Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). I. Identification of the aetiological agent and in vitro modulation of zoosporulation by temperature and salinity

Abstract

Morphological characters of zoosporulation stages and DNA sequence of the internal transcribed spacer (ITS) region and the small subunit ribosomal RNA (SSU rRNA) gene confirmed that the aetiological agent of perkinsosis in the clam Tapes decussatus from Galicia (NW Spain) was Perkinsus atlanticus Azevedo, 1989. In vitro modulation by temperature and salinity of the zoosporulation of the parasite was studied. The optimum temperature range for zoosporulation was 19 to 28°C. The temperature range allowing zoosporulation in vitro was 15 to 32°C, which is broader than previously reported (24 to 28°C) for P. atlanticus, and strongly suggests that zoospores can be produced in Galician Rías, where temperature ranges from 10 to 22°C. Prezoosporangia held at 10°C for 2 mo (similar to winter conditions in Galician waters) gave rise to viable zoospores after they were transferred to higher temperatures. This suggests that prezoosporangia could overwinter and zoosporulate in the next spring. Zoospores could survive for up to 22 and 14 d at 28 and 10°C, respectively. The optimum salinity range for zooporulation was 25 to 35 ‰. Zoospore production was abruptly reduced as salinity decreased. The lowest salinity at which zoosporulation was observed was 10 ‰. The effectiveness of different chlorine concentrations and exposure lengths to kill prezoosporangia and zoospores was tested. No survival of free zoospores, free prezoosporangia and prezoosporangia included in gill tissue was observed after incubation for 1 h with 50, 200 and 3000 ppm of chlorine, respectively.

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

“…P. atlanticus ITS sequences from this study grouped with other P. atlanticus sequences and the P. olseni sequences with 100% bootstrap support in a clade that was sister to ITS sequences from P. marinus. Many of the sequences from the isolate culture DNAs were identical to Perkinsus sequences previously obtained by specific amplification of Perkinsus sequences from P. atlanticus-infected host tissue DNA (Casas et al 2002). …”

“…The primer pair designated 'D' was used to amplify an approximately 675 bp fragment of the ITS region from genomic DNA of the cultured P. atlanticus. PCR amplification, cloning and sequencing was done as previously described (Casas et al 2002).The resulting ITS region sequences were subjected to BLAST searches (Altschul et al 1990) Characterisation: enlargement of cultured cells in RFTM. The enlargement of cultured cells in RFTM was tested for 9 different isolates, 3 established from gill fragments, 3 established from haemolymph and 3 established from hypnospores.…”

Epizootiology of Perkinsus sp. inCrassostrea gasar oysters in polyculture with shrimps in northeastern Brazil

“…(n = 54; Table 1), polymerase chain reaction (PCR) assay was prepared using a pair of Perkinsus genus-specific primers, PerkITS85 / PerkITS750 (CASAS et al, 2002). These primers specifically hybridize to the conserved regions of the ribosomal ribonucleic acid (RNA) gene, including internal transcribed spacer 1 (ITS), the 5.8S gene and ITS2 (ITS rDNA).…”

Perkinsus sp. infecting the oyster Crassostrea rhizophorae from estuaries of the septentrional Northeast, Brazil

“…DNA extraction was achieved with a DNAzol  kit (Invitrogen) following the manufacturerʼs directions, while PCR was performed with the primer set Perk ITS 85/750 (Casas et al, 2002) which specifically hybridizes with conserved regions of the internal transcribed spacers (ITS) from the rRNA gene complex, unique to members of the genus Perkinsus (except for Perkinsus qugwadi incertae sedis). DNA extracted from cells of Perkinsus beihaiensis was used as positive control.…”