Project Topics
Get Complete Project Material File(s) Now! »
Effect biomarkers
Lipofuscin accumulation
Lipofuscins are lipopigments produced after lipid peroxidation, when polyunsaturated fatty acids are damaged by free radical/reactive oxygen species (Au, 2004). These lipofuscins accumulate in lysosomes after pollutant exposure or other stresses (Marigómez et al., 2013b) and are able to bind cations such as: Cu, Zn, Fe, Mg and Mn (Zorita et al., 2006). Previous works have reported an enhanced presence of lipofuscins after pollutant exposure in mollusc tissues, including oysters Crassostrea virginica exposed to metals (Ringwood et al., 1998). The presence of lipofuscins is generally related with the impairment of the correct lysosomal functioning (Moore, 1990), it is also regarded to contribute to the decrease in the degradative capacity of the digestive system (Moore et al., 2006).
Intracellular accumulation of neutral lipids
The accumulation of intracellular neutral lipids in the lysosomes of digestive cells has been classically considered as an exposure biomarker to organic compounds, however it has also been linked to the presence of other non identified stressors (Marigómez et al., 2013b). This increase of neutral lipids in the lysomomes can also occur because of an autophagy of the excess of lipid droplets (Krishnakumar et al., 1994; Lowe, 1988; Moore et al., 1987; Moore, 1988).
Neutral lipids constitute an important storage of nutrients related to growth and reproduction (Holland, 1978). In this context, oysters are known to possess a connective tissue composed by vesicular cells that store glycogen and act as an energy reservoir for energy demanding processes (Jouaux et al., 2013; Thompson et al., 1996). During spawning months neutral lipids are known to be transferred from connective tissue to gonads (Cancio et al., 1999; Dridi et al., 2007) whereas the presence of stress factors can induce their depletion from connective tissue, altering the total energy budget of molluscs (Guerlet et al., 2006).
Tissue level biomarkers
As mentioned above, the digestive gland of bivalves has been usually selected as a target organ for the analyses of tissue level biomarkers, since this organ is known to participate in: food digestion, metabolism processes, reserve storage and in the detoxification and elimination of pollutants among others (Marigómez et al., 2002; Moore and Allen, 2002).
The digestive gland of oysters is composed by several alveolo-tubular units, with secondary ducts connected to primary ducts that finally end up into the stomach (Galtsoff 1964; Langdon and Newell., 1996). In these digestive alveolo-tubular units two main types of cells are present: digestive and basophilic cells. The first ones are in charge of the intracellular digestion of food and materials through their well developed endo-lysosomal system. Once the intracellular digestion has finished waste products are released into the lumen of the tubules as residual bodies. The second type of cells, basophilic cells, are secretory cells related with the synthesis and excretion of proteins (Langdon and Newell., 1996) that could be involved in extracellular digestion and metabolic regulation (Marigómez et al., 2002; Izagirre et al., 2009).
The digestive gland of oysters is a very plastic organ that suffers cyclic changes, even under normal environmental conditions, when bivalves have to face stress conditions these changes can be enhanced (Marigómez et al., 1990; Zaldibar, 2006). Among the observed changes, the most relevant ones are the following: reduction on the epithelium thickness that conform digestive gland tubules (atrophy), changes on proportional ratio among digestive and basophilic cells (Cajaraville et al.,1991; Marigómez et al., 2006) and the increase of connective tissue with respect to digestive tubules, because the latter reduce their amounts or shrink, being surrounded by larger amounts of connective tissue (Brooks et al., 2012, 2011; Garmendia et al., 2011b).
Digestive gland tubule atrophy is caused by several factors including nutrient scarcity or pollutant exposure. Although the atrophy in digestive gland tubules is a reversible pathology (Kim et al., 2006), the epithelial thickness of digestive tubules has been previously used among other tools to determine mussel’s health status in environmental monitoring programs (Garmendia et al., 2011b). In this context, planimetric methods have provided valuable information about mollusc health status, (Garmendia et al, 2011c). In fact, the ICES comparative report includes these measurements as valuable tools for ecosystem health assessments, concluding that the MLR/MET ratio is a more sensitive biomarker for digestive gland atrophy than MET alone (Garmendia et al., 2011c; ICES 2012).
On the other hand, the Connective to diverticula ratio (CTD ratio), corresponds to the relative proportion of connective tissue in front of digestive tubules, has been proposed as a successful biomarker in the digestive gland of molluscs exposed to different stress sources including PAHs or metals (Brooks et al., 2012, 2011; Garmendia et al., 2011b; Múgica et al., 2015).
Histopathology
Histopathological changes have been previously used (including bivalves) in order to assess the individual health status as a sensitive and reliable indicator in several studies (Bignell et al., 2011; Garmendia et al., 2011a).
In oysters as in other bivalve molluscs, the immune system is based on haemocytes (Ottaviani et al., 2010; Renault, 2015) and their main function is to phagocytise biological pathogens, cell debris and foreign particles (De Vico and Carella, 2012). Moreover, haemocytes also participate in other processes such as: tissue reparation (for instance in gonadal follicules after spawning), nutrient digestion, transport and excretion (Cheng, 1981) but also in pollutant detoxification processes through the accumulation of metal or organic compounds in their endolysosomal system (De Vico and Carella, 2012).
The presence of haemocytic infiltrations in bivalves is closely related to the presence of stress conditions (Bayne et al., 1985) including exposure to organic xenobiotics (Auffret, 1988; Bignell et al., 2011; Sunila, 1984), metals or pathogens (Lowe and Moore, 1979; Rasmussen, 1986). In some cases, other types of inflammatory responses known as granulocytomes can appear (Neff et al., 1987; Svärdh and Johannenson, 2002).
Parasite presence in oysters also affects their health status. Gills and digestive gland are the most exposed organs to be parasited due to the filter feeding behaviour of oysters, these organs being almost constantly in contact with sea water (Bignell et al., 2008; Villalba et al., 1993). The impacts produced by these parasite infestations in oysters are multiple and vary according to the pathogen. Some of them, i.e. trematods, cestods or copepods can alter oyster growth. Other pathogens such as protozooans are known to cause severe alterations including even death of oysters.
During the last decades, special attention has been paid to the relationships between infectious diseases and pollutant exposure. In fact, the measurement of parasite prevalence has been included in biomonitoring programs as they can change the sensitivity level to pollutants and endanger host health status (Kim et al., 2008). Thus, the analysis of histopathological alterations provides a non despicable amount of information about individual health status (Bignell et al., 2011; Knowles et al., 2014; Renault, 2015).
The integrative Biological Response Index (IBR)
It is generally accepted that biomarkers used individually do not provide an overall picture of the organism health status and thus the application of several biomarkers, in a battery, is needed to acquire a proper understanding of biological responses to stressors. Consequently, in order to obtain a better understanding of these responses,
biomarkers should be integrated. Different indexes have been proposed for this purpose (Broeg and Lehtonen, 2006; Sanchez et al., 2013). The integration of biomarkers measured at different organisational levels into the Integrative Biological Response (IBR) index has been demonstrated to be a powerful tool that allows obtaining an overall view of the health status of sentinel organisms after pollutant exposure (Beliaeff and Burgeot, 2002; Broeg and Lehtonen, 2006; Serafim et al., 2012). The IBR index was first proposed by Beliaeff and Burgeot, (2002) and it was described as the area of a star plot that allowed the visual integration the biomarker responses.
Since then, the index has been successfully applied in different sentinel species including oysters (Brooks et al., 2011; Cravo et al., 2012; Garmendia et al., 2011a; Marigómez et al., 2013a; Xie et al., 2016). This index has demonstrated its validity in biomonitoring programs being able to discriminate between less and higher impacted sites (Broeg and Lehtonen, 2006; Marigómez et al., 2013a; Serafim et al., 2012; Xie et al., 2016) whereas it has also demonstrated its validity under controlled exposure to pollutants (Asensio et al., 2013; Brooks et al., 2011; Rementeria et al., 2016).
However, caution should be taken when applying and interpreting the IBR index. Indeed two main weak points have been identified for this index. The first one is related to the dependency of the obtained final IBR value to the arrangement of measured biomarkers in the calculation formula. The second weakness, relies on the interpretation of biological responses since only up or down regulation of measured biomarkers are considered (Sanchez et al., 2013). In order to overcome this problems, the calculation of the IBR index has been developed and improved into a new version (Devin et al., 2014; Sanchez et al., 2013) which now includes a simpler formula for its calculation and also a permutation procedure in order to diminish the influence of the biomarker arrangement in the obtained final IBR value.
Table of contents :
I. INTRODUCTION
1. THE BAY OF BISCAY
1.1 Ibaizabal Estuary
1.2 Oka Estuary
1.3 Gironde Estuary
2. METAL POLLUTION IN ESTUARIES
2.1 Copper
2.2 Silver
3. BIVALVES AS SENTINEL ORGANISMS
3.1 Chemical measurements: Stable isotopes application in ecotoxicology
3.2 Cu and Ag interactions in oysters
3.3 Biomarkers
3.3.1 Metal exposure biomarkers
3.3.1.1 Metallothioneins
3.3.1.2 Intralysosomal metal accumulation
3.3.2 Effect biomarkers
3.3.2.1 Lipofuscin accumulation
3.3.2.2 Intracellular acumulation of neutral lipids
3.3.2.3 Tissue level biomarkers
3.3.2.4 Histopathology
3.3.3 The Integrative Biological Response (IBR) index
II. STATE OF THE ART, HYPOTHESIS AND OBJECTIVES
III. RESULTS AND DISCUSSION
Chapter I: Environmental health assessment of 3 estuaries from the Bay of Biscay using cell and tissue level biomarkers in mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas)
Chapter II: Assessment of the effects of Cu and Ag in oysters Crassostrea gigas (Thunberg, 1793) using a battery of cell and tissue level biomarkers
Chapter III: Influence of salinity in Ag and Cu toxicity in oysters (Crassostrea gigas) through the intergrative biomarker approach
Chapter IV: Assessment of the effects exerted by copper and silver in oysters Crassostrea gigas (Thunberg, 1793) after dietary exposure
IV. CONCLUSIONS AND THESIS
V. APPENDIX
Appendix I
Appendix II: Protocols of experimental procedures
