Impairments of Endocrine Functions: Causes and Consequences - Ecological Biomarkers: Indicators of Ecotoxicological Effects - page 202

nuclear receptor present in crustaceans and involved in reproduction. Among more than
80 environmental contaminants tested using this bioassay, including industrial chemi-
cals, pesticides, pharmaceuticals, phytoestrogens, as well as vertebrate steroids, few com-
pounds were defined as ecdysteroid receptor (ant)agonists (Dinan et al. 2001).
In the field, several observations have led to the suspicion of the occurrence of EDCs within
wild crustacean populations. The incidence of intersex individuals among a wide range of
taxa is certainly the most convincing and the most thoroughly documented of these observa-
tions (for a review, see LeBlanc 2007). For example, Jungmann et al. (2004) reported intersex
occurrence up to 24% within a population of Gammarus fossarum (a freshwater amphipod
crustacean). However, and for the reasons discussed above in relation to the available knowl-
edge on the endocrine regulation of reproductive processes in crustaceans, very few specific
molecular biomarkers are available in crustaceans for assessment of exposure to EDCs. By
analogy to the method applied in fish, the measurement of VTG-like proteins has been pro-
posed as a biomarker of AGH pathway disruption (LeBlanc 2007; Matozzo et al. 2008). In
crustaceans, the use of specific methods for VTG assessment in an ecotoxicological context
has been limited to the measurement of gene expression encoding this protein in only a few
species, such as Daphnia magna (Tokishita et al. 2006), the copepod Tigriopus japonicus (Lee
et al. 2008), and Gammarus fossarum (Xuereb et al. 2011). VTG production was more com-
monly assessed at the protein level, using indirect methods such as an ELISA (Sagi et al.
1999; Oberdörster et al. 2000; Volz and Chandler 2004; Ghekiere et al. 2006b) or alkali-labile
phosphate (ALP) measurement (Gagné and Blaise 2004; Huang and Chen 2004; Gagné et al.
2005; Huang et al. 2006). Recently, a method based on LC-MS/MS has been developed allow-
ing absolute quantification of this protein in the freshwater amphipod Gammarus fossarum
(Simon et al. 2010). Among the studies using VTG as a specific ED biomarker, most were con-
ducted on females (Gagné and Blaise 2004; Martín-Díaz et al. 2004; Volz and Chandler 2004;
Gagné et al. 2005; Ghekiere et al. 2006a, 2006b; Huang et al. 2006; Poynton et al. 2007), showing
that VTG synthesis is susceptible to different contaminants (e.g., xenoestrogens, pesticides,
metals, or complex mixtures such as urban and industrial wastes). However, females dis-
play natural fluctuations in VTG levels during their reproductive cycle (Meusy and Junera
1974; Jasmani et al. 2000; Okumura and Aida 2000; Jayasankar et al. 2006). Therefore, accu-
rate use and reliable interpretation of data using VTG measurements in females requires
detailed knowledge of their reproductive cycle to discriminate between natural fluctuations
and chemical-induced variations (Hannas et al. 2011; Xuereb et al. 2011, Jubeaux et al. 2012);
however, this is rarely the case in the literature (Lee and Noone 1995; Gagné and Blaise
2004; Ghekiere et al. 2006b). To our knowledge, only the studies of Huang and Chen (2004)
on the decapod Neocaridina denticulata , Sanders et al. (2005) on Palaemon elegans , and Xuereb
et al. (2011) and Jubeaux et al. (2012) on Gammarus fossarum have developed and used VTG
measurement (i.e., protein or transcript) as an ED biomarker in male crustaceans. In addi-
tion, here again, the vertebrate point of view leading to the interpretation of the induction of
VTG in males as an ED could be debated in invertebrates because of pleiotropic functions of
VTG proteins (e.g., oxidative stress). Indeed, duplications and functional shifts in VTG-like
proteins have occurred during metazoan evolution (Smolenaars et al. 2007). This has been
exemplified in decapods, in which the protein ortholog of VTG in most metazoans plays a
role in clotting, whereas egg yolk protein is produced by a VTG paralog gene belonging to
the subfamily of apolipoproteins (Avarre et al. 2007). Therefore, a functional validation of
such a classical biomarker routinely used in vertebrates has to be undertaken with caution
before application in crustaceans (Xuereb et al. 2011; Jubeaux et al. 2012).
In parallel with development carried out to propose specific molecular biomarkers and as
recommended by Verslycke et al. (2007), several approaches based on the simultaneous use