**4. Interest of** *D. polymorpha* **in aquatic biomonitoring framework**

analyses of these biomarkers have to predict the effects at higher levels of organization, that is, the population. In this context, the hemocytes responses show a particular interest since they are key cells involved in the major physiological functions in invertebrates. Indeed, the hemolymph is a remarkable mussel tissue. With an opened circulatory system, hemolymph circulates through the bodies allowing to bring the nutrients to the various organs. Drawn from the adductor muscle of the bivalves after having carried out a little breach on the shell, hemolymph sampling is not lethal for the individuals and represents a clear advantage for biomonitoring. Circulating cells, the hemocytes, are present in the cellular fraction of the hemolymph and have been recently characterized [68]. These cells, mainly granulocytes and hyalinocytes, provide various functions involved in the essential physiological activities such as the transport and the digestion of the nutrients, breathing, excretion, repairs as well of the tissues and of the shell and, of course, immune defenses [69]. Contrary to vertebrates, which have an *acquired immunity* (or specific), the immunity of the invertebrates is only composed of the *innate immunity* (or not specific) ensured by the hemolymphatic compartment (hemocytes and plasma). In a global context, hemocytes allow the measurement of various markers involved in the physiological processes usually studied in ecotoxicology. Current studies are based mainly on the evaluation of the biomarkers of genotoxicity (measurements of DNA damage in hemocytes [70]), of cellular parameters (oxidative activity, mortality [71]), and immune defense (capacity of phagocytosis [72]). However, all these responses suggest the implication of another component of the immune system and complementary to the cellular responses of the organism, the humoral-mediated responses in plasma [73]. Humoral responses are regrouped in different effectors such as the lysosomal enzymes, lectins, protease inhibitors, or antimicrobial peptides [74]. The phenoloxidase cascade [75] and the fast production of reactive oxygen species (ROS) also take part in organism homeostasis. Both systems interact to give an effective answer relative to the various types of aggressions which

A recent study by Juhel et al. [45] highlights the effect of cyanotoxins on the immune functions of *D. polymorpha*. The authors underline a reduction in the full number of hemocytes per milliliter of hemolymph as well as a modulation of the subpopulations of hemocytes, an inhibition of the phagocytosis capacity and an increase in the concentration of lysozyme after 21 days of *in vivo* exposition to various strains of cyanobacteria. However, this study raises the problematic of the complexity to define the real immune effects based only on cellular approaches. Currently, very few studies take into account the entire hemolymphatic compartment, for example, cells and plasma, in the analysis of the biomarkers. However, the evaluation of the interactions and the relations between these two types of responses would permit to understand the functioning of this complex tissue which maintains actively the homeostasis of organism (for review see [76]). Indeed, considering that a panel of enzymes and hydrolytic molecules can influence the cellular responses, it seems paramount to study the humoral compounds in parallel. Considering the central role of hemolymph in the physiology of the organisms, the comprehension of the different functions it provides in relation to other major biological functions is also necessary to ensure relevant conclusions in ecotoxicological studies. However, to date, very little information is available on the hemocyte implications on the physiological responses other than those of immune functions and defenses. This lack of

the organism undergoes.

52 Organismal and Molecular Malacology

For being usable as early warning system, a sentinel species should be able to accumulate contaminants at much higher levels than those in the environment and/or to show variations in biological parameters at one or several levels of organization. In fact, even if analyses of bioaccumulated pollutants have the great benefit of traducing the bioavailable fraction of xenobiotic, they give no information about the potential biological effects. Therefore, the relationship between exposure to pollutants and associated adverse effects is of growing importance in environmental-risk assessment (ERA) and management. In this way, a sentinel species could be designated as a necessary tool of choice for monitoring both the contamination levels and the associated risks for organisms. The zebra mussel was extensively used as model organism for the quality assessment and the biomonitoring of freshwater systems for large amounts of well-known mineral and organic trace pollutants (for review, see [2]). However, more recently zebra mussel appeared also particularly relevant for the assessment of chemical (pharmaceutical, part 2.1) and biological (protozoa and algae toxins, parts 2.2 and 2.3) emergent contaminants as underlined in this chapter. Similarly, an increasing development of biological responses, called as biomarkers, under controlled laboratory conditions (*in vivo* and *in vitro* approaches considering particularly hemocytes and gill cells) and field studies ([2] and part 3 of this chapter) is observed. Regarding field studies, several passive biomonitoring approaches based on autochthonous populations underline a significant modulation of biological responses not only by environmental parameters (e.g., temperature, conductivity, etc.) but also by intrinsic parameters (e.g., size, age, genetic background, life history according to contamination, and potential adaptive responses) [50, 7]. These confounding factors could induce a misunderstanding in the data interpretation according to the toxicity of pollutants. Also, as discussed in section 4, zebra mussel seems to coexist with another *Dreissena* species and it remains difficult to distinguish between the two species. These potent physiological discrepancies could put into questions the biomarker response interpretation.

An active biomonitoring approach appears as an interesting strategy with the view to improving the usefulness of biomarkers and particularly defining reference and threshold values allowing (i) a qualification of the toxicological effects of water quality on organisms and (ii) a suitable comparison between the monitored sites. In fact, the caging of individuals from the same population (and *a fortiori* the same species) in different sites allows (i) to limit or avoid influence of intrinsic parameters and (ii) to expose organisms for a time-limited period, so recent contamination can be detected. As for contamination level and/or biological effects associated, several studies had underlined the interest of the active biomonitoring approach using the zebra mussel, *D. polymorpha* [28, 52, 53, 77, 78]. In the purpose of improving the ecological relevance of biomarkers as early-warning responses, many studies proposed to apply a multi-biomarkers approach considering physiological processes particularly those implied to maintain an individual and/or its population (e.g., reproduction, energy, and immunity) as indicated in part 3 of this chapter. Therefore, the proposition of an integrated tool is mandatory to ensure the use of zebra mussel (together with developed biological responses) in environmental-risk assessment and management in order to establish environmental quality standards and to favor ecosystem protection. Through the process, several developments and approaches were proposed including "Integrated Biological Response" (IBR) to summarize biomarker responses [79]. This approach takes into account "only" biomarker measurements that may be limiting for an environment quality assessment requiring data from a variety of multidisciplinary sources. Then, the weight-of-evidence (WOE) approach including multiple parameters such as for instance bioaccumulation, biomarkers, bioassays, or life history traits appears particularly relevant (see part 6.2 of this chapter).

### **5. The usefulness of another dreissenid species,** *D. rostriformis bugensis*

Despite its high invasive potential, zebra mussel populations have been recently reported to decline such as in the Rhine River [80] or in the Seine River where some populations seem to have disappeared since a dozen years (personal observations). In the meantime, the closely related quagga mussel *D. rostriformis bugensis* [81] became in turn invasive in Europe and North America. In Western Europe, the first observation of quagga mussels was made in 2006 in the Hollands Diep [82, 83] and since 2008 in the Meuse River [81, 84]. In some sites, *D. rostriformis* seems to have become gradually the dominant species [85], as it has been observed in some German and Dutch rivers [86]. Some researchers tried to explain the dominance shift from zebra to quagga mussels through biological, ecological, and ecophysiological studies. The two species are indeed characterized by differential physiological performances. They disclose different temperature, salinity and low oxygen tolerance levels, byssal thread attachment, growth, respiration rates, assimilation efficiency, or reproduction [87–89]. Quagga mussels reveal a lower tolerance to high temperature, although there is a better adaptability to lower temperature, allowing a development in deeper waters. A higher physiological adaptability of quagga mussel than zebra mussel is also highlighted with higher filtration and lower respiration rates, decreasing the energetic cost in quagga [88, 90]. A higher assimilation efficiency allows quagga mussel to maintain higher growth and fecundity rates even at low food levels [8]. Both species also reveal different reproductive strategies. Quagga mussel spawn earlier in the season and in deeper waters, but zebra mussel releases more eggs than quagga [87, 88, 90]. Quagga mussel seems to invest less energy into reproduction than zebra mussel: its lower fecundity is offset by an earlier maturity [90]. Quagga larvae settle at a larger size as they grow more rapidly. Quagga higher somatic growth rate and size [91] are supposed to contribute to a better stress survivorship. A differential sensitivity to chemicals could also be involved in the replacement of a species by the other. Some studies focused on differential xenobiotics accumulation between the two species, mostly on metals and given scarce or inconclusive information [92–95]. Schäfer et al. [80] also showed differences between *D. polymorpha* and *D. rostriformis* in the bioaccumulation potential of pesticides from resuspended sediments. They showed that quagga mussel accumulates more organochlorine pesticides, with greater DNA damage and lower stress protein hsp70 content. These results suggest a difference of sensitivity to genotoxic stress between the two species that may be related to "threshold" levels of DNA damage or to differential capacity of DNA repair [80]. More recently, Potet et al. [96] exposed zebra and quagga mussels to nickel and chromium at two temperatures in laboratory conditions. They measured a set of 14 biomarkers that revealed differential bioaccumulation patterns, filtration activity, and cellular antioxidant and detoxification responses between the two species, with more marked effect of metals in *D. polymorpha*.

to maintain an individual and/or its population (e.g., reproduction, energy, and immunity) as indicated in part 3 of this chapter. Therefore, the proposition of an integrated tool is mandatory to ensure the use of zebra mussel (together with developed biological responses) in environmental-risk assessment and management in order to establish environmental quality standards and to favor ecosystem protection. Through the process, several developments and approaches were proposed including "Integrated Biological Response" (IBR) to summarize biomarker responses [79]. This approach takes into account "only" biomarker measurements that may be limiting for an environment quality assessment requiring data from a variety of multidisciplinary sources. Then, the weight-of-evidence (WOE) approach including multiple parameters such as for instance bioaccumulation, biomarkers, bioassays, or life history traits

**5. The usefulness of another dreissenid species,** *D. rostriformis bugensis*

Despite its high invasive potential, zebra mussel populations have been recently reported to decline such as in the Rhine River [80] or in the Seine River where some populations seem to have disappeared since a dozen years (personal observations). In the meantime, the closely related quagga mussel *D. rostriformis bugensis* [81] became in turn invasive in Europe and North America. In Western Europe, the first observation of quagga mussels was made in 2006 in the Hollands Diep [82, 83] and since 2008 in the Meuse River [81, 84]. In some sites, *D. rostriformis* seems to have become gradually the dominant species [85], as it has been observed in some German and Dutch rivers [86]. Some researchers tried to explain the dominance shift from zebra to quagga mussels through biological, ecological, and ecophysiological studies. The two species are indeed characterized by differential physiological performances. They disclose different temperature, salinity and low oxygen tolerance levels, byssal thread attachment, growth, respiration rates, assimilation efficiency, or reproduction [87–89]. Quagga mussels reveal a lower tolerance to high temperature, although there is a better adaptability to lower temperature, allowing a development in deeper waters. A higher physiological adaptability of quagga mussel than zebra mussel is also highlighted with higher filtration and lower respiration rates, decreasing the energetic cost in quagga [88, 90]. A higher assimilation efficiency allows quagga mussel to maintain higher growth and fecundity rates even at low food levels [8]. Both species also reveal different reproductive strategies. Quagga mussel spawn earlier in the season and in deeper waters, but zebra mussel releases more eggs than quagga [87, 88, 90]. Quagga mussel seems to invest less energy into reproduction than zebra mussel: its lower fecundity is offset by an earlier maturity [90]. Quagga larvae settle at a larger size as they grow more rapidly. Quagga higher somatic growth rate and size [91] are supposed to contribute to a better stress survivorship. A differential sensitivity to chemicals could also be involved in the replacement of a species by the other. Some studies focused on differential xenobiotics accumulation between the two species, mostly on metals and given scarce or inconclusive information [92–95]. Schäfer et al. [80] also showed differences between *D. polymorpha* and *D. rostriformis* in the bioaccumulation potential of pesticides from resuspended sediments. They showed that quagga mussel accumulates more organochlorine pesticides, with greater DNA damage and lower stress protein hsp70 content. These results suggest a difference of sensitivity to genotoxic

appears particularly relevant (see part 6.2 of this chapter).

54 Organismal and Molecular Malacology

In most of the comparative studies, the morphological criterion is chosen to identify the two species. However, the two species show high morphological similarities [97, 98] that prevent from morphological consistent species identification [99]. Mussel's "angularity" has been proposed anyway to improve morphometric identification with discriminating thresholds for zebra and quagga mussels [100]. Molecular genetic markers have then been developed to differentiate *Dreissena* species [97, 98, 101], but most of them are based on restriction fragment length polymorphism (RFLP) of cytochrome-C oxidase subunit I (COI gene). Such mitochondrial genetic tool does not allow identifying potential interspecific hybrids [97]. If natural hybridization has been reported only once by Voroshilova et al. [97] using allozyme patterns, no evidence exists that the two species are unable to hybridize in natural environment. In ecotoxicological studies, a reliable identification of *Dreissena* species is a key step to ensure the efficiency of the use of such freshwater sentinel species and their associated biomarkers to reflect exposure and toxic effects of xenobiotics. It is currently essential to deepen our knowledge on the levels of responses and sensitivity of these two species with a view to their use in biomonitoring projects.
