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    J Comp Physiol B DOI 10.1007/s00360-017-1089-2

    ORIGINAL PAPER

    Temperature increases, hypoxia, and changes in food availability affect immunological biomarkers in the marine mussel Mytilus galloprovincialis

    M. G. Parisi1 · M. Mauro1 · G. Sarà2 · M. Cammarata1 

    Received: 2 November 2016 / Revised: 1 March 2017 / Accepted: 15 March 2017 © Springer-Verlag Berlin Heidelberg 2017

    the stability of the lysosomal membrane was altered under conditions of thermal stress and food change, under nor-moxic and anoxic conditions. Overall, environmental stress factors affected immune biomarkers of Mediterranean mus-sels, and the level of food acted as a buffer, increasing the thermal resistance of the specimens.

    Keywords Environmental multiple stressors · Mytilus galloprovincialis · Immunobiology · Biomarkers · Enzymes · Lysosomal membrane

    Introduction

    Effectors of internal defense systems allow the maintenance of the biological integrity of animals, from single-celled organisms to mammals, during invasion by pathogens or parasites, injury, and self/non-self-recognition (Ballarin and Cammarata 2016). Disease outbreaks among marine organisms, and impairment of host immunity may be caused either by introduction of new pathogens or a change in the environment (Harvell et  al. 2001; Lafferty et  al. 2004; McCallum et al. 2004). Environmental stressors can alter organism functions, and elicit coordinated physiologi-cal responses in an attempt to return to normal homeosta-sis. Maintaining homeostasis requires proper functioning of all physiological processes within the body, including the molecular and cellular immune system networks, which are influenced by environmental conditions (water temperature, salinity and pH) (Mydlarz et  al. 2006), pathogen expo-sure (bacteria, viruses, and parasites) (Belkaid and Hand 2014), genetic makeup (Cantet et al. 2012), chemical pol-lutants (pesticides, heavy metals and hydrocarbon) (Luma-ret et  al. 2012; Spada et  al. 2013; Guardiola et  al. 2015),

    Abstract Temperature increases, hypoxia, and changes in food availability are predicted to occur in the future. There is growing concern for the health status of wild and farmed organisms, since environmental stressors alter organism functions, and elicit coordinated physiological responses for homeostasis. Mussels are good bioindica-tors of environmental conditions. Their ability to maintain unaltered immunosurveillance under adverse environmen-tal conditions may enhance their survival capability. Few studies are currently concerned with the relationships and feedback among multiple stressors. Here, food concentra-tion, temperature, and oxygenation treatments were evalu-ated for their effects on immune enzymatic parameters of Mytilus galloprovincialis detected in the digestive gland and the lysosomal viability by neutral red uptake. Mus-sels were exposed to three temperatures (12, 20, and 28 °C) under normoxic (8  mg  O2l−1) and anoxic conditions and specimens were fed with six food concentrations, ranging 0.2–5  g chlorophyll l−1. Temperature increases affected esterase and alkaline phophatase enzyme functionality, and addition of food buffered detrimental effects generated by harsh conditions, such as those provided by low oxygen concentrations. Kinetics of the phenoloxidase was nega-tively correlated with increasing temperature. In this case, food had a buffering effect that counteracted the limiting temperature only under normoxic conditions. In addition,

    Communicated by I.D. Hume.

    * M. G. [email protected]

    1 Marine Immunobiology laboratory, University of Palermo, CONISMA, Via Archirafi 18, 90128 Palermo, Italy

    2 Dipartimento di Scienze della Terra e del Mare, University of Palermo, Viale delle Scienze Ed. 16, 90128 Palermo, Italy

    http://crossmark.crossref.org/dialog/?doi=10.1007/s00360-017-1089-2&domain=pdf

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    physiological conditions (Zuk and Stoehr 2002) (reproduc-tion and starvation), and food availability (Houston et  al. 2007).

    Bivalves are abundant in marine habitats and are largely accessible in the field. Given that the correlation between the immune response and disease outbreaks in mollusc farming, the physical stressor impact on immunologi-cal response may be paralleled by associated changes in bivalve disease status. Here, our target species was the Mediterranean mussel, Mytilus galloprovincialis, which is among the most important commercial cultivated organ-isms in the Mediterranean and is one of the most com-mercialized in global seafood markets. Together with other members of the same genus, this species has become a model study organism due to its economic value in shellfish farming (Gueguen et al. 2011) and its role as a bioindicator (Ostapczuk et al. 1997; De Donno et al. 2006; Bankhment et  al. 2009). As for other marine species, the changes in abiotic factors strongly influence mollusc immune param-eters, but the interactive effects of different types of stress-ors (e.g., increasing temperature and hypoxia) have rarely been investigated in aquaculture species, even though this could impact on socio-economic factors of mollusk farm-ing. Most studies, to date, have investigated the effects of single stressors on biological and ecological responses; however, this can be misleading and generates unrealistic conclusions (Crain et  al. 2008; Gunderson et  al. 2016). Nonetheless, the majority of research currently proceeds on a single issue basis, with scant consideration for the rela-tionships and feedback between multiple stressors. On the other hand, the relationship between multiple environmen-tal conditions and immunomodulation mechanisms plays a role in the susceptibility of each species to infections (Mydlarzet al. 2006; Buchmann 2014). Multiple stressors can interact, producing three main effects: additive, inde-pendent interaction; antagonistic, interactive interaction with performance decrease; and synergistic, interactive interaction with performance increase (Gunderson et  al. 2016). Multiple stressors, co-occurring in time and space, expose marine organisms and ecosystems to increasingly simultaneous impact sensu Matzelle et  al. (2015). The recognition of the differential manifestations of any indi-vidual stressor is fundamental when considering the scale at which they can be managed or mitigated (Murray et al. 2015). In general, global stressors (e.g., acidification) tend to change slowly over long periods of time, although their intensity and effects can be contingent on local conditions. Local stressors, instead, have rapid changes over shorter spatial and temporal scales and interactions among stress-ors transversally generates unexpected effects from local to global scale (Helmuth et al. 2014). In this context, it is cru-cial to understand if and how living organisms cope with these changing stress conditions, considering that defense

    capabilities differ among species. Few studies to date have investigated the effects of multiples stressors on biological traits, such as enzymes and cellular mechanisms involved in mussel innate immune defense. Nevertheless, it is easy to predict how the action of a global driver (e.g., increas-ing temperature due to climate change increasing the like-lihood of heat waves, etc.) may synergistically generate a direct effect on organisms through the impairment of these traits, sensu Sarà et al. (2014). Thermal stress in combina-tion with a second stressor (e.g., hypoxia or acidification) may worsen organism conditions, compromising immunity and facilitating new disease outbreaks in marine animals.

    Since bivalve hemocytes are both osmo- and thermo-conformers, functional cell-mediated immune responses are affected easily by exogenous environmental factors (Pail-lard et al. 2004; Gagnaire et al. 2006). Biomarkers enable the detection of deviation from physiological homeostasis. In mussels, the stability of the lysosomal membrane (LMS) represents one of the simplest, most sensitive, and low-cost biomarkers for evaluating the physiological status of the organisms subjected to stress. Moreover, the enzymes produced in the digestive gland are well known to assist, modulate, and accelerate immunological processes in hemocytes. The digestive gland is a source of innate immu-nity molecules (Smith 2001), and is involved in pathogen clearance, antigen processing and infection-induced meta-bolic changes (Luchtel et al. 1997; Alday-Sanz et al. 2002).

    In addition, the hydrolase enzymes, normally involved in detoxification, inflammatory and digestive processes, and the phenoloxidase (PO) cascade, are recognized as immune parameters potentially affected by environmental factors (Hellio et al. 2006).

    Here, we investigated biomarkers belonging to the innate immunity effectors, i.e., phenoloxidase (PO) and hydro-lase (esterases and phosphatases) kinetics enzymes in the digestive gland of Mytilus galloprovincialis and changes in lysosomal membrane permeability, under multiple stressor effects of temperature increases, anoxia, and food concen-tration availability.

    Materials and Methods

    Animals and experimental plan

    Specimens of M. galloprovincialis were collected from Lake Pantano Piccolo (38°15′53″N 15°38′33″E), and were transported in jute bags soaked with water from the lake, in thermal containers to maintain a wet environment and a stable temperature. In the laboratory, animals were accli-mated in a flow-through oxygenated system (by aeration; dissolved oxygen [DO] was about 8 mg l−1) at 28‰ salin-ity and at 12 ± 2 °C. Mussels of size class 4.5–6.5 cm were

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    separated, (corresponding to commercial size) and, after being cleaned by epiphytic, they were placed in 36 grids with 22 specimens in each. Assuming a possible 10% natu-ral mortality, six extra grids were provided to replace any dead or unresponsive animals. Each grid was then placed in a large glass-resin bath, containing oxygenated and con-stantly filtered sea water at 12 °C. Animals were acclimated for 1 month and were fed regularly with a monoalgal cul-ture of Isochrysis galbana. The specimens were subjected to multiple stressors by exposure to three different tem-peratures (12, 20, and 28 °C), six different concentrations of food (I. galbana = 0.2, 0.9, 1.8, 2.5, 3, 5, 5 g CHLal−1) at each temperature, both in anoxic (0 mg O2l−1) and nor-moxic (~8  mg  O2l−1) conditions. The whole experiment lasted 6 weeks (2 weeks for each temperature).

    For each treatment, the experimental procedure was carried out as follows: inside the glass-resin bath, two 17 l tanks for each concentration of food (a total of six tanks) were inserted. For each concentration of food, the two tanks were maintained at different oxygen concentrations: nor-moxic (~8  mg  O2l−1), which acted as control, and anoxic (0 mg O2l−1), obtained by bubbling nitrogen (N2) and kept constantly covered by a transparent film to prevent gaseous exchange with the environment (Fig. 1).

    Hemolymph and digestive gland collection

    Mytilus galloprovincialis specimens were removed from aquaria, and the hemolymph (0.8 ml for mussel) was col-lected from the posterior adductor muscle with a 1  ml disposable syringe containing 0.2  ml of anticoagulant modified Alsever’s solution (Na3C6H5O7 27  mM, d-glu-cose 115  mM, NaCl 336  mM, EDTA 9  mM). Hemocyte

    counts were carried out using the Neubauer chamber and, for each sample, the number of cells was adjusted to 1 × 106 cells ml−1. The digestive glands were removed from each specimen using a scalpel and stored at −80 °C until the preparation of protein extracts. Tissues were homog-enized using a tissue homogenator (Bioneer Corporation, Daejeon, Korea) in one volume of PBS (NaCl 137  mM, KH2PO4 1.76 mM, Na2HPO4 8.1 mM, KCl 2.7 mM, CaCl2 1.19 mM, and MgCl2 1.05 mM). The homogenate was cen-trifuged at 9000×g for 25 min at 4 °C, and the supernatant was stored at −80 °C. The proteins concentration was deter-mined according to the Bradford (1979) method using the Coomassie blue assay.

    A grand total of 154 animals were sampled for collect the hemolymph and the digestive gland (72 maintained in normoxia and 72 in anoxia).

    Neutral red (NR) uptake assay

    Hemolymph was withdrawn into an equal volume of antico-agulant buffer, and 100 µl aliquots of each sample were dis-pensed into three replicate wells of a flat-bottom microplate (Nunc). The aliquots (10 µl) of 0.33% neutral red solution (C15H17ClN4, Sigma-Aldrich) in PBS containing 2% NaCl were added to each well, and the plate was incubated for 1 h at 10 °C. After centrifugation at 252×g for 5 min, the supernatant was removed and the microplates were washed twice in PBS buffer. Subsequently, a solution of 1% acetic acid in 50% ethanol was added to the wells and incubated for 15 min at 20 °C in the dark. The absorbance (Abs) was then measured at 550  nm (RT-2100C Microplate Reader Rayto). The uptake of neutral red was expressed as ODT mg−1  ml−1 protein, in reference to the hemocyte contents (Repetto et al. 2008).

    Phenoloxidase assay

    Gland phenoloxidase (PO) activity was measured spectro-photometrically by recording the product obtained from the reaction between DOPA-quinone and 3-methyl-2-benzothi-azolinone hydrazone hydrochloride (MBTH). In the phe-noloxidase assay, 50  µl of digestive gland protein extract was added per well to a 96-well flat-bottom plate with 50 µl of zymosan (1  mg  ml−1, dissolved in PBS) and 50  µl of MBTH (0.0019 g L-DOPA, 0.0026 g MBTH dissolved in 400 µl of CH3CH2OH, and 1.6 ml of DW). Three controls were performed with (1) 50 µl of PBS, 50 µl of zymosan, and 50 µl of MBTH, (2) 100 µl of PBS and 50 µlof MBTH, and (3) 150  µl of PBS. After 60  min of incubation, the reaction product was detected at optical density (OD) of 540  nm (OD540). The PO activity was expressed as units (U) per min where 1 U = 0.001 ΔA540 min−1 mg−1 protein.

    Fig. 1 Multiple stressor experiment scheme. Mytilus galloprovin-cialis specimens were acclimated for 1 month and were fed regularly with a monoalgal culture of Isochrysis galbana. After acclimatiza-tion, the animals were subjected to multiple stressors: exposure to anoxic (0 mg O2l−1) and normoxic conditions (~8 mg O2l1), three dif-ferent temperatures (12, 20, and 28 °C), and six different concentra-tions of food (I. galbana = 0.2, 0.9, 1.8, 2.5, 3.5, 5 g CHLal−1). The whole experiment setup lasted 6  weeks (2  weeks for each tempera-ture)

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    Alkaline phosphatase activity

    Alkaline phosphatase activity was measured by incubating equal volumes (50 µl) of digestive gland extract and 4 mM p-nitrophenyl liquid phosphate (Sigma) in 100 mM ammo-nium bicarbonate buffer containing 1 mM MgCl2 (pH 7.8, 30 °C) in 96-microwell plates, as described by Ross et  al. (2000). The OD was continuously measured at 5-min inter-vals over 1 h at 405 nm in a plate reader. The initial rate of the reaction was used to calculate the activity. The values of Abs thus obtained were processed using Sigma Plot to obtain the value for Abs/min. Enzyme activities (U ml−1) are expressed as: {(Abs/min) × (1000/Eb) × (Vf/Vi)}. Eb was a constant value of 18.45. One unit of activity was defined as the amount of enzyme required to release 1 µmol of p-nitrophenol product in 1 min.

    Esterase activity

    Esterase activity was determined according to the method by Ross et  al. (2000). Equal volumes of digestive gland and 0.4 mM p-nitrophenyl-myristate substrate in 100 mM ammonium bicarbonate buffer containing 0.5% Triton X-100 (pH 7.8, 30 °C) were incubated at room tempera-ture. The OD was measured at 5-min intervals over 1  h at 405  nm in a plate reader. The initial rate of the reac-tion was used to calculate the activity. The values of Abs thus obtained were processed using Sigma Plot to obtain the value for Abs/min. Enzyme activities (U ml-1) are expressed as: {(Abs/min)×(1000/Eb)×(Vf/Vi)}. Eb for esterase was 16.4. One unit of activity was defined as the amount of enzyme required to release 1  µmol of p-nitro-phenol product in 1 min.

    Statistical analysis

    A multifactorial ANOVA was applied to different factors. Variables were PO, alkaline phosphatase, esterase activi-ties, and neutral red uptake, while the factors were the availability of oxygen (α), temperature (β), and diet (γ). Differences between means were considered significant for *p < 0.05, **p < 0.01 and ***p < 0.001. To assess mul-tiple comparisons, a parametric one-way analysis of vari-ance (ANOVA) was performed on the data, with a post hoc Tukey test. The analyses were carried out using STATIS-TICA 10.0 (StatSoft Inc. USA).

    Results

    This experiment (Fig. 1) allowed the evaluation of enzy-matic activity involved in immunity responses and the

    pinocytosis activity by lysosomes in the samples main-tained under multiple factors of oxygenation (normoxia and anoxia), temperature (12, 20, and 28 °C), and food (ranging 0.2–5 μg1−1 of chlorophyll).

    Overall, the enzymatic kinetic of esterase decreased from 12 to 20 °C until reaching the lowest values at 28 °C, both in anoxic and normoxic conditions (Fig.  2). As shown in Table 1, varying the food concentrations (I. gal-bana monoalgal culture) significantly influenced esterase production in all experimental temperatures (p < 0.001, ANOVA). Thus, esterase units detected in normoxic and anoxic organisms maintained at 20 and 28  °C increased in the highest food concentration (5.5 g CHLal−1). Con-versely, the oxygenation factor significantly regulated the expression of esterase at 28 °C (p < 0.001, ANOVA).

    The alkaline phosphatase enzyme showed a high kinetic at 12 °C compared to that detected at 20 and 28 °C (Fig.  3). Food concentration significantly influenced the phosphatase production, except in organisms maintained at 28 °C (p > 0.05), while oxygenation affected phos-phatase enzyme production at 12 and 28 °C (p < 0.001, ANOVA) (Table 1).

    The phenoloxidase enzyme (PO) showed a slight decrease in expression from 12 to 20 °C, except at 5.5 g CHLal−1 of food at 20 °C, and for 1.8 g CHLal−1 at 28 °C (Fig. 4).

    Statistical analysis showed that PO was significantly affected by food concentration at 12 °C, while the oxy-genation condition significantly modulated the enzyme production at 20 and 28 °C (Table 1, p < 0.001, ANOVA).

    The uptake of neutral red (NR) was most elevated at 12 °C (Fig.  5), where it was significantly regulated by food (Table 1, p < 0.001, ANOVA). At 20 and 28 °C, the lysosomal membrane stability marker was decreased in all concentrations of food. Only in specimens maintained in normoxia at 20 and 28 °C, and fed, respectively, with 5.5 and 1  g  CHLal−1 of food, the enzyme maintained a slightly higher level. Moreover, a significant influence of oxygenation was detected (Table  1, p < 0.001, ANOVA) at 20 and 28 °C.

    Effects of the three factors [availability of oxygen (α), temperature (β), and diet (γ)] and interactions of first, second, and third orders were calculated (Fig.  6). Thus, a multifactorial analysis of variance revealed a statisti-cally significant effect of temperature and food on ester-ase, alkaline phosphatase, and phenoloxidase enzymatic responses (p < 0.005, Multifactorial ANOVA) in interac-tions of first, second, and third orders. Oxygen availabil-ity did not modulate the esterase and phosphatase enzyme production but did modulate the phenoloxidase response (p < 0.005, Multifactorial ANOVA).

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    Discussion

    This work describes the first step in exploring the compli-cated interactive effects of multiple environmental changes in marine intertidal systems that are expected to occur due

    to climate change. These changes may occur as tempera-ture shifts, oxygenation changes, and differences in food availability.

    Research suggests that as ocean water temperatures increase due to climate change, marine productivity may decrease (O’Reilly et  al. 2003). Thus, depending on the timing of phytoplankton blooms, it is possible for marine bivalves to be exposed to reduced food conditions and elevated temperatures similar to those in this study, espe-cially in the high intertidal zone. Moreover, it is necessary to investigate species tolerant to a range of thermal regimes with varying food availabilities.

    Changes in water temperature are thought to be the cause of observed biogeographic shifts in many taxa (Thomas and Lennon 1999; Parmesan and Yole 2003) including mussels. Understanding the effects of temperature on the Mytilus genus can be complex, especially for intertidal specimens exposed to the aerial environment at low tide.

    Moreover, it is possible that variation in a single envi-ronmental parameter can induce a different cell response in contrast to those obtained under a combination of environ-mental factors.

    Bivalve humoral and cellular immune-related parameters are known to be sensitive to the disturbance effects of envi-ronmental factors, such as water temperature, salinity, dis-solved oxygen, nutrients, and parasites, that exhibit short- and long-term fluctuations (Auffret et al. 2004; Giron-Perez 2010). The crossed treatments applied to M. galloprovin-cialis specimens (three temperatures, oxygen availability,

    Fig. 2 Kinetics of esterase enzyme in M. galloprovincialis digestive gland, expressed as U mg−1 protein, at three different temperatures (12, 20, and 28 °C) during the administration of six different food concentrations (I. galbana = 0.2, 0.9, 1.8, 2.5, 3.5, 5  μg  CHLal−1) under anoxic (0 mg O2l−1) and normoxic conditions (~8 mg O2l−1). Data represent the mean ± SE (n = 4 replicates). Significant differ-ences were detected by Tukey post hoc test: *p < 0.05, **p < 0.01, and ***p < 0.001. At food concentrations of 2.5 μg CHLal−1 at 20 °C, values of enzymatic activity were too low to be detected, while at 28 °C, data were absent due to 100% mortality of specimens

    Table 1 ANOVA analysis on immunological biomarkers. Univariate results of enzymatic and lysosomal stability variations (phosphatase, esterase, peroxidase, and uptake of neutral red markers) from speci-mens maintained at the three experimental temperatures

    The significance of food and oxygenation factors was calculated for each temperature. The analyses were carried out using the STATIS-TICA rel. 10.0 (StatSoft Inc., USA). Differences between means were considered significant for p < 0.05 are indicated in bold

    Biomarkers Tem-perature (C°)

    [Food] Oxygenation

    F value P value F value P value

    Esterase 12 4.14 0.00001 1.608 0.2120 24.55 0.00001 3.49 0.07028 11.28 0.00001 17.25 0.000018

    Phosphatase 12 6.36 0.00024 8.50 0.0006020 22.91 0.0000 0.70 0.40828 2.37 0.057 15.64 0.00033

    Phenoloxidase 12 6.97 0.00012 1.40 0.2420 3.58 0.010 0.0003 0.9828 2.09 0.088 28.88 0.000004

    Uptake of neutral red

    12 53.117 0.00001 2.42 0.12720 9.23 44.58 0.000013 0.0000128 0.894 0.49 27.58 0.000006

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    and six food concentrations) differentially modulated enzy-matic parameters in the digestive gland and affected the biomarker of lysosomal membrane stability.

    Hydrolase endogenous enzymes are well known to modulate and accelerate immunological processes in hemo-cytes, such as phagocytosis, and are also involved in nutri-ent transport and digestion (Chen et al. 2007). Phosphatase is involved in various functions such as food ingestion and shell mineralization (Carballal et al. 1998; Jing et al. 2006).

    The increase in temperature, from 12 to 28 °C, caused a decrease in the kinetics of esterase and alkaline

    phosphatase both in anoxic and normoxic conditions. Slight increases were detected only in specimens fed with the highest concentration of food (5 g CHLal−1) at 20 and 28 °C for esterase, and at 28 °C for phosphatase under con-ditions of anoxia. This indicates that food can buffer the detrimental effects generated by harsh conditions, such as those provided by low oxygen concentrations.

    Nutrients, as denoted by the concentration of proteins and lipids, or the assumption of various components of the algae cell wall and vitamins, can affect immune functions of the invertebrate digestive gland (Goimier et al. 2006). In

    Fig. 3 Kinetics of phosphatase activity expressed as U mg−1 protein, in the digestive gland of M. galloprovincialis after treatment at three temperatures (12, 20 and 28 °C), and six different food concentra-tions (0.2–5  μg  CHLal−1) under anoxic (0  mg  O2l−1) and normoxic conditions (~8 mg O2l−1). Data represent the mean ± SE (n = 4 repli-cates). Significant differences were detected by Tukey post hoc tests: *p < 0.05, **p < 0.01 and ***p < 0.001

    Fig. 4 Kinetics of phenoloxidase activity expressed as U mg−1 pro-tein, in the digestive gland of M. galloprovincialis after treatment with three temperatures (12, 20, and 28 °C) and six different food concentrations (0.2–5  μg  CHLal−1) under anoxic (0  mg  O2l−1) and normoxic conditions (~8  mg  O2l−1). Data represent the mean ± SE (n = 4 replicates). Significant differences were detected by Tukey post hoc tests: *p < 0.05, **p < 0.01 and ***p < 0.001

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    particular, it is known that the amount of administered food increases the enzymatic activities in molluscs, such as alka-line phosphatase (Noomhorm et al. 2014).

    In invertebrates, phenoloxidase (PO) is an important humoral defense system and it can be activated by non-self material (Asokan et al. 1997). Normally, PO is present in mollusc plasma in the inactive prophenoloxidase (proPO) state, and PO activation leads to the melanization reaction, leading to the entrapment of foreign material in a melanin

    capsule (Coles and Pipe 1994). Changes in the level of this key component of the immune system may affect the sur-vival of the organisms when challenged within infectious pathogens (Cajaraville et al. 1996; Jing et al. 2008).

    Our results showed that the kinetics of phenoloxidase negatively correlated with increasing temperature. Moreo-ver, in this case, the food explains a buffer effect to coun-teract the limiting temperature for the production of the enzyme in normoxic specimens fed with 5 g CHLal−1 and maintained at 20 °C and in those fed with 3 g CHLal−1 and maintained at 28 °C. In addition, in clams C. farreri, high values of PO and alkaline phosphatase during a period of favorable water temperatures, and low values during repro-duction completion and high water temperatures were found by Cajaraville et al. (1996).

    These results suggest that if the phytoplankton concen-tration drops and intertidal temperatures increase, localized extinction could be accelerated in some locations. While the mechanisms underlying these patterns of mortality remain to be fully explored, recent studies strongly suggest that interactions between body temperature and oxygen demand may underlie at least some of the lethal effects of body temperature.

    Numerous environmental factors are known to have impacts on the hemocyte parameters of bivalves (Duch-emin et al. 2007). Among them, high water temperature can depress hemocyte parameters, including number, motility, viability, adhesive capacity, phagocytic ability, membrane permeability, and intracellular enzyme activity, which result in a weakened ability for an effective immune defense (Le Moullac and Haffner 2000; Yao and Somero 2012). The uptake of neutral red assay has been confirmed to be a biomarker to monitor the health of marine environments (Martinez Gomez et al. 2008; Franzellitti et al. 2010). The lysosomal membrane stability (LMS) responded to changes in environmental parameters in a dose-dependent manner and is determined by the stress types, their levels or dos-ages and exposure durations.

    As an enzymatic biomarker, the LMS value decreased from 12 to 28 °C both under anoxia and normoxia. All stud-ied factors, i.e., temperature, food, and oxygen availability, modulated the LMS of M. galloprovincialis hemocytes. Specifically, temperature was found to modulate the stabil-ity of lysosomes at 12 °C, while at higher temperatures, the response of the marker was significantly modulated by the oxygenation level.

    Various studies have demonstrated that the endocytotic activity of bivalve hemocytes can be affected by both expo-sure to xenobiotics and changes in environmental param-eters (Matozzo et al. 2011). In other bivalves, such as the clam Mactra veneriformis (Yu et  al. 2009) and mussels M. californianus (Yao and Somero 2012), the viability of hemocytes decreased under acute temperature stress. In

    Fig. 5 Uptake of neutral red from the lysosomal membrane, expressed as ODT mg−1 ml−1 hemocyte protein. The hemolymph was withdrawn individually from the adductor muscle of M. galloprovin-cialis specimens maintained at three temperatures (12, 20, and 28 °C) and six different food concentrations (0.2–5  μg  CHLal−1) under anoxic (0  mg  O2l−1) and normoxic conditions (~8  mg  O2l−1). Data represent the mean ± SE (n = 4 replicates)

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    Cassostrea gigas, experiments of both gradual and rapid changes in water temperature (from 15 to 5 or 25 °C) sig-nificantly reduced hemocyte lysosomal membrane stabil-ity (Zhang et  al. 2006). However, our results showed that high food levels (2.5–5  g  CHLal−1) counterbalanced the

    down-regulation caused by the temperature increase under conditions of normoxia, and especially of anoxia, at 28 °C. The food-derived materials can act on different immune cells, likely improving at least some parameter of innate immunity. Similarly, M. trossulus and M. galloprovincialis

    Fig. 6 Multifactorial ANOVA analysis results. Statistically sig-nificant effects of the following variables: availability of oxygen (α), temperature (β), and diet (γ) and interactions of first, second, and third orders for phenoloxi-dase, phosphatase, and esterase enzymatic responses and uptake of neutral red (NR) in cells from M. galloprovincialis hemolymph were investigated. The analyses were carried out using the STATISTICA rel. 10.0 (StatSoft Inc. USA). Differences between means were consid-ered significant for p < 0.05 are indicated in bold

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    showed lowered resistance to elevated temperatures in low food regimes (Schneider et al. 2010).

    Since the mollusc immune response is composed of an integrated process of phagocytosis and lysosomal degra-dation, multiple stressors that induce dysfunction of these processes may suppress immunocompetence. In conclu-sion, we found a lowered efficiency of immunological bio-markers in elevated temperatures and anoxic conditions in low food regimes. High food levels could counterbal-ance the effects of multiple environmental stressors. The knowledge of the additive, synergistic, interactive interac-tion (with increased or decreased) effects of environmental stressors or biotic factors on mollusc immuno competence is an important topic, especially when considering that interspecific differences in response to the same environ-mental stressor can often occur among mollusc species and among different invertebrate species in general.

    Thus, by investigating the relationship between environ-mental conditions and immune mechanisms in bivalves, this study was able to explain the susceptibility to external stress and infections. Moreover, the knowledge of the effec-tors involved in immune modulation will be increasingly important to develop management and conservation pro-grams for predicted climate change.

    Acknowledgements This work was supported by PRIN TETRIS 2010 Grant n. 2010PBMAXP_003, funded by The Italian Ministry of Education, University and Research (MIUR) supported GS and FFR-Cammarata at the Scientific Research University of Palermo (2014).

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    Temperature increases, hypoxia, and changes in food availability affect immunological biomarkers in the marine mussel Mytilus galloprovincialisAbstract IntroductionMaterials and MethodsAnimals and experimental planHemolymph and digestive gland collectionNeutral red (NR) uptake assayPhenoloxidase assayAlkaline phosphatase activityEsterase activityStatistical analysis

    ResultsDiscussionAcknowledgements References