting in aneuploidy (low viability or lethality) or even triploidy depending on the number of chromosomes lost (Guo & Allen 1997, McCombie et al. 2005). With regard to sex, the other factor affecting the generational maintenance of a tetraploid line, it is known that tetraploid Pacific oysters retain the same sexual characteristics than those observed in diploids, having an equal proportion of males and females with a low percentage of hermaphrodites (Guo & Allen 1997).

For monoecious or true functional hermaphroditic molluscs as are some Pectinidae, triploidy effects have been extensively studied (Ruiz-Verdugo et al. 2000, 2001, Maldonado-Amparo & Ibarra 2002a, 2002b. Maldonado-Amparo et al. 2004), but little is known about the effects of having a higher ploidy level, such as tetraploidy, on the development of gamete cells and on the hermaphrodite condition or the development of the characteristic sexually divided gonad sac, where both ovary and testis are present simultaneously in defined proportions in diploids, and it is probably an inheritable characteristic because it is retained from one generation to the next (Mason 1958, Beninger & Le Pennec 2006), indicating that there must be a genetic determination/differentiation system involved in the gonad structuration.

In this study the effects of the tetraploid condition on the macro-, micro-, and ultrastructure of the gonad and on the sizes of reproductive cells of the functional hermaphroditic Catarina scallop Argopecten ventricosus when contrasted with diploids are reported. Based on the effects that the tetraploid condition has on the gonad structure, an XX-chromosome mode of sex determination might be present for this species.

MATERIAL AND METHODS

Origin of Diploid and Tetraploid Scallops

The tetraploid scallops used in this study were progeny from the only family reported in Maldonado-Amparo et al. (2003) that resulted in five tetraploid scallops. In short, the first tetraploid scallops were produced as in Guo and Allen (1994b), by inhibiting the first polar body with 0.5 mg/L of cytochalasin-B in eggs from partially fertile triploids, fertilized with haploid sperm. Those scallops that reached adult age (1-y age, 6 cm shell height) were evaluated for their ploidy condition through flow cytometry analyses: 0.1-0.2 mL hemolymph was extracted from the adductor muscle, stained with DA PI solution (1 mL), and analyzed with a Ploidy Analyzer (Partec, Germany). Two of the scallops found to be tetraploids were conditioned for maturation (Ramirez et al. 1999) during 17 days and spawned. Cytometry analysis (Allen 1983, Allen & Bushek 1992) was done to establish ploidy on the resulting D-larvae, obtaining 100% tetraploid larvae. Spat produced from that larva were reared to 12-mo age and then six tetraploid adults were randomly chosen to be processed for histological analysis. Four diploid scallops of the same age (12 mo) were simultaneously sampled also for histological analysis from a different batch.

Macro-, Micro-, and infrastructure Analyses of Gonad

Macroscopic Gonad Structure of Diploids and Tetraploids

All scallops sampled were visually inspected to establish the gonad macrostructure and to obtain a drawing of the characteristic gonad of each ploidy group.

Light Microscopy of Diploid and Tetraploid Gonads

All diploid and tetraploid scallops sampled were used to measure gametic cell stages. A portion of each gonad was fixed according to standard methods used for electronic microscopy studies (Komaru et al. 1994) with the purpose of obtaining thin (2-[micro]m) cross sections, with the following modifications: the gonads were fixed in 2% glutaraldchyde in phosphate buffer (0.2 M) with an adjusted pH of 7.4 for 24 h. After this, all samples were washed twice, each for 30 min, in a washing solution (9 g NaCl, 0.14 g KC1, 0.12 g Ca[Cl.sub.2], 0.2 g NaHC[O.sub.3], and 2 g glucose in 1,000 mL distilled water). Samples were then progressively dehydrated by passing them consecutively from 30% alcohol to absolute alcohol. The rest of the histological process was the same as is used for hematoxylin and eosin (Humanson 1972).

Electron Microscopy of Diploid and Tetraploid Gonads

Electron microscopy work was carried out at the Universidad Autonoma de Mexico in the Instituto de Fisiologia Celular. For transmission electron microscopy (TEM) and for scanning electron microscopy (SEM), the methodology followed is the same as described by Komaru et al. (1994). Briefly, gonad samples of approximately 2- and 4-[mm.sup.2] for TEM and SEM, respectively, were fixed by immersing in a near neutral (pH 7.5) solution of glutaraldehyde and Sorensen's phosphate buffer during 120 min at 4[degrees]C. Both SEM and TEM gonad tissue samples were washed with Sorensen's phosphate buffer three times to remove excess glutaraldehyde and fixed with Os[O.sub.4] at 4[degrees]C for 1 h, consecutively dehydrated with ethanol and propylene oxide for 20 min in each step. For the TEM gonad sample, the Kit Embed 812 Electron Microscopy Sciences was used following the manufacturer instructions, and 900 [Angstrom] slices were obtained with an ultramicrotome (Model OMU3; Reicher), mounted and stained, and microphotography was carried out with JEOL JEM-1200EXII TEM. For the SEM, the preparation procedure of the samples is identical to that for TEM up to dehydration with alcohol/propylene oxide. In addition drying was carried out for the preparation of SEM samples with a dryer (SAMDRI-PVT-3P) and coated in gold with a sputter (EDWARDS S150B) to be able to visualize the samples in the SEM.

Oogenic and Spermatogenic Cells Measurements

A microscope (Olympus BX-41) with an integrated camera (CoolSNAP-Pro Color) was used to take microphotographs that were digitalized. The contours of spermatogenic and oogenic cells for the stages described by Dorange and Le Pennec (1989a, 1989b) were used to estimate mean diameters. Thirty cells of each type and gonad sex were measured per organism sampled within each ploidy condition using the image analysis program SigmaScan Pro5.

Differences in diameters of spermatogenic (spermatogonium, spermatocyte, spermatid, and spermatozoa) and oogenic stages (oogonia, previtellogenic oocyte, vitellogenic oocyte, and postvitellogenic oocyte) between diploids and tetraploids were analyzed by means of analysis of variance (Statistica v.8). Means of cells from diploids and tetraploids were compared using Newman--Keuls tests. Significance was established as P< 0.05.

RESULTS

Macro-, Micro-, and Ultrastructure of Gonad in Diploids and Tetraploids

The characteristic gonadal sac in diploids was morphologically different in tetraploids (Fig. 1A, B, respectively). In particular, the testis part of the gonad organ was considerably smaller in tetraploids (Fig. 1B) when contrasted with diploids (Fig. 1A). In diploid organisms, the testis occupies approximately one-third of the total gonad sac, it begins close to the foot and runs along the length of the external gonad sac surface (Fig. 1A). In tetraploid organisms, the testis area was, besides smaller, restricted to a small portion close to the foot (Fig. 1B). Further light microscopy analysis corroborated the macroscopic observations of an unusually small testis area in contrast with diploids. The first notable difference found was that in diploid organisms large numbers of male acini are present in the testis region of the gonad (Fig. 1C), whereas in tetraploid organisms, most of the gonad was occupied by female acini, and only a reduced portion of the gonad contained an area with male acini (Fig. 1D). Another difference found was that in diploid organisms the female and male acini lay laterally but separately from each other with no abnormal morphology present (Fig. 1E), whereas in tetraploids, the female acini closest to the male acini shows a large number of atresic oocytes, and the separation of male and female gonad parts is not as marked as in diploids, with male acini projecting into the female acini area (Fig. 1F).

Microphotographs from the ovary part of the gonad organ and the cell types present in diploids and tetraploids are presented in Figure 2. All oocytes types (previtellogenic. vitellogenic, and postvitellogenic) are present in both ploidy groups, but oocyte sizes appear to be visually larger in tetraploids than in diploids (Fig. 2A, B), a difference that is more readily observable at higher magnification and resolution when images arc obtained by TEM (Fig. 2C, D).

Acini in the testis part of the gonad in diploids and tetraploids follows the characteristic concentric arrangement (Fig. 3A, B), where spermatogonia lays adjacent to the acinus wall and spermatocytes lay near the inner of the wall, followed by spermatids and spermatozoa, located toward the center of the acinus. Transmission electron microscopy microphotographs depict the spermatozoa ultrastructure in diploid and tetraploid organisms (Fig. 3C, D), where the size differences between diploids and tetraploids can be noted. Scanning electron microscopy microphotographs show the acinus in diploid and tetraploid testis (Fig. 3E. F). where spermatozoa and their flagella are evident.

Gametic Cells Sizes in Diploid and Tetraploid Scallops

The comparative analysis on diameters of gametic cell stages from diploid and tetraploid Catarina scallop indicated that cell size was significantly larger in tetraploids than in diploids for all oogenic and spermatogenic stages (Table 1), corroborating the microphotograph observations. The estimated percentage increase in cell diameters was generally larger for spermatogenic cells when compared with oocyte cells: 100% for spermatozoa and 30% for postvitellogenic oocytes (Table 1).

DISCUSSION

The tetraploid scallops analyzed in this study represent a second generation of reproductively viable tetraploid scallops (Maldonado-Amparo et al. 2003). When considering only diploids, there is a strong agreement with previous evaluations of gamete cells sizes in this same species (Maldonado-Amparo & Ibarra 2002a, 2002b). In tetraploids, the gamete cell sizes were significantly larger in comparison with diploids for all measured cells, although the increase in diameters varied for male and female gamete cells. Only the spermatozoa had a 100% increase in diameter in tetraploids when compared with diploids, whereas oocyte diameters in tetraploids only increased 19% for vitellogenic oocytes or 30% for postvitellogenic oocytes of diploids. This is similar to the small increase in egg size found for the tetraploid Pacific oyster, which increased in size by 17% (Guo & Allen 1997). Furthermore, when diploid and triploid Catarina scallop gamete cells were compared, the percentage increase in size for spermatogenic cells was also larger than that observed for oocyte cells (Ruiz-Verdugo et al. 2001, Maldonado-Amparo & Ibarra 2002a, 2002b).

Regarding the hermaphrodite sex condition, tetraploid Catarina scallops were still hermaphrodites, but had a strikingly different gonad morphology than that observed in diploids, where the testis part of the gonad organ was found to be significantly smaller when compared with diploid hermaphrodites. The same gonad morphology was observed in three generations of Catarina scallop tetraploids. indicating the inheritance of this new hermaphrodite gonad structure in tetraploids with a largely reduced testis area. In other tetraploid molluscs as the protandric Pacific oyster Crassostrea gigas, changes in sex or sex ratios have not been observed although fecundity/fertility decreases were observed for females by Guo and Allen (1997) and for males by Dong et al. (2005), associated with the increased volume in oocytes or sperm when compared with diploids. For nonmollusc invertebrate species, such as the hermaphrodite nematode Caenorhabditis elegans (Maupas, 1900), the tetraploid condition does not result in an alteration of sex because it has been reported that tetraploids still develop as hermaphrodites (Nigon 1949b, cited by Madl & Herman 1979). On the other hand, similarly to the Pacific oyster, in C. elegans Madl and Herman (1979) found that tetraploid (4A:4X) hermaphrodites had lower fertility than diploids, with an average of 84 eggs for tetraploids, whereas for diploids, it was about 300 eggs, and egg viability from tetraploids was 87% in contrast to almost 100% in diploids. The testis area or the amount of spermatozoa produced was not reported; hence, it is not possible to know whether the area is smaller and production of spermatozoa was reduced as observed in the Catarina scallop.

The reduction in testis area or increased ovary area observed for tetraploid Catarina scallops indicates an effect of ploidy on the fecundity/fertility of maleness versus femaleness, with the testis being largely and negatively affected. There is limited understanding of sex determination in scallops. Recently Llera-Herrera et al. (2013) found genes known to be involved in sex determination and differentiation that are similar to model organisms as Drosophila melanogaster and Caenorhabditis elegans in the transcriptome of another functional hermaphrodite, the lion-paw scallop Nodipecten subnodosus (Sowerby I, 1835). Catarina scallop has not been reported to be affected by environmental conditions in their sex determination, suggesting the possibility that in some Pectinids, sex determination and differentiation might involve sex chromosomes. In fact, some evidence for the presence of sex chromosomes in scallops is already emerging. For example, in Chlamys farreri (Jones et Preston, 1904), gonochoric or dioecious scallop having both male and female organisms; Li et al. (2005) suggested that females were the heterogametic sex (WZ) after finding one sex-linked marker that mapped only to the maternal linkage map (presumably on the W chromosome). Also for C. farreri, more recently Jiao et al. (2014) found highly significant quantitative trait loci for sex on one linkage group (one chromosome) that is present in both males and females (possibly on the Z-chromosome). The two previous works mentioned earlier, although not conclusive, provide strong support to the hypothesis that sex is chromosomally determined in dioecious scallops.

Among other Pectinidae species, the presence of a low frequency number of hermaphrodite scallops has been reported before, for example, for Placopeeten magellanicus (Gmelin, 1791) by Merrill and Burch (1960) and Worms and Davidson (1986), for Patinopecten yessoensis (Jay, 1857) by Yamamoto (1964, cited by Silina 2016) and Osanai (1975), for Patinopecten caurinus (Gould, 1850) by Hennick (1971), and for Chlamys nobilis (Reeve, 1852) by Komaru and Wada (1988). The rare occurrence of hermaphrodites within some scallop species can be explained by considering that some of those species can be classified as protandric hermaphrodites because they develop first when young, as males, and a proportion of those males change later when older to females (Barber & Blake 2006, Otani et al. 2017). On the other hand, for scallops that are presumably strictly gonochoric or dioecious as C. nobilis, the observation of some hermaphrodites can be explained by some type of gene expression alteration resulting from a mutation on a key sex-differentiation gene or as a consequence of triploidy. In this species, sex determination/differentiation of genes as dmrt2 (doublesex/mab-3 related family of transcription factors) has been characterized and found to be highly expressed at key developmental stages as blastula and during male gonad maturation (Shi et al. 2014). In some frogs, fish, and avian species, Dmrt genes are known to be the sex-determining gene (see review by Kopp 2012), and it is known that dosage alterations resulting from a triploid condition (ZZW) in chickens induce development of hermaphrodites (Fitzgerald & Cardona 1993). Whether triploid scallops can occur naturally in the wild is not known, but the frequency of the C. nobilis hermaphrodites found by Komaru and Wada (1988) was higher than expected (4.8%--14.3%) for a newly occurring mutant phenotype.

Conversely, for true functional hermaphrodite scallop species, there is at least one previous report indicating the presence of an abnormal gonad sex. For example, Mason (1958) found that occasionally for Pecten maximus (Linnaeus, 1758), "the gonad could be almost exclusively either male or female." For the Catarina scallop, an abnormal gonad has been observed occasionally, finding either a gonad that appeared to be only testis (completely white-cream in coloration) with no ovary area (orange-red color), or gonads with only ovary and no testis area. This observation, together with that from Mason (1958), allows to propose that for the Catarina scallop, a sex-determination system similar to that present in the worm Caenorhabditis elegans (Nigon 1949b, cited by Haag 2005) exists.

In the nematode Caenorhabditis elegans, sex determination is a direct function of X-chromosome dosage, with XX being hermaphrodites if the ratio of X-chromosomes to autosomes (A) is balanced (2X/2A = 1), and X0 being male (Hodgkin 2002). Males occur at a very low frequency (1%) because of nondisjunction of the two X-chromosomes (1X/2A) during meiosis (Nigon 1949b, cited by Haag 2005). Tetraploid C. elegans have been produced, and it was found that when they have a balanced sex and autosome ratio (4X:4A), they are still hermaphrodites, and as with diploids, a low percentage of those still produce approximately 1% male progeny (Nigon 1949a, 1949b, 1951a, 1951b, cited by Madl & Herman 1979).

The occasional occurrence of scallops otherwise known to be true hermaphrodites with an abnormal sex gonad development as those with only a male gonad can be explained by the possible very low frequency occurrence of nondisjunction of the two X-chromosomes expected in hermaphrodites, resulting in scallops inheriting only one X-chromosome (XO). On the other hand, the presence of hermaphrodite scallops developing only or mostly a female gonad might be the result of inheriting the two X-chromosomes from the nondisjunction (XXX), as well as possibly from random mutations in other key-genes involved in the sex-determination cascade. For example, in Caenorhabditis elegans mutations in several genes (tra-1 and tra-2, and fern-1 to fern-3) are known to feminize not only XX hermaphrodites but also XO males (Hodgkin 1986).

In this model organism, during early embryogenesis the two X-chromosomes are upregulated. resulting in the overexpression of X-linked genes that are associated with the hermaphrodite sex. Downregulation of the two X-chromosomes is accomplished by the dosage compensation complex. Thereafter, the X:A ratio provides a signal that directs sexual fate and does so by binding competitively the promoter of the sex-determination switch gene xol-1 with X-signal elements (XSE = several genes located on the X-chromosome that are expressed early) and autosomal-signal elements (=genes located on autosomes also expressed early). 1X:2A organisms develop as males because the expression of the xol-1 gene (a male sex-determining gene) is not reduced by the XSE as it occurs with 2X:2A organisms; this is because of the higher expression of XSE associated with the presence of 2X-chromosomes resulting in low transcript levels of xol-1 (for reviews see Meyer 2010, Lau & Csankovszki 2015). This mechanism in which the number of X-chromosomes is associated with expression of genes on it could explain the change in gonad morphology of Catarina scallop, especially if a gene similar to xol-1 is present, and its expression is largely reduced because of an overexpression of genes on the 4X-chromosomes in the tetraploid condition.

In support of this hypothesis, it is important to mention that in another functional hermaphrodite species for which we are currently doing transcriptomic work, the lion-paw scallop, although at present we have not found a gene for which BLAST searches indicates significant similarity to xol-1, other genes involved in early sex determination and known to be part of the XSE have been found, such as sex-1 (Galindo-Torres et al. 2017). In Caenorhabditis elegans sex-1 is located on the X-chromosome, and it has been confirmed to be a key gene in the negative regulation of transcription of xol-1 (see http://www.wormbase.org/species/c_elegans/gene/WBGene00004786#0-9g-3).

For other molluscs, different models for sex determination involving putative sex chromosomes have been proposed and have been inferred after observing the sex ratios in gynogenetic progeny and triploids (Allen et al. 1986, Guo & Allen 1994a). For example, for the dioecious softshell clam Mya arenaria (Linnaeus, 1758) no triploids developed as males, and most triploids were females with a few being undifferentiated, which led to the proposal of a sex-determination mechanism similar to that found for Drosophila, in which triploids XXX and XXY are all-female regardless of the Y-chromosome. In dioecious dwarf surfclam Mulinia lateralis (Say, 1822), it has been also found that its gynogenetic progeny are all females indicating that they must be the homogametic sex (carrying 2X-chromosomes), and triploids showed both genders, males and females, such that it was proposed that they were XXX and XXY, with the Y-chromosome being dominant over the X-chromosome. On the other hand, triploids from the protandric dioecious Pacific oyster Crassostrea gigas are not affected in their sex, with the proportions of males and females being similar as those found with diploids (Allen & Downing 1990), and thus no sex chromosomes can be inferred to be present from their sex ratios. Guo et al. (1998) proposed a one-locus model with two genotypes. This model explains the development of more males at first maturation and the increase in females in later maturations. Later, Hedrick and Hedgecock (2010) elaborated more on the one-locus model, proposing that a three-genotype model also explains the sex ratio variability observed in the data by Guo et al. (1998), and provided support in explaining the variability observed between half-sibs from a single male. Recently, a search of the oyster genome for genes known to be involved in sex determination in other organisms found several genes (Zhang et al. 2014), although their function in C. gigas has not been evaluated.

In conclusion, whereas the decrease in fertility in other tetraploids has been mostly associated with increases in gamete size, in the hermaphrodite tetraploid Catarina scallop, the decreased fertility in the male gonad part is unique and not directly caused by an increase in size of gametes but most probably is the result of a genetic mechanism involved in sex determination.

ACKNOWLEDGMENTS

This research was supported by CONACYT (grant No. 28256-B to A. M. Ibarra). We thank Carmen Rodriguez-Jaramillo and Eulalia Meza-Chavez from CIBNOR for histological support, and Dr. Mark West, Rodolfo Paredes, and Jorge Sepulveda from the Institute of Cellular Physiology of the UN AM for their support to carry out the electron microscopy. Gerardo Hernandez-Garcia edited the microphotography prints. We thank Dr. Michael V. Cordoba, a native English editor, for his comprehensive revision of the manuscript.

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ANA M. IBARRA, (*) JOSE L. RAMIREZ AND ROSALIO MALDONADO

Aquaculture Genetics and Breeding Laboratory, Centro de Investigaciones Biologicas del Noroeste, S.C., A. P. 128, La Paz, Baja California Sur 23096, Mexico

(*) Corresponding author. E-mail: aibarra@cibnor.mx

DOI: 10.2983/035.036.0313
TABLE 1.
Mean diameter in [micro]m (SE) of male and female gametic cells in
diploid (n = 4) and tetraploid (n = 6) Catarina scallop (Argopecten
ventricosus).

                         Mean diameter, [micro]m (SE)
Spermatogenic             Diploid           Tetraploid      Tetraploids
cells                                                       % increase
                                                            diameter

Spermatogenic
cells
 Spermatogonia (Spg)    6.49 (0.09) (a)    7.95 (0.13) (b)      22
 Spermatocyte (Spc)     3.93 (0.05) (a)    5.81 (0.07) (b)      48
 Spermatid (Spm)        2.84 (0.05) (a)    4.41 (0.05) (b)      56
 Spermatozoa (Spz)      1.68 (0.02) (a)    3.36 (0.04) (b)     100
Oogenic cells
 Ovogonia (Ovog)        4.64 (0.08) (a)    5.34 (0.08) (b)      15
 Previtellogenic
 (Prev)                 6.96 (0.11) (a)    8.10 (0.08) (b)      19
 Vitellogenic
 (Vit)                 32.62 (1.37) (a)   38.88 (2.12) (b)      19
Postvitellogenic
(Postv)                44.09 (0.57) (a)   57.47 (1.34) (b)      30

Different superscript letter between columns indicate significant
differences at the pre-established level.
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Author:Ibarra, Ana M.; Ramirez, Jose L.; Maldonado, Rosalio
Publication:Journal of Shellfish Research
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Date:Dec 1, 2017
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