ABSTRACT It is expected that in triploid organisms the energy normally used for reproduction would be allocated to growth. However, not all triploid molluscs are completely sterile, and in some cases even gametes are produced. The aim of this study is to assess the gonadal development in the native scallop Argopecten purpuratus induced to triploidy. Argopecten purpuratus Lamarck 1819, is a functional hermaphrodite, the male portion of the gonad being creamy-white, located proximal to the foot, and the distal female gonadal portion is bright orange-red. They were induced to triploidy with 6-dimethylaminopurine (6-DMAP). Treated (= induced) and control scallops were processed for histology using routine methods. At the age of 11 too, when the control scallops were mature, some treated scallops had a gonad, which showed a uniform brown color. These were true triploids, assessed by chromosome counts. They showed the tendency of reducing the "ripeness" of the female gonad, only few acini with oocytes were observed, associated to hemocytes, presumably phagocytosing them. Female acini were mostly empty. The male gonad was relatively more developed, but the spermatocytes, spermatids and spermatozoan-like cells showed a highly abnormal morphology. This evidence strongly suggests that these gametes are incapable of viable fertilization. Triploid A. purpuratus did not lose their hermaphroditic condition, which was different from another functional hermaphrodite scallop, Argopecten ventricosus, whose triploid gonad turned into only female.
KEY WORDS: Argopecten purpuratus, triploid, scallop, functional hermaphrodite, abnormal gametogenesis, sterility
The Northern Chilean scallop or "ostion del Norte" Argopecten purpuratus Lamarck, 1819, is a functional hermaphrodite, that lives on sandy bottoms of the Pacific Ocean from Paita, Peru, to Valparaiso, Chile (Pena 2001). Since in 1986 there is a complete fishing ban on this species in Chile, and now it is an important cultivated bivalve, mainly in the 3rd and 4th regions, which account for 97% of the total national production of 18,039 tons (Anonimo 2002).
Trying to find ways of faster growth of A. purpuratus, triploid induction was performed, using 6-dimethylaminopurine (6-DMAP) (Desrosiers et al. 1993, Gerard et al. 1999). Triploid animals can be partially or totally sterile because of the extra set of chromosomes (Allen et al. 1986). Some triploid bivalves studied so far, the clam Mya arenaria (Allen et al., 1986), the mussel Mytilus galloprovincialis (Kiyomoto et al. 1996), the scallops Chlamys nobilis (Komaru and Wada 1989), Argopecten ventricosus (Ruiz-Verdugo et al. 2000) and Nodipecten subnodosus (Maldonado-Amparo et al., 2004) were found to be sterile. However, other triploid bivalves, like the Japanese oyster (Crassostrea gigas) and the pearl oyster (Pinctada fucata), showed a limited number of aneuploid gametes (Allen & Downing 1990, Komaru & Wada 1994). In the Japanese oyster Crassostrea gigas, sperm and oocytes were fully capable of fertilization (Guo & Allen 1994). For the pearl oyster, crosses could be made between oocytes from triploid females, and spermatozoa from diploid males (Komaru et al. 1994). For species that are cultivated, it is especially important to study the gametogenesis of triploid animals, because the production of aneuploid viable gametes in an environment where natural populations of these species live could have disastrous genetic and reproductive consequences.
In this study, the gametogenesis of triploid A. purpuratus was analyzed, and compared with the gametogenesis of control diploid scallops from September 2001 to September 2002, which includes a whole reproductive season that extends from spring (September) through early fall (March), plus the start of a second reproductive season in September 2002.
MATERIALS AND METHODS
Argopecten purpuratus were spawned, fertilized, and induced to triploidy with 6-DMAP. Treated and control juvenile scallops were grown in La Herradura Bay, Universidad Catolica del Norte, located in the 4th Region. The control scallops were assessed periodically using histology, and when they started to show gonadal maturation in spring (age 11 mo), treated and control animals were sampled.
The treated and control animals were analyzed using Image Proplus software and a Nikon Eclipse 600 microscope. The individual DNA content was determined by integrated optical density (IOD) of Dapi-stained haemocyte nuclei (manuscript in preparation). Those individuals showing much higher IOD values than the diploid (2n) control animals, were considered potential triploids, and processed for histology. The triploidy was confirmed by chromosome counting in gill cells. A total of 34 diploid scallops and 72 potential triploids were examined histologically. After counting chromosomes, 26 out of the 72 potential triploids were true triploids. The sampling ages were: 11, 12, 13, 14, 15, 16, 17, 21 and 22 mo. The usual harvest size is reached at about 18 mo of culture.
The gonads were fixed in Davidson fluid (Shaw & Battle 1957) and prepared using routine histologic methods. Five-[micro]m-thick sections were cut and stained with hematoxilyn and eosin (H & E).
For scanning electron microscopy (SEM), sections from selected wax blocks were cut at 7-15 [micro]m, and mounted on cover-slips. Sections were de-waxed in three changes of xylene, passed through three changes of 100% ethanol and critical point dried using C[O.sub.2] (Lohrmann et al. 2002). Samples were mounted with nail polish on bronce stubs, and ion sputtered with gold. The sections were viewed and photographed using a JEOL TS 300 microscope.
For analyzing the gametogenic stage of the sampled scallops, a maturity scale slightly modified from Patinopecten yessoensis (Maru 1976) and from Argopecten ventricosus (Ruiz-Verdugo et al. 2000) was established:
Early growth, female acini with oogonia and early oocytes; male acini with spermatogonia and early spermatocytes. Abundant connective tissue between the acini.
Late growth, female acini with oocytes initiating vitellogenesis; male acini with spermatocytes. Some connective tissue between the acini.
Maturation. Vitellogenic oocytes almost filling the acini, late spermatids or spermatozoa almost filling the acini. Very little connective tissue present.
Mature, ready to spawn. Mature, polygonal oocytes and spermatozoa filling the acini, connective tissue absent.
Spent. Most gametes have been liberated, some remnant oocytes and spermatozoa, hemocytes present. Some gonia sticking to the interior of the acini walls.
In some cases, the female and the male portion of the gonald did not have the same maturity in one individual, so the female gonad was assessed separately from the male gonad.
The gonad of triploid scallops was significantly smaller than in diploid scallops, and showed a brownish-transparent color at the macroscopic level, where the male and female portions could not be distinguished. The triploid gonad was also different from a spent diploid gonad, which was transparent, not brownish, and the male and female portions still showed some of their original color: cream-white for males, and orange for females. The potential triploids that had been processed for histology, but were diploids, did all show a normal (diploid) histologic aspect. Figure 1A compares the macroscopic aspect of the gonad of a triploid (3 n) and a diploid (2 n) scallop, both 17 mo old. The diploid gonad was fully mature (stage IV), which is histologically shown in Figure 1B. The gonad of triploids was histologically clearly different from that of diploids (Fig. 1C), and differed also from a spent diploid gonad (stage V), which is shown for the male (1D) and for the female portion (1E).
[FIGURE 1 OMITTED]
As the triploid gonad differed histologically from the diploid gonad, it could not be assigned to any of the stages of the maturity scale used for diploid scallops. So, a new scale, based on the analyzed triploids, was established:
Acini almost empty, with oogonia attached to the walls (Fig. 2A). Spermatogonia and few spermatocytes in the male acini (Fig. 2B).
[FIGURE 2 OMITTED]
Acini with few vitellogenic oocytes, up to half filled, some of these had a normal aspect, other were degenerating. Numerous hemocytes surrounding and inside the acini (Fig. 2C). Male acini half filled with spermatocytes. No flagella were detected (Fig. 2D).
Acini completely filled with oocytes, some postvetellogenic, some degenerating, with numerous hemocytes (Fig. 2E). Male acini almost full of spermatocytes and early spermatids. Hemocytes phagocytosing sperm cells inside the acini and picnotic nuclei, these acini have a "moth-eaten" appearance, hemocytes also present around the acini (Fig. 2F).
Histologic sections of diploid and triploid male gonads were observed with the scanning electron microscope (SEM). Diploid spermatozoa exhibited an anterior acrosome, head, four mitochondria and a flagellum (Fig. 3A). In the triploid gonad abnormal shaped cells, presumably spermatocytes or early spermatids could be observed (Fig. 3B). Flagellated, spermatozoan-like cells that had not been observed with the light microscope (LM), were seen in some of the triploid male gonad acini (Fig: 3, C and D). The sperm cell shown in Figure 3C had been sectioned, and in its interior neither chromatin nor mitochondria could be distinguished. Mitochondria could also not been observed externally (3D). The morphology of these spermatozoan-like cells from triploid scallops, looks different from a normal spermatozoon as are those shown in Figure 3A. Note the size difference between diploid and triploid germ cells comparing Figures 3A, C and D that have exactly the same magnification.
[FIGURE 3 OMITTED]
The maturity stages of the female and male gonad of diploids (Figs. 4A and B) and triploids (Fig. 5A and B) along the sampling period (from month 11 to month 22) is shown. Early in maturation the triploid gonad was more mature than the diploid one, but afterward, from age 14-22 mo, most of the female (4A) and male diploids (4B) were either fully mature (stage IV) or spent (Stage V). The female triploids (5A) were mostly at the less developed stage (I') from month 15 through 22, showing a clear tendency to present earlier stages of development with older age. The male acini (5B) tended to be riper, however not as ripe as those from diploids (4B), during most of the time. Nevertheless, all triploid A. purpuratus sampled were clearly hermaphrodites.
[FIGURE 4 OMITTED]
The results of this study are similar to those reported for the gonochoristic or dioecious scallop Chlamys nobilis (Komaru & Wada 1989), and coincide in some aspects, differing in others, from the other two functional hermaphrodite scallops induced to triploidy and studied histologically so far, the catarina scallop, Argopecten ventricosus (Maldonado-Amparo & Ibarra 2002, Ruiz-Verdugo et al. 2001, Ruiz-Verdugo et al. 2000) and Nodipecten subnodosus (Maldonado-Amparo et al. 2004).
The gonadal sac of triploid A. purpuratus and A. ventricosus was similar in presenting a brownish color, which makes the identification of triploids possible by visual inspection. However, there was a difference between both species: in A. purpuratus the gonadal sac was significantly smaller than in diploids, but for A. ventricosus it was larger than in diploids (Ruiz-Verdugo et al. 2000).
The gametes produced by triploid A. purpuratus do not seem capable of fertilization. Most of the few oocytes found in triploids were degenerating, presumably being phagocytosed, as evidenced by numerous hemocytes compared with normal diploids. The triploid female gonad showed vitellogenic and postvitellogenic stages early, at the beginning of the maturity period of diploids, but then started showing less mature stages with increasing age. This was also observed in triploid Pacific oysters Crassostrea gigas, where mature ova was found from the first sampling period on, and stayed arrested at that stage (Allen & Downing 1990). In the hermaphroditic scallop Nodipecten nodosus the oocytes were arrested at the previtellogenic stage, both in the first and the second reproductive season (Maldonado-Amparo et al. 2004).
Histologically, the mature male acini of diploid A. purpuratus are filled with spermatozoa arranged in a neat radial pattern, very different from the "untidy" appearance of the advanced (III') triploid acini. This latter aspect is caused by the presence of degenerating male germ cells, the process of them being phagocytosed and also the absence of spermatozoa. A similar image can be seen in other male triploid gonads, such as Mytilus galloprovincialis (Kiyomoto et al. 1996), the hard clam Mercenaria mercenaria (Eversole et al. 1996), and the scallop Chlamys nobilis (Komaru & Wada 1989).
At the SEM level, the spermatozoan-like cells observed in A. purpuratus showed a bigger size than the diploid sperm cells, which has also been reported for spermatozoa from triploid catarina scallops (Maldonado-Amparo & Ibarra 2002) and from triploid Pacific oysters (Komaru et al. 1994). The spermatozoa of triploid catarina scallop showed a normal external morphology, as shown in a SEM photograph (Maldonado-Amparo & Ibarra 2002). The authors state that these spermatozoa had the same number of cross-sectioned mitochondria than diploids, although it is not shown. Similarly, the spermatozoa of triploid Pacific oysters exhibited a completely normal morphology, viewed with SEM and TEM (Komaru et al. 1994). This is completely different for A. purpuratus: the spermatozoan-like cells showed an abnormal shape, and they lacked mitochondria. These organelles could neither be seen externally in SEM images (where they can be observed in diploid spermatozoa) nor in one of the spermatozoan-like cells that had been sectioned. The mitochondria of triploid spermatozoa from the Pacific oyster had a normal morphology at SEM and TEM level, being the only organelle not showing an increased size in triploid spermatozoa (Komaru et al. 1994).
In some species, a change of sex ratio has been observed in triploids: for the gonocoric soft shell clam Mya arenaria (Allen et al. 1986) the sex ratio shifted to female in triploids, and triploid mussels Mytilus galloprovincialis were all identified as males (Kiyomoto et al. 1996). In Mercenaria mercenaria instead, no change in sex ratio caused by triploidy was observed (Eversole et al., 1996). The same was determined for the triploid scallop C. nobilis (Komaru & Wada 1989). For A. purpuratus the hermaphroditic condition was not lost, the 26 true triploids had female and male portions in their gonad. The only difference observed, was that triploid females tended to be less ripe with growing age, but the female portion of the gonad was always present. This contrasts with the findings in the functional hermaphrodite catarina scallop A. ventricosus, where a gradual reduction of the male portion of the gonad was reported, ending up with 96-100% of the scallops as only females (Maldonado-Amparo & Ibarra 2002, Ruiz-Verdugo et al. 2001, Ruiz-Verdugo et al. 2000). This is an important difference between triploid A. purpuratus and A. ventricosus, both being functional hermaphrodites and belonging to the same genus. The other hermaphroditic scallop studied (Maldonado-Amparo et al. 2004), Nodipecten subnodosus, does not lose this condition, showing less mature stages of the triploids with increasing age (during the second maturation peak), as it occurs in A. purpuratus. It is necessary to undertake studies on other hermaphroditic scallop species for getting an understanding of the meaning of these differences.
From the evidence presented in this study, it seems very unlikely that triploid A. purpuratus can produce viable oocytes or spermatozoa up to an age of 22 mo. During this period two reproductive seasons were covered, though not completely. It was not possible to sample triploid scallops until the age of 27 mo, which would have covered two complete reproductive seasons. It might be possible that a very late maturation of the gametes could occur in triploids, however, the tendency seems to be opposite to this. The female part of the triploid gonad showed the most mature oocytes, postvitellogenic, only early in the reproductive season, at the same time as the diploids were starting to mature. With increasing age they showed less advanced stages, and the same tendency, but less marked, occurred in the male part of the gonad.
Because cultivated A. purpuratus are harvested at the age of 18 mo, it can be considered safe to grow triploid scallops in the environment, where normal diploid scallops are cultivated, because no release of viable, aneuploid gametes has to be feared.
The authors thank Cristian Gallardo for image analysis, Catherine Cruz for assistance in histology and Pedro Jara for chromosome analysis. This research was supported by FONDEF (Chile) grant D-981-1044.
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KARIN B. LOHRMANN * AND ELISABETH VON BRAND Universidad Catolica del Notre, Facultad de Ciencias del Mar, Larrondo 1281, Coquimbo, Chile
* Corresponding author. E-mail: firstname.lastname@example.org
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