Osmoregulatory responses to cadmium in reference and historically metal contaminated Gammarus fossarum (Crustacea, Amphipoda) populations

Highlights

• Osmoregulatory responses to Cd were studied in 3 Gammarus populations.

• One population lives in a Cd-contaminated river, the others inhabit Cd-free sites.

• High inter-individual variations in osmoregulation were noted in 2 populations.

• Gills of Cd-impacted animals showed histopathology, ion transporters overexpression.

• No obvious correlation was found between osmoregulatory responses and sampling sites.

Abstract

In order to better understand the variable sensitivities of crustaceans to metals, we investigated the impact of cadmium exposure in 3 populations of Gammarus fossarum from different rivers of France. The first population lives in a Cd-contaminated river from a geochemical background, while the others inhabit Cd-free sites. Osmoregulation, a relevant biomarker to evaluate crustacean health following metal contamination, was used as a proxy to evaluate the intra- and inter-populationnal sensitivities to Cd. Specimens from each population were experimentally exposed to 9 μg Cd²⁺/L Cd for 7 days and hemolymph osmolality (HO) was then individually measured. In exposed populations, high inter-individual variations in HO values were noted, resulting in their separation into non-impacted and slightly or highly Cd-impacted (with lower HO) animals. In gills of impacted organisms, deep histopathological alterations and protein overexpression of Na⁺/K⁺-ATPase and V-H⁺-ATPase were observed through histology and immunolocalization, while non-impacted animals showed profiles comparable to controls. Moreover, the osmoregulatory processes in the population living in the Cd-contaminated site were impacted by acute Cd exposure in the laboratory as much as for one of the two populations originating from Cd-free sites. The observed changes did not reveal any obvious adaptive osmoregulatory phenomena at the population scale, but they may be due to differences in fitness between individuals and between populations in relation to the features of their respective environments, unrelated with the presence of the metal.

Introduction

The aquatic environment is subjected to increasing contamination by metals that, in addition to geochemical backgrounds, is mainly due to past and present mining activities and to the continuous industrialization of watersheds since the industrial revolution. In France, some cadmium-contaminated streams show mean annual concentrations of up to 22 μg/L (e.g. Riou Mort, France; Morin et al., 2008). In crustaceans, water-borne cadmium is known to induce a decline of the overall performance in feeding rate, locomotor and ventilation activities, metabolic rate and mating (Lawrence and Poulter, 1998, Maltby et al., 2002, Wallace and Estephan, 2004, Wu and Chen, 2005, Mills et al., 2006). Cadmium is also known to severely impact osmoregulation in crustaceans (review in Charmantier et al., 2009) and among them in amphipods (Felten et al., 2008, Issartel et al., 2010). Osmoregulation is a vital physiological mechanism conditioning the maintenance of the overall metabolism and plays a major role in the distribution of aquatic organisms. In freshwater crustaceans, body fluids are strongly regulated hyperosmotic to the external environment, thus against water invasion and ion loss. Compensatory active uptake of ions from the water is performed through the gills into body fluids. Ion transport is performed by specific transmembrane enzymes and ion channels, highly expressed in specific cells of the gill epithelium, the ionocytes. Two major ionocyte enzymes involved in osmoregulation play a key role in ion uptake: the basolaterally located Na⁺/K⁺-ATPase (NKA) and, particularly in fresh water, the apically located V-H⁺-ATPase (VHA) (review in Charmantier et al., 2009, McNamara and Faria, 2012).

The freshwater amphipod crustacean Gammarus fossarum (Koch, 1836) is commonly found in West Paleartica streams (Karaman and Pinkster, 1977). Previous studies have revealed that G. fossarum sampled from a reference population (River Bourbre, France) and exposed to 15 μg Cd/L for 3 and 7 days showed a decrease in hemolymph osmolality (Felten et al., 2008, Issartel et al., 2010), resulting from a strong alteration of the gill tissues (collapse of the gill epithelium resulting in the disappearance of the hemolymph canals, hyperplasia), along with a disturbed expression of NKA (Issartel et al., 2010). Interestingly, though exposed groups showed significant differences with controls, a high inter-individual variability was noted in response to Cd exposure, with different levels of impact at the sub-individual scale suggesting the existence of a high inter-individual sensitivity to Cd.

In the case study of Cd impact in Gammarus fossarum, we identified one population (Ardillats: Ard) that reproduces and spends its entire life cycle in a French stream chronically exposed to Cd due to a geochemical background and low water hardness (Vigneron et al., 2015). Taking this opportunity to further assess the response of crustacean populations to Cd environmental contamination, the present study aimed at investigating the different sensitivities to sub-lethal Cd stress in populations of the amphipod G. fossarum, and explore the importance of pre-exposure of populations in the field using osmoregulation, which allows to accurately determine the physiological status of contaminated organisms at the individual scale. For this, we investigated the osmoregulatory responses to a sub-lethal Cd exposure in individuals sampled from three populations of Gammarus fossarum of southeastern France: first the population (Ard) identified as chronically exposed to Cd due to geochemical background (Vigneron et al., 2015); and secondly two populations inhabiting Cd-free sites, one in the River Bourbre flowing in a weakly and sporadically cultivated area (station called Tour du Pin: TdP) which is the population used in our previous studies (Felten et al., 2008, Issartel et al., 2010), and one in a brook spring in a pristine environment (Bastide: Bas). After sampling in the field, all animals were exposed in the laboratory in their river water to Cd concentrations yielding to 9 μg Cd²⁺/L for 7 days. Hemolymph osmolality measurements, histological observations of the gills, and immunolocalization of NKA and VHA were performed as a proxy to highlight potential difference in sensitivities to Cd.