The neonicotinoid insecticide imidacloprid, but not salinity, impacts the immune system of Sydney rock oyster, Saccostrea glomerata

Highlights

• Imidacloprid causes stress to Sydney rock oysters at concentrations <100 μg/L⁻.

• Exposed SRO showed oxidative stress and significant changes in hemolymph proteins.

• Circulating hemocytes increased but the percent of aggregation significantly reduced.

• There were no synergistic effects of imidacloprid with reduced salinity.

• This implies exposure to pesticides alone can compromise oyster health.

Abstract

The broad utilisation of neonicotinoids, particularly imidacloprid (IMI), in agriculture has led to unplanned contamination of aquatic systems around the world. The sublethal effects of individual pesticides on the immune system of oysters, as well as their combined effects with other environmental stressors that fluctuate in estuarine environments, such as salinity, are yet to be investigated in ecotoxicology. We investigated the acute (4 d) toxicity of IMI in two salinity regimes on the immune parameters of Sydney rock oysters (SRO), including total hemocyte counts (THC), differential hemocyte counts (DHC), phagocytosis and hemocyte aggregation (HA), hemolymph protein expression and enzyme (catalase (CAT), glutathione S-transferase (GST) and acetylcholinesterase (AChE)) activities. Environmentally relevant concentrations of IMI were found to cause an increase in THC, induce GST activity, reduce HA, and inhibit AChE activity. However, DHC, CAT activity and phagocytosis were not significantly impacted at any test concentration at either salinity. IMI concentrations ≥0.01 mg/L significantly altered the expression of 28 proteins in the hemolymph of SRO, including an increase in the relative expression of extracellular superoxide dismutase, severin, ATP synthase subunit beta, as well as stress response proteins (heat shock proteins, serine/threonine-protein kinase DCLK3 and peroxiredoxin-1), and a decrease/absence of collagen alpha-4 (VI) and alpha-6 (VI) chain, metalloendopeptidase, L-ascorbate oxidase, transporter, CEP209_CC5 domain-containing protein and actin. This study indicates that the immune system of SRO can be impacted at environmentally relevant concentrations of IMI, but reduced salinity does not appear to influence the toxicity of this insecticide.

Introduction

The increasing production and utilisation of neonicotinoids in agriculture, and for domestic purposes, is causing the contamination of aquatic systems around the world (Jeschke et al., 2011; Sanchez-Bayo and Hyne, 2014; Anderson et al., 2015; Hook et al., 2018). As a result, evaluating their biological impacts on aquatic organisms, particularly keystones species, is essential (Saraiva et al., 2017; Ewere et al., 2019a; Ewere et al., 2019b; Stara et al., 2020). The effects of these chemicals on aquatic organisms could be exacerbated in estuarine environments (Hano et al., 2019) due to fluctuations in salinity, which frequently occur in coastal water bodies, along with the input of land-based contaminants, especially after flood events. Therefore, investigation of the synergistic impacts of neonicotinoids under conditions of normal and hyposalinity are required to simulate freshwater run-off events and gain a deeper insight into potential neonicotinoid toxicity.

Neonicotinoids, including imidacloprid (IMI), are very soluble in water (Jeschke and Nauen, 2008; Jeschke et al., 2011; Anderson et al., 2015) and have been reported to enter waterways as run-off from agricultural crops along with dust-drift during seed planting and harvesting operations, and leaching from contaminated soil (Jemec et al., 2007; Morrissey et al., 2015). These insecticides act on nicotinic acetylcholine receptors (nAChRs) in invertebrates, causing various biological effects (Tomizawa and Casida, 2005; Alexander et al., 2007; Tomizawa et al., 2007; Tomizawa et al., 2008; Azevedo-Pereira et al., 2011; Tison et al., 2016). Due to neonicotinoid solubility, filter-feeders (e.g. bivalves) can be considered as a significant risk group, as they can passively accumulate pesticides from the water (Jacomini et al., 2006; Zorita et al., 2015). In fact, a range of sublethal effects have been documented for the effects of neonicotinoids in bivalve tissues, including inhibition of acetylcholinesterase activity in the gills, digestive gland and adductor muscles of oysters (Moncaleano-Niño et al., 2018; Ewere et al., 2019b) and significant differences in the expression of genes, including the possible impact on immune gene regulation, in the digestive gland of oysters (Ewere et al., 2019a) and mussels (Dondero et al., 2010). However, there are no reported studies on the impact of IMI or any neonicotinoid on the immune system of oysters.

The immune system of vertebrates and invertebrates, including oysters, has been acknowledged as a sensitive target for a wide range of pesticides (Corsini et al., 2008; Gangemi et al., 2016; Mitra et al., 2019). While bivalves lack adaptive immunity, they have a potent innate immune system that responds appropriately to environmental perturbation. In oysters, the essential cells involved in immunoregulation are the hemocytes, which play a role in digestion and nutrient transport (Cao et al., 2007), as well as providing a focal part in the molluscan immune guard (Cao et al., 2007; Renault et al., 2011). These cells remove or deactivate foreign particles in the hemolymph by encapsulation, aggregation and phagocytosis (Renault et al., 2011; Sureda et al., 2013; Stara et al., 2020). This process is accomplished by granulocytes and hyalinocytes, cells that are thought to aggregate at the point of injury and are mainly responsible for wound repair in bivalves (Hégaret et al., 2003; Stara et al., 2020). The viability and functional capabilities of these hemocytes influence their capacity to respond to pathogenic challenges. Therefore, the evaluation of the whole or individual hemocyte biomarkers in oysters may reveal alterations in immune function in response to neonicotinoid exposure, possibly leading to changes in oyster health.

Among the cell-mediated biomarkers in bivalve toxicology, hemocyte numbers and functions have been observed microscopically and by flow cytometry (Hégaret et al., 2003; Donaghy et al., 2009; Hong et al., 2014). These biomarkers have been shown to respond to different stress treatments, including a decrease in hemocyte viability after 12 d treatment of oysters with organochlorine pesticides (Anguiano et al., 2007), increased in hemocyte counts of mussels exposed to air for 12 to 14 min (Renwrantz et al., 2013) and reduction of total and viable hemocyte counts and phagocytosis activity in clams that were stressed with salinity and temperature (Reid et al., 2003; Christine et al., 2004). Humoral biomarkers for immune health in invertebrates include the proteome, which enables a wholistic analysis of which proteins in the hemolymph are influenced by stress (Dondero et al., 2010; Muralidharan et al., 2012; Mukherjee et al., 2013; Li et al., 2014; Timmins-Schiffman et al., 2014). Proteome assessment can provide a robust approach for the evaluation of a wide range of biological implications from exposure of organisms to environmental perturbation. For example, Yang et al. (2013) reported differential expression of 52 proteins, including glutathione S-transferase (GST) and glucosyl/glucuronosyltransferase in the entire tissue of sweet potato whitefly, Bemisia tabaci, that were exposed to the neonicotinoid, thiamethoxam. It is not currently known if exposure to IMI will impact the protein expression profile of Sydney rock oyster (SRO) hemolymph. However, IMI was shown to alter the digestive gland proteome of the mussel, Mytilus galloprovincialis (Dondero et al., 2010), and heat shock protein genes and GST activity in the tissues of SRO (Ewere et al., 2019a; Ewere et al., 2019b). Here we investigate the impacts of IMI, in combination with reduced salinity on the hemocytes of SRO in terms of total hemocyte and differential hemocyte counts, aggregation, phagocytosis, hemolymph protein expression and enzyme activities.