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Food deprivation and modulation of hemocyte activity in the zebra mussel (Dreissena polymorpha).

ABSTRACT Bivalve hemocyte responses are modulated by numerous biotic and abiotic environmental factors. This study investigates how nutritional stress can modulate two hemocyte functions classically studied in bivalves, i.e., phagocytic and oxidative activities. For this purpose, we exposed zebra mussels (Dreissena polymorpha) to three nutritional conditions, for 42 days: (1) one group was fed with 1 X 106 algal cells per mussel and per day, (2) a second group was fed with 10% of the previous food intake (1X 105 cells per mussel and per day), and (3) the third one was deprived of food. Hemocyte responses were assessed by flow cytometry every week for 42 days. Food deprivation was associated with a significant decrease in granulocyte size. Phagocytic activity increased in mussels exposed to the three diets, but it was more pronounced in mussels partially deprived of food (10% food intake). Mussels exposed to stressful nutritional conditions (10% and 0%) displayed significantly decreased oxidative activity from 14 days of exposure, whereas those fed on a normal diet displayed stable intracellular oxidative activity throughout the experiment. This study shows that nutritional conditions affect hemocyte morphometry and activity. Zebra mussel sensitivity to the nutritional conditions seemed low, even with total food deprivation for 42 days. It is necessary to estimate the physiological consequences of these food conditions, especially on the energetic status of this bivalve organism but also on its ability to resist to infection.

KEY WORDS: hemocyte, stress, Dreissena polymorpha, cytometry, phagocytosis


The zebra mussel Dreissena polymorpha (Pallas) is a freshwater Ponto-Caspian bivalve spreading throughout most of Europe because the end of the 18th century due to building new waterways to favor trade exchanges and tourism (Akopian et al. 2001). It lives fixed to a substratum in great densities, up to several hundred thousand per square meter. Therefore, it strongly affects ecosystems in novel areas by filtering water and removing large quantities of suspended matter, including phytoplankton, and forming numerous shelters and providing food for benthic invertebrates and molluscivorous fish (Reeders et al. 1989, Mackie 1991, Kobak 2010). It is counted among the most efficient ecosystem engineers because it modifies its own environment and also living conditions for other organisms (Karatayev et al. 2002). It also has a negative influence on various branches of economy by fouling and damaging hydrotechnical appliances such as water intakes, industrial cooling systems, ship hulls, fishing nets, etc. (O'Neill 1997, Khalanski 1997). It is one of the worst invasive species in the world, particularly because its invasion is still in progress in some regions (such as North America, Spain, Ireland, and the Czech Republic) (McMahon 1996).

The bio-ecological traits of the zebra mussel (e.g., abundance, wide distribution, filtering activity, and high xenobiotic bioaccumulation ability) make it an interesting sentinel species more and more often proposed as the counterpart of the blue mussel Mytilus edulis L., in mussel watch programs for freshwater environments (Mersch et al. 1992, Minier et al. 2006, Bourgeault et al. 2010). The zebra mussel has been frequently used for monitoring water quality in European lakes and rivers for many years (Bervoets et al. 2005, Zorita et al. 2006, Guerlet et al. 2007, Bacchetta & Mantecca 2009, Bourgeault et al. 2010). In this ecotoxicological context, numerous health status biochemical and physiological biomarkers have been developed for Dreissena polymorpha for environmental assessment (De Lafontaine et al. 2000, Minier et al. 2006, Contardo-Jara & Wiegand 2008, Palais et al. 2011, 2012).

In the physiological system of bivalve molluscs, hemocytes occupy a central position because they circulate within the open vascular system and across all epithelial boundaries (Cheng 1996). Although hemocytes differ according to molluscan species, two main categories are broadly described (Soares-daSilva et al. 2002): (1) granulocytes, with cytosolic inclusions and a low nucleo-cytoplasmic ratio, and (2) agranular cells called hyalinocytes (Bachere et al. 1991, Giamberini et al. 1996, Li 2008). Zebra mussels possess these two categories of hemocytes (Giamberini et ai. 1996). Hemocytes are not only very important for bivalve internal defense, but are also involved in wound healing, shell recalcification, excretion, and heavy metal sequestration (Cheng 1996, Ahearn et al. 2004). They are also involved in storage and distribution of nutritive material (Russo et al. 2001, Soares-da-Silva et al. 2002) and tissue formation (Van de Braak et al. 2002). When foreign particles (pathogens or not) enter into the body, they induce hemocyte migration by chemotactic processes, followed by phagocytosis (Pipe 1990). Molluscan hemocytes engulf and encapsulate particles. This process is associated with nonspecific lysis especially by oxygen-dependent activity corresponding to reactive oxygen species (ROS) production in the phagosome (Pipe 1992, Winston et al. 1996, Xu & Faisal 2010). Phagocytosis and oxidative activities are central to bivalve internal physiology. These activities provide quick and sensitive adaptive responses linked to the general health status.

Like other organisms, the zebra mussel may be exposed to environmental stress in natural freshwaters. In molluscan species, changes in the number of hemocytes and their relative abundance appear to be a response to environmental stress, particularly heat stress and chemicals (Renwrantz 1990, Perez & Fontanetti 2011). Changes in environmental factors, related to global climate changes, strongly influence mollusc hemocytic activities, e.g., phagocytosis and oxidative activities (Fisher 1988, Cajaraville & Pal 1995, Giamberini & Cajaraville 2005, Salo et al. 2005, Gagnaire et al. 2006, Matozzo & Marin 2011). Many environmental factors other than pollution (e.g., development stage, reproduction, parasitism, and seasonal condition) can influence these physiological responses and make it difficult to interpret results from an ecotoxicological point of view (Lemaire et al. 2006, Minguez et al. 2009, Morley 2010). Most of the studies performed up to now concern marine bivalves, whereas data about freshwater species are scarce. The zebra mussel seems to exhibit high ecological tolerance to abiotic and biotic stress compared with other molluscan species, but the adaptive capacities of this invasive bivalve species are poorly studied (Gu & Mitchell 2002, Guerlet et al. 2007). Some physiological features could explain the remarkable ability of zebra mussels to survive in numerous and different freshwater ecosystems (Molloy et al. 1997, Emblidge & Dybdahl 2006). Unfortunately, little is known about Dreissena polymorpha physiological resistance to environmental stressful conditions.

This study addresses for the first time the effects of intense nutritional stress on hemocyte activities in this freshwater bivalve, with special focus on ecotoxicology and ecology. It is of interest for ecotoxicologists to understand how physiological parameters of this potential sentinel species may be modulated by natural factors other than chemicals; in parallel, it is of interest for ecologists to have more information about the modulation of hemocyte physiological responses by nutritional conditions in an invasive freshwater bivalve species.


Mussel Collection

Mussels were sampled from a ship canal, at Commercy (Meuse, France, N48[degrees] 46'55"; E5[degrees] 35'07") at the end of February 2012.

As bivalve size may influence hemocyte morphological and functional characteristics (Mosca et al. 2011), the length of each individual was measured (18.54 [+ or -] 2.96 mm).

In laboratory, mussels were maintained in six tanks (100 mussels per tank) containing each 5 1 of aerated spring water (14 [+ or -] 1[degrees]C; 8.4 [+ or -] 0.4 mg [O.sub.2]/L; 432 [+ or -] 34 [micro]S/cm) (Aurele, Ardennes, France) with a 16 h:8 h light:dark photoperiod. After each sampling point, the water volume in each tank was adapted to the number of remaining mussels. Mussels were acclimatized in laboratory conditions for 5 wks before starting the experiments. Throughout the acclimation phase, mussels were fed, every other day, with two microalga species: Scenedesmus obliquus (Meyen) and Chlorella pyrenoidosa (Beij.) (500,000 microalgae of each species per mussel). A bialgal diet was preferred to a single algal diet because a bialgal diet can provide more energy required for bivalves' needs compared with a single algal diet (Langdon & Waldock 1981, Soudant et al. 1995, Utting & Millican 1997, Parrish et al. 1998, Brown & Stickle 2002, Delaporte et al. 2006).

Experimental Procedure

One third of the acclimated mussels was fed daily with 1 x 106 algal cells per mussel and per day, another third was fed with 10% of the previous food intake (1 X 105 cells per mussel and per day), and the last remaining third was deprived of food. The ratio of 1 X [10.sup.6] cells per mussel and per day corresponds to a mean nutritional supply for zebra mussel under laboratory conditions (Mersch et al. 1996, Kraak et al. 1997, Navarro et al. 2011). The three groups of mussels were submitted to their respective diets for 42 days. Biological analyses were performed after 0, 7, 14, 21, and 42 days of experimental procedure.

Hemolymph Collection and Structure of Hemocytes Populations

For each sampling time, hemolymph was withdrawn from the posterior adductor muscle sinus using a 0.5 mL syringe with a 0.33 X 12 mm needle.

Hemocyte biological analyses were performed on five pools of hemolymph samples (n = 5 for statistical analyses). Each hemolymph pool was made of five hemolymph volumes sampled from five distinct individuals. These samples were then centrifuged (400 X g, 10 min, 20[degrees]C) and cell pellets were resuspended in fresh phosphate-buffered saline (PBS, Dulbecco's phosphate 0.0095 M, Lonza, Basel, Switzerland) buffer. Cell suspensions were kept on ice to reduce hemocyte aggregation, and cell concentrations were evaluated using a Malassez hemocytometer.

Hemolymphatic cells were characterized by flow cytometry and light microscopy. For cytometry analyses, 1 X [10.sup.5] cells were kept in 250 [micro]L of PBS and analyses were performed with a FACScalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Size- and forward-scatter parameters were studied to assess cell granularity and size, respectively. For light microscopy analyses, hemolymph samples were cytocentrifuged (200 X g, 5 min) and hemocytes monolayers were stained with May Griinwald-Giemsa reagent.

Observations were conducted with a light microscope (1000X), and 1000 hemocytes per group were counted for each treatment. Maximal cell diameter was measured with the image processing software (Archimede 5.6.0., 2008, Microvision Instruments, Evry, France). The procedure was undertaken fast to limit cellular changes.

Hemocyte Cytometry Analyses

Potential effects of nutritional conditions on zebra mussel hemocyte functions were assessed from hemolymphatic samples. Cytometry analyses were carried out on granulocytes because they were the only cell population systematically visible on the dot plots of all the hemolymphatic samples (Fig. 1). Hemocyte activities were measured by flow cytometry and 10,000 events were counted for each hemolymph sample. Data were analyzed with WinMDI 2.9 software (2000).

Cell Viability

Percentages of dead cells were assessed using propidium iodide (Sigma-Aldrich, Saint-Louis, MO), which permeates only through the membrane of dead cells and then stains nucleic acids. Hemocytes (1 X 105 cells/250 [micro]L PBS) were incubated in the dark with propidium iodide at 0.1 mg/mL for 15 min. Dead cell fluorescence was measured using FL3 (red fluorescence) and the related percentages of dead cells were recorded. Percentages of cell viability were expressed as cell viability = 100--cell mortality.

Phagocytic Activity

Hemocyte suspension (1 X [10.sup.5] cells/250 [micro]L PBS) was incubated for 1 h with fluorescent latex microbeads (FluoSpheres carboxylate-modified microspheres, diameter 1.712 [micro]m, Life Technologies, Carlsbad, CA) in the dark at room temperature with a 100:1 bead:cell ratio. The fluorescence of phagocytic cells was determined using FL1 (green fluorescence). Positive phagocytic cells were defined as cells containing three or more fluorescent microspheres. Results were expressed in percentages of phagocytic cells.

The mean numbers of ingested beads per cell (granulocytes) were calculated by dividing the mean fluorescence units (FU) of cells that had ingested three beads or more (positive phagocytic cells) by the mean fluorescence of cells that had ingested only one bead.

Intracellular Oxidative Activity

Oxidation of the probe dichlorofluorescein diacetate (DCFHDA, Sigma-Aldrich) was used to detect basal intracellular ROS formation in hemocytes (Brousseau et al. 1998). Cell suspensions (1 X 105 cells/250 pi PBS) were incubated for 30 min with 5 pM DCFH-DA in the dark, at room temperature. During incubation, the probe diffuses into the cell where nonspecific esterases cleave off the acetate moiety and release the intact substrate. If ROS, particularly hydrogen peroxide, are produced inside the cells, they cause the nonfluorescent probe to fluorescent by oxidizing it. The resulting fluorescence is proportional to the intensity of the oxidative activity. This biomarker have been measured using FL1 (green fluorescence), and was expressed in FU.

Statistical Analyses

Data obtained for each nutritional treatment and for each exposure length were obtained from n = 5 observations for immune function measurements and from n = 50 for hemocyte morphometric analyses. Prior to statistical analyses, data distribution was checked for normality with Shapiro-Wilk's test, and variance homogeneity with Bartlett's test. When variances were heterogeneous, Kruskal-Wallis and

Dunn tests were performed and were associated with a Bonferroni correction. When variances were homogeneous, two-way analysis of variance and Tukey's test were performed. Statistical tests were performed using Excel Stats software (2012). Differences were significant at P [less than or equal to] 0.05. Results were graphically treated in box plots (median, 25th and 75th percentiles).


The low mortality rate (<5%) we observed throughout the experiment did not vary with the different exposure conditions. Cell suspensions were

separated into one or two dot clouds according to cell size and cell complexity criteria (Fig. 1). As granulocyte-like cells were always visible in all hemolymph samples, the 10,000 events recorded by flow cytometry were systematically gated on the granulocyte area.

Variations in Hemocyte Morphometry

A significant decrease in granulocyte size was evidenced by flow cytometry in the hemolymph from zebra mussels submitted to the strongest nutritional stress (0% food intake) for 7 days (421.86 FU median value for 0% food intake versus 474.63 FU median value for 100% food intake, P = 3.70 X [10.sup.-3] Table 1, Fig. 2). From 7 days of experiment onward, the median value of hemocyte maximum diameter was 19.61 [micro]m for cells from mussels fed with the maximal level of diet (100% food intake) down to 15.48 [micro]m for cells from mussels under total fasting conditions (0% food intake) (P < 1.00 X [10.sup.-4]; Table 1). The same observation was made at the other sampling times (Table 1). This decrease in hemocyte size was time dependent. The lowest hemocyte sizes were recorded from unfed mussels (0% food intake) after 21 and 42 days (Table 1). At the end of the experiment, hemocyte size also decreased among mussels fed with the 10% diet (Table 1). Despite this smaller granulocyte size, neither hemocyte complexity nor concentrations changed with nutritional status.

Variations in Hemocyte Cytometric Parameters

Cell Viability

Cell viability ranged from 82.88% to 93.60% for hemocytes from 100% fed mussels, and from 84.84% to 93.2% for hemocytes from 100% food-deprived mussels (Fig. 3). Cell viability increased significantly during the exposure period for mussels submitted to the 10% diet (Fig. 3) (P = 4.46 X [10.sub.-2]). Overall, cell viability was not reduced by the different diet conditions.


In the control group (100% food intake), phagocytic activity values ranged from 0.84% to 10.51% (median value: 3.65%) (Fig. 4). Phagocytic activity significantly increased throughout the experiment in all mussels submitted to the different diet conditions (median values between TO and T42: 1.97%-5.19% with 100% food intake, P = 2.78 X [10.sup.-2] ; 1.97%4.38% with 10% food intake, P = 6.40 X [10.sup.-3]; 1.97%-4.00% with 0% food intake, P = 1.17 X [10.sup.2]; Fig. 4). After 14 days of exposure, 100%-fed mussels displayed significantly lower phagocytic activity than 10%-fed mussels (median values: 2.95% and 6.57% respectively, P = 1.33 X [10.sub.-2]). Regarding the increase in phagocytic activity over the experiment, mussels deprived of food for 21 days exhibited a significantly lower phagocytic activity compared to fed mussels (median values: 2.18%, 3.22%, and 4.17% for 0%, 100%, and 10% food intakes, respectively, P = 1.28 X [10.sup.-3]; Fig. 4).

These increases in phagocytic activity characterized 100%-fed and 10%-fed mussels, in parallel, the mean number of ingested beads per hemocyte was stable throughout the experiment (P = 8.53 X [10.sup.-1] with 100% food intake, P = 2.43 X [10.sup.-1] with 10% food intake; Fig. 5). The hemocytes of 0%-fed mussels ingested higher numbers beads after 21 days of exposure as compared with T0 (median values: 4.75 at TO and 5.62 after 21 days, P = 3.05 X [10.sup.-2]; Fig. 5).

Intracellular Oxidative Activity

In mussels fed 10 and 0% of normal food intake, basal oxidative activity significantly decreased between 7 and 42 days after the experiment started (median values: 207.85 down to 91.24 with 10% food intake, P= 1.16 X [10.sup.-2]; 185.64 down to 65.09 with 0% food intake, P = 7.10 X [10.sup.-3]; Fig. 6). After 14 and 21 days, stressed mussels displayed a lower oxidative activity compared with the control group (median values at T21: 111.22 for 10% food intake; 217.05 and 135.40 for 100% and 0% food intake, respectively, P = 8.89 X [10.sub.-3]; Fig, 6). Although mussels deprived of food exhibited the lowest oxidative value after 42 days (median value: 65.09), it was not statistically significant when compared with the control group (median value: 98.99 for 100% food intake, P = 1.51 X [10.sup.-1]; Fig. 6). This result can be related to the high variability of data observed in fed mussels at the end of the 42 days of exposure (Fig. 6).


This study tested the impact of a nutritional stress on zebra mussel hemocyte parameters to contribute to determining how food deprivation modulates their physiological hemocytic responses.

Although there exist a wide range of opinions about numbers and types of hemocyte subpopulations among bivalves, all agree with the presence of a granulocyte population (Auffret & Oubella 1995, Ashton-Alcox & Ford 1998, Hine 1999, Soares-da-Silva et al. 2002). Granulocytes are easily identifiable among phagocytic cells (Auffret & Oubella 1995, Cheng 1996). Granulocytic hemocytes display the greatest phagocytic activity compared with other hemocyte types (Carballal et al. 1997, Mosca et al. 2011). That is why we focused on granulocytes.

We observed a significant decrease in granulocyte size in zebra mussels exposed to the two low-food conditions. Delaporte et al. (2006) did not record any change in hemocyte size in unfed oysters (Crassostrea gigas, Thunberg), but Hegaret et al. (2004) evidenced a decrease in size and complexity of small hyalinocytes and granulocytes in starved oysters (Crassostrea virginica, Gmelin). Other studies show changes in hemocyte morphology in bivalve species, but under other stress origins. Hegaret and Wikfors (2005) evidenced a decrease of oyster hemocyte size and complexity with time of exposure to Prorocentrum minimum (Pavillard), a Dinophyceae alga. Galimany et al. (2008) tested the effect of a toxic algal treatment on blue mussel hemocytes: hemocyte size and complexity decreased, probably in relation to the cell apoptotic response. In this study, even if cell viability did not vary among nutritional conditions, induction of the hemocyte apoptotic response related to a decrease in cell size may explain the results. In an ecotoxicological context, the size of hemocyte from a gastropod, Haliotis tuberculata (L.), decreased during in vitro zinc exposure (Mottin et al. 2010). This decrease in size can be related to a red uction of cell cytoplasmic extensions. Maity et al. (2012) applied a nutritional stress on Diporeia, which resulted in lower metabolism of many essential amino acids, some of which are important for cell growth. A lack of amino acids can cause reducer cell size in Escherichia coli (Grossman et al. 1982). In this study, the smaller size of Dreissena polymorpha hemocytes may have been caused by a deficiency in essential amino acids related to fasting conditions.

Hemocyte phagocytosis varied from 0.84% to 10.51% in this study. Phagocytic activity can vary greatly, depending on species, experimental conditions (field, laboratory, etc.) and the methodological conditions used to analyze this immune parameter (Delaporte et al. 2006, Lemaire et al. 2006). Phagocytic activity increased in mussels exposed to the three diets, but it was highest in partially food-deprived mussels (10% food intake). Similarly, phagocytosis was higher in oysters (Crassostrea virginica) fed with a ratio of 12% of oyster dry weight in algal dry weight per day compared with oysters fed with a ratio of 4% (Delaporte et al. 2006). The significant increase in phagocytosis in mussels exposed to 10% food intake could be due to an adaptive physiological process aimed at augmenting the ability of mussels to ingest more food particles. For instance, Asian clams Corhicula fluminea (Muller) displayed such an adaptive response intensifying their ventilatory activity in relation to lower food availability (Fournier et al. 2005). Hemocytes capable of phagocytosis can affect nutrition by producing and storing glycogen (Cheng 1975, 1976, Donaghy et al. 2009). This is true for several bivalve species (Cajaraville & Pal 1995, Harris-Young et al. 1995, Lopez et al. 1997, Donaghy et al. 2009) and in a mollusc (Travers et al. 2008a). Hemocyte glycogen content rises in bivalves after food intake, but it is low without food supply (Donaghy et al. 2009). Different glycogen contents according to mussel nutritional status could be responsible for the smaller hemocyte size we observed. The high phagocytic activity could be related to higher granulocyte numbers in hemolymph samples (Donaghy et al. 2009). In this study, the increase in phagocytosis was significant in mussels exposed to the 10% food intake diet, yet it was true for all three experimental groups. This observation can reveal a natural evolution of this cellular activity in zebra mussels over the time course of this experiment (February-March). Nevertheless, in marine bivalves, the phagocytic index grows lower and lower from autumn (September) to the spawning period (June-July) (Duchemin et al. 2007, Travers et al. 2008b). The increase in phagocytosis in the three groups of mussels could be due to unknown stressful parameters in these experimental conditions (Malagoli et al. 2007). In another experiment, oysters (Crassostrea virginica), starved for 6 wks had a less robust immune response associated with low phagocytosis compared with fed oysters (Hegaret et al. 2004). In the same line, we measured the lowest phagocytic activity values in mussels unfed for 21 days.

In the literature, upward or downward variation in phagocytic responses in unfed bivalves could be due to numerous different factors (diet composition, seasonal variations, parasitological status of organisms, etc.) (Ashton-Alcox & Ford 1998). For instance, the immune profile of oysters (Crassostrea virginica) depends on the algal species that compose their diet, and some algae can provide "better" immune profiles than others. This can be related to the fatty acid composition of algal species (Hegaret et al. 2004). As hemocyte parameters (mobility, abundance, and total area) can be seasonally modulated, it would be interesting to see how these possible seasonal variations in hemocyte activities relate to the reproductive cycle of zebra mussels (McCormick-Ray & Howard 1991, Nalepa et al. 1993, Svardh & Johannesson 2002, Delaporte et al. 2006, Flye-Sainte-Marie et al. 2009). Similarly, as the parasitic status of organisms can influence hemocyte activities, it should be considered to improve the understanding of the hemocyte responses evidenced by this study (Oubella et al. 1994, Hooper et al. 2007, Lambert et al. 2007).

Granulocyte oxidative activity significantly decreased in oysters (Crassostrea virginica and Crassostrea gigas) exposed to nutritional stress (Hegaret et al. 2004, Delaporte et al. 2006). The results confirm this finding in zebra mussels exposed to nutritional stress (10% and 0% food intake) after the 14th day of exposure, whereas mussels fed with 100% food intake had a stable intracellular oxidative activity throughout the experiment. Reactive oxygen species production by bivalves can be influenced by numerous factors, such as nutritional conditions (Lambert et al. 2007). Food availability is one of the most probable explanations for the differences observed in oyster ROS production levels (Lambert et al. 2007). The high variability in basal ROS production noted in the 100%-fed group over 42 days may be because of the differences in access to food among individuals under the experimental conditions. The low basal oxidative activity measured in hemocytes from unfed mussels can be logically associated with low food availability. This condition caused a lack of many essential metals and nutrients normally provided by food, which modulate and stimulate the cellular oxidative metabolism (Hegaret et al. 2004, Lambert et al. 2007). Starvation impacts amino acid metabolism in invertebrates (Maity et al. 2012).

Cell viability was not affected by total starvation or by normal food intake, but it was enhanced in mussels exposed to 10% food intake throughout the experiment (P = 4.46 X 10~2). As cell viability increased over the experiment, mussels were not under stressful conditions. Hegaret et al. (2004) proposed to define the "unhealthy" immune response profile of unfed oysters as displaying low phagocytosis and a high respiratory burst. In their study, nutritional stress was associated with significantly higher oxidative response and hemocyte mortality, and lower phagocytosis. In this context, it can be considered that unfed zebra mussels, under the experimental conditions, were relatively "healthy" and unaffected by nutritional conditions, as revealed by greater phagocytosis and lower cellular oxidative activity. Hemocyte functional activities are probably related to the overall metabolic activity of mussels. The mussels were collected in winter, a reproductively quiescent period with high glycogen contents. This may have buffered the short-term effects of nutritional variations on metabolic activity and contributed to a relatively low influence of food deprivation on hemocyte parameters (Palais et al. 2011). Laboratory acclimation can modulate basal levels of hemocyte responses (Hurtado et al. 2011). The recorded responses may have been mitigated by acclimation and could therefore lead to underestimated field results.

Nonetheless, the increase in phagocytosis and the decrease in oxidative activity were significantly reduced in 0%-fed mussels, suggesting an ecophysiological effect of food deprivation on starved mussels.


Nutritional stress is expected to increase susceptibility to diseases and parasites by compromising defense mechanisms. Yet, zebra mussel susceptibility to the nutritional conditions of this study seemed low, even with a total food deprivation for 42 days.

It will be necessary to estimate the physiological consequences of low food availability, particularly on the energetic status of this bivalve, but also on its ability to tolerate or resist to infection.


This work was supported by the French CNRS-INSU (Programme EC2CO, IPAD project) and the Programme Interdisciplinaire de Recherche sur l'Environnement de la Seine (PIREN-Seine). We are grateful to Annie Buchwalter for improving English language.


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UMR-102 (INERIS-Universite Reims Champagne-Ardenne, Universite du Havre) SEBIO Stress environnementaux et Biosurveillance des milieux aquatiques, Universite de Reims Champagne-Ardenne, Campus Moulin de la Housse, BP 1039 Reims Cedex 2, France

* Corresponding author. E-mail:

DOI: 10.2983/035.034.0226

Granulocyte size measured by flow cytometry (n = 5)
and by microscopy (n = 50) according to nutritional stress.

Exposure conditions

Exposure                      Food
time                       intake (%)

T0                             100
T7                             100
T14                            100
T21                            100
T42                            100

Flow cytometry                                 Microscopy
FU                                          Maximum diameter
Mean [+ or -] SEM             size       Mean [+ or -] SEM

487.04 [+ or -] 12.87          --        19.41 [+ or -] 0.52
474.63 [+ or -] 11.00          --        20.29 [+ or -] 0.49
462.11 [+ or -] 8.20           --        19.77 [+ or -] 0.45
421.86 [+ or -] 5.82 **        --        16.25 [+ or -] 0.33 ***
473.92 [+ or -] 9.22           --        20.87 [+ or -] 0.40
460.12 [+ or -] 8.48           --        19.87 [+ or -] 0.42
380.80 [+ or -] 10.72 **       --        16.60 [+ or -] 0.34 ***
446.75 [+ or -] 13.30          --        20.05 [+ or -] 0.41
431.09 [+ or -] 4.36           --        20.83 [+ or -] 0.32
391.94 [+ or -] 2.43 **    P = 1.27 X    17.10 [+ or -] 0.27 ***
435.75 [+ or -] 11.19          --        20.93 [+ or -] 0.33
297.85 [+ or -] 4.75 **    P = 1.30 X    19.47 [+ or -] 0.29 **
314.21 [+ or -] 4.71       P < 1.00 X    17.44 [+ or -] 0.29 ***

Values are means [+ or -] SD (SEM). P values correspond to
significantly different groups between sampling times and TO.

Asterisks correspond to significant differences between two levels of
food intake for the same sampling time (** P value [less than or
equal to] 0.01;  *** P value [less than or equal to] 0.001).
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Date:Aug 1, 2015
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