A molecular method to detect and identify the native species of southwestern Atlantic Crassostrea (Mollusca: Ostreidae)

Abstract

Among oysters, species of Crassostrea (Sacco, 1897) are the most attractive to aquaculture. In Brazil, the genus is represented by C. rhizophorae (Guilding, 1828) and C. brasiliana (Lamarck, 1819). Because the maturation and breeding technology is not well developed for these species, aquaculturists need a reliable method to decide the correct time to place spat collectors in the field, and to identify both species, which are morphologically similar. In this study a specific Multiplex PCR protocol was developed, using one pair of universal primers from 18S rDNA as a positive control and a pair of specific primers for each target species. The sensitivity and specificity of the protocol was evaluated. It detected C. rhizophorae DNA in low concentrations, and C. brasiliana DNA in even lower concentrations. Further, the Multiplex PCR proved efficient in detecting DNA in concentrations equivalent to that of a single larva of each species, either separated or combined, when mixed with total DNA extract of a plankton sample representing 1000 L of filtered water. Field tests confirmed the applicability of the protocol, which holds the promise to become an important tool for aquaculture or conservation programs, allowing for the continuous monitoring of the life cycle of C. brasiliana and C. rhizophorae, by detecting the right periods of larval release and settlement

Gonad morphology and reproductive cycle of the mangrove oyster Crassostrea brasiliana (Lamarck, 1819) in the baía de Guaratuba, Paraná, Brazil

“…Oysters used in this research were identified initially by morphological characteristics and by the position they occupy in the water column, as described by Castilho-Westphal (2012). This preliminary identification was confirmed by sampling (n = 263) and further application of molecular protocols developed by Ludwig et al (2011). Of these, 59% belonged to the C. brasiliana species, 38% belonged to C. rhizophorae, and 3% were classified as Crassostrea sp.…”

“…This preliminary identification was confirmed by sampling ( n = 263) and further application of molecular protocols developed by Ludwig et al . (). Of these, 59% belonged to the C. brasiliana s pecies, 38% belonged to C. rhizophorae , and 3% were classified as Crassostrea sp.…”

“…The adductor muscle fragments were preserved in a dimethyl sulphoxide (DMSO)-ethylenediaminetetraacetic acid (EDTA) buffer to identify the species via the polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) protocol developed by Ludwig et al (2011). Of the animals collected for gonadal histology, 58% were used to confirm their species identity.…”

Identificação Molecular De Ostras Nativas Na Costa Do Maranhão, Brasil

“…Multiplex PCR techniques have been developed specifically for the identification of oysters of the genus Crassostrea (LUDWIG et al, 2011;MELO et al, 2013), with the objective of optimizing the taxonomic identification of these organisms, providing an important tool for aquaculture and fishery operations. In addition to this approach, a fragment of approximately 650 base pairs of the 5' extremity of the mitochondrial Cytochrome oxidase subunit I (COI) gene has been adopted as a universal identification system for animal species, including the oysters of the Atlantic Ocean.…”