Research paperEffects of cyanobacteria Synechocystis spp. in the host-parasite model Crassostrea gasar–Perkinsus marinus
Introduction
Perkinsosis is a disease caused by protozoan parasites from the Perkinsus genus (Alveolata, Protalveolata, Perkinsidae) (Adl et al., 2012). The disease affects bivalve molluscs from wild and cultured populations and causes mortality in their hosts (Choi and Park, 2010, Villalba et al., 2011). Two species of Perkinsus (P. marinus and P. olseni) were responsible for huge economic losses and are listed by the World Organisation for Animal Health (OIE, 2017) for imminent declaration. In Brazil, Perkinsus spp. have been recently described (Sabry et al., 2009, Sabry et al., 2013), and two species, P. beihaiensis and P. marinus, are often found infecting native oysters, Crassostrea gasar and C. rhizophorae, including cultured populations (da Silva et al., 2014, da Silva et al., 2013, Dantas-Neto et al., 2015, Luz and Boehs, 2016, Queiroga et al., 2015). By contrast, P. olseni has been reported occasionally in Northeast Brazil (da Silva et al., 2014, Queiroga et al., 2015), and Uruguay (Cremonte et al., 2005). In Brazil, due to its recent discovery, the pathology caused by Perkinsus spp. in bivalves is still poorly understood. Facing the threat of this disease to Brazilian oyster farming, there is a growing interest in studying the biology of this parasite (da Silva et al., 2016, da Silva et al., 2013) and associated diseases, as well as in understanding the role of the environment on this host-parasite association.
Bivalves rely on several mechanisms to prevent infectious diseases (for reviews see Allam and Raftos, 2015, Bachère et al., 2015, Schmitt et al., 2011, Song et al., 2010); and in turn, Perkinsus spp. parasites display several mechanisms of evasion that enable them to survive inside the host (Fernández-Boo et al., 2015a, Fernández-Boo et al., 2015b, Fernández-Boo et al., 2014, Hasanuzzaman et al., 2016, Pales Espinosa et al., 2014, Soudant et al., 2013). Assessing the host immune responses to infection by Perkinsus spp. may help identify the impact on the host. There is a large body of information on cellular and humoral host immune responses of bivalves to the two most studied Perkinsus species from temperate regions (Villalba et al., 2011). By contrast, in Brazil, only one study has addressed the immune responses of the native oyster species, C. gasar, to Perkinsus spp. infection, and showed the impairment of some important immune parameters, such as phagocytic capacity and ROS production (Queiroga et al., 2013).
It is well known that (i) the host immune system, (ii) Perkinsus spp. ability to invade the host, and (iii) the host-parasite interaction, are modulated by abiotic environmental factors, such as temperature and salinity, and biotic agents, such as harmful algal blooms (for reviews see Allam and Raftos, 2015, Guo et al., 2015, Renault, 2015). Temperature and salinity are the most important factors controlling the seasonal patterns of infection of P. marinus and P. olseni (Bushek et al., 2012, Oliver et al., 1998, Villalba et al., 2005), and can also affect haemocytes, oyster immune cells (Donaghy and Volety, 2011, Gagnaire et al., 2006, Hégaret et al., 2003a, Hégaret et al., 2003b). Concerning exposure to harmful algal blooms, the parasite P. olseni is sensitive to the presence of toxic dinoflagellates, such as Karenia selliformis and Prorocentrum minimum (da Silva et al., 2008) (Hégaret et al., 2009); although within the host Ruditapes philippinarum tissues it does not appear to be affected by K. selliformis, K. mikimotoi or Alexandrium ostenfeldii (Hégaret et al., 2007, Lassudrie et al., 2014). Alongside this, haemocytes exposed to artificial harmful algal blooms and their toxins (Lassus et al., 2014) may undergo different effects, depending on the species of microalgae and bivalves (Astuya et al., 2015, da Silva et al., 2008, Hégaret et al., 2009, Hégaret and Wikfors, 2005, Lassudrie et al., 2016, Lassudrie et al., 2014, Mello et al., 2013, Simões et al., 2015). To date, there is no study addressing the effects of cyanobacteria on host-parasite interactions.
Harmful algal (Hallegraeff et al., 2004) and cyanobacterial blooms (Backer et al., 2015, Rastogi et al., 2015) are common and growing events in coastal regions worldwide, and have also been reported in the Brazilian literature (Castro et al., 2016, Mafra-Junior et al., 2006, Sacilotto Detoni et al., 2016). The Brazilian government recently created a monitoring programme to survey harmful marine algal blooms and their toxins in Santa Catarina state (Southern Brazil), where the largest national production of bivalves is concentrated (Santos and Costa, 2016). So far, this monitoring network focuses mainly on the well-known and characterized toxic diatoms and dinoflagellates; however, little is known about the marine cyanobacteria blooms and their toxins (Golubic et al., 2010). Conversely, freshwater cyanobacteria blooms are well-studied worldwide, as they are responsible for the contamination of water bodies and, consequently, human consumer poisoning (Bláha et al., 2009, Catherine et al., 2013, C. Moreira et al., 2013). Cyanobacteria can proliferate very quickly causing blooms (Backer et al., 2015); their toxins can be accumulated in the tissues of aquatic animals, including those used for human consumption (Berdalet et al., 2016, Mulvenna et al., 2012) and produced in aquaculture farming (Smith, 2008). Problems caused by cyanobacteria are mainly related to hypoxia and the presence of toxic compounds, such as microcystins, nodularins, cylindrospermopsins, saxitoxins, anatoxins, lyngbyatoxins, antillatoxins, jamaicamides, among others (Catherine et al., 2013, Golubic et al., 2010, C. Moreira et al., 2013). Nevertheless, the characterization of these toxic compounds and their ecological impacts remain scarce (Rastogi et al., 2015).
In Brazil, an ongoing project prospecting marine microalgae from the NE region revealed the occurrence of several species of marine and estuarine cyanobacteria (Synechocystis, Synechococcus, Romeria, Phanktonlyngbya, Cyanothece, and Phormidium) on the Paraíba coast, including regions where oysters are produced (information from the Reef and Biotechnology of Microalgae Laboratory, from Federal University of Paraíba). Cyanobacteria of the genus Synechocystis (Order Chroococcales) have been demonstrated to produce cytotoxic compounds. More recently, methanolic extracts from Synechocytis spp. showed cytotoxic effects on marine invertebrate cells, such as nauplii of Artemia salina, and embryos of sea urchin Paracentrotus lividus and mussels Mytillus galloprovincialis (Martins et al., 2007).
Mollusc haemocytes are somatic cells that can be used to evaluate the effects of various bioactive compounds (Aladaileh et al., 2008, Bouchard et al., 1999, Jeong and Cho, 2005, Mohamed, 2011), including toxins from microalgae (Astuya et al., 2015, Mello et al., 2013, Nunez-Acuna et al., 2013). To date, some studies have evaluated the effect of cyanobacteria on the immune system cells of freshwater bivalve species, showing modulatory effects (Gélinas et al., 2014; Juhel et al., 2015).
In the present work, the effect of intact marine and estuarine cyanobacteria of the genus Synechocystis and their extracellular products were evaluated by flow cytometry on oyster defence cells (haemocytes) and its intracellular parasite (trophozoites of P. marinus).
Section snippets
Perkinsus marinus and cyanobacteria isolates and in vitro culture
A culture of P. marinus trophozoites isolated from Brazilian oysters (CR-PB192; Queiroga et al., 2016) was propagated in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DME-Ham’s F12, 1:1, Sigma, Saint Louis, Missouri, USA; Gauthier and Vasta, 1995) supplemented with 5% fetal bovine serum (FBS), at a salinity of 20, and at 25 °C. P. marinus was propagated in DME-Ham’s F12 for five days before being used for assays, from an initial inoculum of 106 cells mL−1. Then cells were rinsed
Short term in vitro effects of cyanobacteria on Perkinsus marinus
The short exposure of P. marinus trophozoites to different WCs of cyanobacteria led to a slight increase in cell viability (Fig. 1A), which was significant (P < 0.0001) for all isolates when compared to the control (Conway medium, 95.4 ± 0.5%). The isolate M60C caused the highest increase in viability (3.5%), followed by isolates M3C (2.9%) and M129C (2.8%); while the lowest change was induced by isolate M62C (1.3%). No ECP significantly affected the viability of the parasite (mean of all
Discussion
The present study evaluated the in vitro interaction between isolates of marine and estuarine cyanobacteria, Synechocystis spp., and the trophozoites of the parasite, P. marinus, as well as the immune cells (haemocytes) of the oyster, C. gasar.
To date, the cytotoxic effects of cyanobacteria have not been tested on Perkinsus spp. cells. However, the effect of harmful marine microalgae has previously been shown for the parasite P. olseni infecting Manila clams R. philippinarum. The direct (in
Author contributions
Conceived and designed the experiments: PMS and FRQ. Performed the experiments: FRQ, NDF, LNS. Analysed the data: FRQ and HH. Contributed reagents/materials/analysis tools: PMS, RS. Wrote original draft: PMS, FRQ. Wrote review & editing: HH, LFMS, PMS, RS.
Acknowledgments
The last author would like to acknowledge financial support provided by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) (Project: CIMAR 2202/2014, No. 23038.004302/2014-39). FR Queiroga, ND Farias, LN Santana were supported by fellowships provided by the Brazilian research agencies CAPES and CNPq. We also thank to the oyster producer Sebastião L Costa. The authors thanks the Laboratório de Farmacologia e Aplicação de Produtos Bioativos (LFAPBIO/IDEP) for allowing the use of
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