First record of Perkinsus marinus infecting Crassostrea sp. in Rio Grande do Norte, Brazil, using real-time PCR

Abstract

The introduction of pathogens into disease-free areas or sanitary-controlled regions is one of the main threats to the maintenance of coastal ecosystems and the health of commercially important fishery resources. Bivalve molluscs are one of the aquatic organisms most affected by diseases caused by microorganisms, a number of these caused by protozoans of the genus Perkinsus, which can cause serious mortality events producing significant economic losses (Villalba et al., 2004). When they do not cause death, these protozoa can impair reproductive performance and host health generally, reducing growth and condition and weakening defenses, which favors opportunistic secondary infections (Montes et al., 2001, Lee et al., 2001, Itoïz et al., 2021). Perkinsus marinus is particularly pathogenic notifiable to the World Organisation for Animal Health (WOAH, 2022), having been responsible for mass mortalities of oyster populations since the 1940 s in the Gulf of Mexico (Ray, 1996). This species has been spreading along the North American coast (Ford and Chintala, 2006, Ford and Smolowitz, 2007) and in Mexico with infections in C. corteziensis (Escobedo-Fregoso et al., 2015, Villanueva-Fonseca et al., 2020) and Saccostrea palmula (Cáceres-Martínez et al., 2012).

In Brazil, the first record of Perkinsus sp. infecting native C. rhizophorae oysters was in the estuary of the Pacoti River, Ceará (Sabry et al., 2009), but without parasite species identification. From this record, new research was conducted and diagnostic techniques were improved in order to identify the species detected in Brazil. Da Silva et al. (2013) identified P. marinus for the first time on the Brazilian coast in C. rhizophorae, in the estuary of the Paraíba River, Paraíba. This protozoan has also been detected in C. rhizophorae of natural banks in Baia De Camamu, Bahia (Pinto et al., 2017); in C. gasar in the São Francisco River estuary, Sergipe (da Silva et al., 2014, Scardua et al., 2017); in the estuary of the Mamanguape River, Paraíba (Queiroga et al., 2015); and putatively infecting C. gigas and C. gasar oysters in Santa Catarina (Luz Cunha et al., 2019, Leibowitz et al., 2019).

Although it is not presented as an official diagnostic method recommended by the World Organization for Animal Health (WOAH, founded as OIE), the real-time polymerase chain reaction (qPCR) technique has been developed over the years as a fast and safe alternative to identify and quantify P. marinus in host tissues or in the environment (Audemard et al., 2004, Gauthier et al., 2006, Faveri et al., 2009, Marquis et al., 2020). In this study, we used the qPCR technique as a confirmatory diagnostic tool for the detection of P. marinus in Crassostrea sp. oysters from estuaries of Rio Grande do Norte, Brazil.

2. Materials and methods Oysters of the Crassostrea genus (n = 1200) were collected in the estuaries of the Potengi River (S05o45′51.48″, W035o12′42.16″), and the Guaraíras lagoon (S06o12′38.62″, W035o08′08.27″) in the state of Rio Grande do Norte (Fig. 1). The collections were carried out in 2019, March and September in Potengi and April and October in Guaraíras, a total of 300 oysters/month/estuary. Seawater temperature and salinity were measured during sampling. The average height of the oysters was 52.94 ± 8.65 mm and 64.45 ± 8.28 mm respectively in the Potengi River and the Guaraíras Lagoon estuaries. The temperature and salinity of the water ranged from 25 to 26 °C and 8 to 34‰ in the Potengi River and from 26 to 27 °C and 1 to 36‰ in the Guaraíras lagoon. The oysters were collected manually in natural banks from the roots of Rhizophora mangle and were then transported to the laboratory. Rectum and gill tissues of each animal were incubated in Ray's fluid thioglycollate medium (RFTM), in the presence of antibiotics (penicillin G/streptomycin, 100 U ml –1/µg –1) and antifungal (nystatin, 100 U ml –1) for 7 days in the dark at room temperature. Then the tissues were processed and observed by optical microscopy to verify the presence or absence of Perkinsus sp. (Ray, 1954). Infection intensity was established according to a modified semiquantitative Mackin scale (Sabry et al., 2009) and mean infection intensities and standard deviations (±SD) were calculated. Simultaneously, tissue samples from gills were preserved in 95 % alcohol and stored at −20 °C for DNA extraction and confirmatory molecular diagnosis.