In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Abstract

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.

The paralytic shellfish toxin, saxitoxin, enters the cytoplasm and induces apoptosis of oyster immune cells through a caspase-dependent pathway

“…Altogether, these data support a mechanism for PST-induced apoptosis in bivalve hemocytes that is independent of ROS production. This observation is consistent with previous studies showing that exposure of C. gigas hemocytes to the PSTproducer, Alexandrium minutum, and purified STX decreased ROS production (Hegaret et al 2011;Mello et al 2013). Thus, the PST-induced death of hemocytes may result from a ligandreceptor interaction rather than a response to oxidative stress.…”

“…Previous studies have shown that PSTproducing algae increase the mortality of C. gigas hemocytes both in vitro (Hegaret et al 2011;Lassudrie et al 2016) and in vivo (Medhioub et al 2013). However, fundamental features of this process, including key mechanistic details of how specific PSTs cause toxicity, remained poorly characterized.…”

“…Moreover, STX and its analogs have been shown to modulate immunological parameters and gene transcription in mollusc hemocytes (Estrada et al, 2010;Núñez-Acuña et al 2013;Astuya et al 2015;Detree et al 2016). Toxic algae have also been reported to compromise oyster immune cell function by inhibiting cell adhesion and phagocytosis (Hegaret et al 2011). …”

Effects of Alexandrium minutum exposure upon physiological and hematological variables of diploid and triploid oysters, Crassostrea gigas

“…Encapsulation of P. minimum cells by Ruditapes philippinarum hemocytes has been also suggested by Hégaret et al (2009). Similarly, aggregates of Mercenaria mercenaria hemocytes surrounding P. minimum cells when exposed in vitro have been observed by Hégaret (2007) and Hégaret et al (2008b). Results of the present and previous studies support the hypothesis that hemocytes migrate into the lumina of digestive organs to remove A. minutum cells and/or to protect tissues from toxicity.…”

Combined effects of a parasite, QPX, and the harmful-alga, Prorocentrum minimum on northern quahogs, Mercenaria mercenaria

“…Several studies have highlighted effects of harmful-algal species upon bivalve hemocytes (Hégaret and Wikfors, 2005a, b;Hégaret et al, 2007a, b;da Silva et al, 2008;Ford et al, 2008;Galimany et al, 2008a, b). Specifically, the dinoflagellate Prorocentrum minimum has been shown to cause morphological and functional changes in hemocytes of several bivalve species (Hégaret and Wikfors, 2005a, b;Galimany et al, 2008a;Hégaret et al, 2008bHégaret et al, , 2009). This dinoflagellate is present throughout the world (Heil et al, 2005) and has been reported to affect filtration, growth, survival, or organ and tissue development of northern quahogs, bay scallops and juvenile eastern oysters (Leibovitz et al, 1984;Shumway et al, 1985;Luckenbach et al, 1993;Wikfors and Smolowitz, 1993, reviewed in Wikfors (2005).…”