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Early Gametogenesis of Kumamoto oyster (Crassostrea sikamea) Gametogénesis temprana en el ostión Kumamoto (Crasssotrea sikamea) Jorge Cáceres-Martínez,1,2 Rebeca Vásquez-Yeomans2 and Yanet Guerrero-Rentería1 1 Centro de Investigación Científica y de Educación Superior de Ensenada. Departamento de Acuicultura. Carretera Ensenada-Tijuana No. 3918, Zona Playitas. Ensenada, Baja California, 22860. México 2 Instituto de Sanidad Acuícola, A. C. (ISA). Calle 15 #265, entre Obregón y Moctezuma, Zona Centro. Ensenada, Baja Cáceres-Martínez J., R. Vásquez-Yeomans and Y. Guerrero-Rentería. 2012. Early Gametogenesis of Kumamoto oyster (Crassostrea sikamea). Hidrobiológica ABSTRACT
crosatellite DNA markers, concordant differences in 16S rDNA The Kumamoto oyster, Crassostrea sikamea, starts gametogen- and allozymes (Banks et al., 1993, 1994; Hedgecock et al., 1999; esis as young as 71 days old from spawning (35 days from post- Sekino et al., 2003; Reece et al., 2008; López-Flores et al., 2010). settlement) with a mean shell height of 3.0 mm. This information Additionally, there are phenotypic and physiological differences constitutes a new record in age-size for gametogenesis in oysters between C. gigas and C. sikamea, the later has slower growth for commercial importance and adds another biological differ- rate, smaller size, smaller eggs, a more deeply cup-shaped left ence comparing this species with the Pacific oyster Crassostrea valve, and a highly wrinkled or ridged shell (Amemiya, 1928; Nu- machi, 1978). The age and size for gametogenesis could also be different among genetically related species. The smaller size for Key words: Crassostrea sikamea, gametogenesis, gonadal devel-
opment, Kumamoto oyster, sexual differentiation.
gametogenesis in C. gigas is between 16 to 39 mm of shell height (Buroker, 1983), while for C. virginica (Gmelin, 1791) it is smaller than 35 mm of shell height, and as young as 42 days from post- El ostión Kumamoto, Crassostrea sikamea, inicia la gametogéne- settlement (Stafford, 1913; Thompson et al., 1996). The oyster C. sis a una edad de 71 días después del desove (35 días después del rizophorae (Guilding, 1828) begins gametogenesis at the small- asentamiento) con un promedio de altura de la concha de 3.0 mm. est sizes; from 5 to 9 mm of shell height and less than 42 days old Esta información constituye un nuevo registro en la edad y talla from post-settlement (Vélez, 1976). Until now, there is no informa- en la cual se inicia la gametogénesis en ostiones de importan- tion about the age and size for the beginning of gametogenesis in cia comercial. Adicionalmente, pone en evidencia otra diferencia C. sikamea. During a histological survey of Kumamoto oysters of biológica respecto al ostión Japonés Crassostrea gigas.
< 4.5 mm of shell height and 35 days old from post-settlement, sur-prisingly, we observed gametogenesis. Results from this survey Palabras clave: Crassostrea sikamea, desarrollo gonadal, dife-
renciación sexual, gametogénesis, ostión Kumamoto.
In August 2007, one sample of 120 Kumamoto oysters from The Kumamoto oyster, Crassostrea sikamea Amemiya, 1928, was a commercial hatchery in Sinaloa, Northwest Mexico, was sent introduced for commercial culture into bahía de San Quintín, Baja alive to the Laboratory of the Instituto de Sanidad Acuícola, A.C. California, Mexico around 1970 from the West coast of the U.S.A. for histological analysis. The sample was composed by oysters as a variety of the Pacific oyster Crassostrea gigas (Thunberg, of 71 days old from spawning and 35 days from post-settlement, 1793) (Gobierno del Estado de Baja California Sur, 2010). Nowa- cultured at 24 oC. A second sample of 80 oysters of similar age and days, it is known that the Kumamoto oyster is a separate species cultured at the same temperature was sent a month later to the from the Pacific oyster based on molecular characterization, mi- same laboratory. The oysters were cleaned using running water Figure 1A-F. A. Live juvenile Kumamoto oyster. B. Undifferentiated oyster of 2.5 mm of shell height. There is no follicular develop- ment in the connective tissue (Ct) surrounding the digestive gland. C. Male oyster of 2.7 mm of shell height with sperm (S) sur- rounding the digestive gland. D. Female oyster of 3.5 mm of shell height with ovocytes (Ov). E. Hermaphrodite oyster of 3.00 mm of shell height. F. Detail of hermaphrodite (encircled), showing ovogonies (Og) attached to the germinal wall of the follicle and spermatids and spermatozoids (Sp) in the central area of the follicle.
to eliminate debris. From each sample, 54 and 58 organisms were The shell height of the oysters from the first subsample chosen respectively due to best appearance and entire shells. ranged from 2 to 4.1 mm, the mean value was 2.99 mm (± 0.51 These organisms were considered as subsamples and were SD). In Figure 1A is showing a live oyster of about 3 mm of shell measured using a micrometer placed in a stereo microscope, height. In 64% of individuals from this subsample, we do not de- considering the shell height (distance from the umbo to the distal tect gonad development and this sub-group was considered as posterior border of the shell; Helm et al., 2006). The whole oysters undifferentiated (Fig. 1B); however, 36% of these oysters showed were fixed by 24 h in Davidson’s fixative solution (Shaw & Battle, development of reproductive follicles with sperm (Fig. 1C). The 1957) and washed in running water. Later, oysters were decalci- fied by immersion in a solution of 10% EDTA for approximately 12 shell height of this sub-group varied from 2 to 4 mm with a mean h (Howard et al., 2004) and washed in running water to eliminating value of 2.95 mm (± 0.52 SD). There was no significant difference excess of EDTA. Thereafter, oysters were placed in histocassetes between the shell heights of both subgroups (t-test p > 0.05). The for histological process according to Shaw and Battle (1957) and shell height of oysters from the second subsample varied from 2 they were checked for tissue alterations and general condition. to 4.4 mm, the mean value was 3.19 mm (± 0.58 SD), 62% of the The size of oysters from different subsamples and reproductive oysters showed gonad development, from which 80.55% were conditions was compared for possible significant differences us- males, 13.89% females (Fig. 1D) and 5.56% hermaphrodites (Figs. There was no significant difference between the shell height Buroker, n. e. 1983. Sexuality with respect to shell length and group size of both subsamples (t-test, p > 0.05); however, there was a trend Japanese oyster Crassostrea gigas. Malacologia 23 (2): 271-279.
of larger oysters in the second subsample.
cooper, k. r. & m. Wintermyer. 2009. A critical review: 2,3,7,8 -tetrachloro- Results indicate that gametogenesis of cultured Kumamoto dibenzo-p-dioxin (2,3,7,8-tcdd) effects on gonad development in bi- oyster starts in very young and small size. This finding adds an- valve mollusks. Journal of Environmental Science and Health - Part other biological difference between C. gigas and C. sikamea, C. Environmental Carcinogenesis and Ecotoxicology Reviews 27 (4): the later starts gamete production at about 3 mm of shell height versus 16 mm in C. gigas. It is important to carry out studies on GoBierno Del estADo De BAJA cAliforniA sur. 2010. Plan Rector: Sistema natural populations of C. sikamea for determining if this early de- velopment of gametes takes place in nature or if it was influenced by the hatchery operation, possible due by use of chemicals dur- HeDGecock, D., m. A. BAnks & Z. kAin. 1999. Occurrence of the Kumamoto ing culture (Cooper & Wintermyer, 2009). Presence of gametes in oyster Crassostrea sikamea in the Ariake Sea. Japan. Marine Biol- these oysters do not means that they are physiologically viable. Moreover, there was not observed empty follicles or the pres- ence of hemocytes which could indicate spawning or reabsorp- elm, m. m., n. Bourne & A. lovAtelli (eDs.). 2006. Cultivo de bivalvos en criadero. Un manual práctico. FAO Documento Técnico de Pesca tion process. Specific studies are needed for determining if these small oysters may reproduce successfully and if so, how this early reproduction accounts for oyster population dynamics in nature, HoWArD, D. W., e. J. leWis, B. J. keller & c. s. smitH. 2004. Histological and how this information may impact management practices in techniques for marine bivalve mollusks and crustaceans. NOAA Technical Memorandum NOS NCCOS 5: 1-218.
The fact that the percentage of oysters showing gametogen- lópeZ-flores, i., c. ruiZ-reJón, i. cross, l. reBorDinos, f. roBles & r. nA- esis was greater in oysters from the second sample could be re- vAJAs-péreZ. 2010. Molecular characterization and evolution of an lated to their slightly larger size; in this sense, Quayle (1988) noted interspersed repetitive DNA family of oysters. Genética 138 (11): that in C. gigas sexual maturity appears to be a function of size rather than age. This information constitutes a new record in the size of gametogenesis in oysters of aquaculture importance, plac- ikHAilov, A. t., m. torrADo & J. ménDeZ. 1995. Sexual differentiation of reproductive tissue in bivalve molluscs: identification of male asso- ing the Kumamoto oyster (Crassostrea sikamea) as the most pre- ciated polypeptide in the mantle of Mytilus galloprovincialis Lmk. Int. cocious species among cultivable oysters in the world. Precocity International Journal of Development Biology 39: 545-548.
in bivalve mollusks has been documented in Mytilus galloprovin-cialis Lamarck, 1819 (Mikhailov et al., 1995; Paz et al., 2001).
numAcHi, k. 1978. Japanese species, breed, and distribution. In: Imai, T. (Ed.), Aquaculture in shallow seas: progress in shallow sea culture, ACKNOWLEDGEMENTS
part II, Chapter 1, Biologial research on the oyster. Tokyo: Koseisha Koseikakn Publishers, pp. 123-126.
The authors would like to thank Ing. Daniel Carreño Montreal from Sea Farmers, S. A. de C. V. for providing us with the oysters for the pAZ, m., A. mikHAilov & m. torrADo. 2001. Sexual differentiation of the study and allowing us to publish the data obtained.
somatic gonad tissue in marine bivalve mollusks: esterase- and fi-bronectin-like recognition signals. International Journal of Develop-ment Biology 45: 9911-9912.
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QuAyle, D. B. 1988. Pacific oyster culture in British Columbia. Canadian AmemiyA, i. 1928. Ecological studies of Japanese oysters, with special Bulletin of Fisheries and Aquatic Sciences 218: 1-241.
reference to the salinity of their habitats. Journal of the College of Agriculture 9: 333-382.
reece, k. s., J. f. corDes, J. B. stuBBs, k. l. HuDson & e. A. frAncis. 2008. Molecular phylogenies help resolve taxonomic confusion with Asian BAnks, m. D., D. J. mcGolDrick, W. BorGeson & D. HeDGecock. 1993. Discrim- Crassostrea oyster species. Marine Biology 153: 709-721.
ination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large subunit rRNA. sekino, m., m. HAmAGucHi, f. ArAnisHi & k. okosHi. 2003. Development of Molecular Biology and Biotechnology 2: 129-136.
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BAnks, m. D., D. J. mcGolDrick, W. BorGeson & D. HeDGecock. 1994. Ga- metic incompatibility and genetic divergence of Pacific and Kuma- sHAW, B. l. & i. H. BAttle. 1957. The gross microscopic anatomy of the di- moto oysters. Crassostrea gigas and C. sikamea. Marine Biology 121: gestive tract of the oyster Crassostrea virginica (Gmelin). Canadian Journal of Zoology 35: 325-346.
stAfforD, J. 1913. The Canadian Oyster. Its Development, Environment véleZ, A. 1976. Crecimiento, edad y maduréz sexual del ostión Crassostrea and Culture. Commission of Conservation, Committee on Fisheries, rhizophorae de Bahía Mochima. Boletín del Instituto Oceanográfico Game and Fur-bearing Animals. The Mortimer Company, Otawa. de la Universidad de Oriente 15 (1): 65-72.
ZAr, J. H. 1974. Bioestatistical Analysis. Prentice Hall. Englewood Cliffs, tHompson, r. J., r. i. e. neWell, v. s. kenneDy & r. mAnn. 1996. Repro- ductive process and early development. In: V. S. Kennedy & R. I. E. Newell (Eds.). The Eastern Oyster Crassostrea virginica. Maryland Recibido: 24 de febrero de 2012.
Sea Grant Book. College Park, Maryland, pp. 335-370.
Aceptado: 03 de mayo de 2012.

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