Food-type may jeopardize biomarker interpretation in mussels used in aquatic toxicological experimentation
Food-type may jeopardize biomarker interpretation in mussels used in aquatic toxicological experimentation
Esther Blanco-Ray o?n 0 1
Anna V. Ivanina 1
Inna M. SokolovaID 1
Ionan Marigo? mezID 0 1
Urtzi Izagirre 0 1
0 CBET Research Group, Department of Zoology and Animal Cell Biology, University of the Basque Country (UPV/EHU) , Leioa, Basque Country , Spain , 2 Research Centre for Experimental Marine Biology and Biotechnology (Plentzia Marine Station; PiE-UPV/EHU), University of the Basque Country , Plentzia, Basque Country , Spain , 3 Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, North Carolina, United States of America, 4 Department of Marine Biology, Institute for Biosciences and Department of Maritime Systems, Interdisciplinary Faculty, University of Rostock , Rostock , Germany
1 Editor: Amitava Mukherjee, VIT University , INDIA
To assess the influence of food type on biomarkers, mussels (Mytilus galloprovincialis) were maintained under laboratory conditions and fed using 4 different microalgae diets ad libitum for 1 week: (a) Isochrysis galbana; (b) Tetraselmis chuii; (c) a mixture of I. galbana and T. chuii; and (d) a commercial food (Microalgae Composed Diet, Acuinuga). Different microalgae were shown to present different distribution and fate in the midgut. I. galbana ( 4 ?m ?) readily reached digestive cells to be intracellularly digested. T. chuii ( 10 ?m ? and hardly digestible) was retained in stomach and digestive ducts for long times and extracellularly digested. Based on these findings, it appeared likely that the presence of large amounts of microalgal enzymes and metabolites might interfere with biochemical determinations of mussel's biomarkers and/or that the diet-induced alterations of mussels' digestion could modulate lysosomal and tissue-level biomarkers. To test these hypotheses, a battery of common biochemical, cytological and tissue-level biomarkers were determined in the gills (including activities of pyruvate kinase, phosphoenolpyruvate carboxykinase and cytochrome c oxidase) and the digestive gland of the mussels (including protein, lipid, free glucose and glycogen total content, lysosomal structural changes and membrane stability, intracellular accumulation of neutral lipids and lipofuscins, changes in cell type composition and epithelial thinning, as well as altered tissue integrity). The type of food was concluded to be a major factor influencing biomarkers in short-term experiments though not all the microalgae affected biomarkers and their responsiveness in the same way. T. chuii seemed to alter the nutritional status, oxidative stress and digestion processes, thus interfering with a variety of biomarkers. On the other hand, the massive presence of I. galbana within digestive cells hampered the measurement of cytochemical biomarkers and rendered less reliable the results of biochemical biomarkers (as these could be attributed to both the mussel and the microalgae). Research to optimize dietary food type, composition, regime and rations for toxicological experimentation is urgently needed. Meanwhile, a detailed description of the food type and feeding conditions should be always provided when reporting
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This work was funded by the Spanish
Ministry of Economy and Finance
(BMWCTM2012-40203-C02-01), the University of the
Basque Country UPV/EHU (UFI 11/37) and the
Basque Government (Consolidated Research
Groups Grant IT810-B). EB-R was recipient of a
doctoral fellowship (PRE2013 1 640) financed by
the Department of Education of the Basque
Government (Eusko Jaurlaritza).
Competing interests: The authors have declared
that no competing interests exist.
aquatic toxicological experiments with mussels, as a necessary prerequisite to compare
and interpret the biological responses elicited by pollutants.
Mussels are widely used sentinel organisms in pollution monitoring programs to assess the
biological effects of pollutants. There is an urgent need to develop consensus standardized
procedures for biomarker determinations [
]. Recently, a large effort has been directed towards
development of the Best Available Practices for mussel sampling and processing in field studies
and monitoring programs [
]. Together with field studies, laboratory experiments are
crucial to gain understanding of the biological effects of pollutants and to develop a reliable
toolbox of biomarkers for environmental monitoring and assessment. Some experimental
variables such as temperature, photoperiod, salinity, water renewal, and dosing are recognized as
key conditions to correctly perform laboratory experiments and routinely reported in
publications and research reports. However, less attention has been paid to the potential effects of the
food type and feeding strategy. Although digestion and food type may modulate biomarker
], to our knowledge, there is no guidelines dealing with recommended
food types and feeding strategies to keep mussels during laboratory experiments. Thus, a large
variety of food types and feeding strategies are used in aquatic toxicological experiments with
mussels; these include absence of additional food supply, supply of diverse commercial food
products or a variety of live microalgae either in monocultures or in mixtures. Moreover, in
many cases no mention is made to the food type or feeding conditions. For example, in a
nonexhaustive literature mining in which 75 classical and recent manuscripts were selected, a 16%
of the papers provided no indication of whether mussels were fed during experimentation,
11% maintained mussels without additional food supply (particularly during short-term
experiments), 40% of the studies used live microalgae in monoculture (29%) or in mixtures
(11%), and a 33% used commercial food of diverse origins (14 manufacturers) and/or
naturederived food (such as lyophilized algae or flour) (Table 1). Moreover, the rations and regime of
food availability (e.g., continuous flow vs. pulses) were also different. If, the food type and
feeding strategy influence the levels and responsiveness of biomarkers, the generalizations and
comparisons between these experiments would be difficult.
To test the hypothesis that the feeding regime might affect the toxicologically important
biomarkers, the present investigation was aimed at determining the influence of food type on a
battery of biomarkers frequently analysed in mussels (Mytilus galloprovincialis).
As the initial step, the distribution and fate of the microalgae of different sizes and
biochemical composition (Isochrysis galbana, Tetraselmis chuii and their mixture) were investigated in
the mussels? midgut by light and fluorescence microscopy following ad libitum feeding for 5
min, 2 h and 5 d. These algal species are commonly used as food for bivalves in aquatic
toxicological experimentation (Table 1). We then exposed the mussels to different diets (I. galbana;
T. chuii; I.galbana and T.chuii microalgae mixture; and commercial Shellfish Diet microalgae
blend, Acuinuga) for a week and investigated a battery of biomarkers commonly employed for
biological effect assessment in marine pollution monitoring. These biomarkers included
activities of key metabolic enzymes (cytochrome c oxidase, pyruvate kinase, phosphoenolpyruvate
carboxykinase), oxidative lesions of proteins and lipids, lysosomal membrane stability, and
tissue-level markers for the integrity and health of digestive epithelia. Cytochrome c oxidase
(COX) was used as a marker of mitochondrial capacity that commonly correlates with
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PLOS ONE | https://doi.org/10.1371/journal.pone.0220661
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Types of biomarkers depending on their endpoint and technology: (1) Functional and in vitro assays; (2) Biochemistry and molecular biology; (3) Cryotechnology and
cytochemistry; (4) Histo(path)ology; (5) Biometry and physiology.
mitochondrial activity and oxygen consumption rates [
]. Pyruvate kinase (PK) and
phosphoenolpyruvate carboxykinase (PEPCK) channel the glycolytic substrate (pyruvate) to
the aerobic (PK) vs. anaerobic (PEPCK) pathways [
], so that the PK/PEPCK ratio is
commonly used as a measure of the relative aerobic/anaerobic capacity of the organism .
Increased levels of protein carbonyl groups (CO) are signs of early oxidative damage (protein
oxidation) whilst increased levels of malondialdehyde (MDA) and 4-hydroxy-2-nonenal
(HNE) indicate later oxidative damage (lipid peroxidation) [
]. Lysosomal enlargement
and membrane destabilization in mussel digestive cells are widely used as pollution effect
]. Intracellular neutral lipid accumulation has been related to organic
xenobiotic exposure, non-specific stress and nutritional status [
]. The relative proportion
of basophilic cells is known to increase in the digestive gland epithelium under stress
4 / 25
]. Atrophy of the digestive epithelium and loss of digestive gland histological
integrity occur in response to pollutant exposure [
]. Therefore, investigation of the
battery of these biomarkers provided a comprehensive insight into the potential impact of the
altered diet quality on the integrated metabolic and stress response of the mussels.
Material and methods
Experimental design and sample processing
Intertidal mussels (M. galloprovincialis) of 3.5?4.5 cm shell length were collected from the low
tide-mark level (0.5?1.0 m) in Plentzia (Basque Coast; 43?260N; 2?550W) in September 2014.
Permits by the Directorate of Fishing and Aquaculture of the Department of Economical
Development and Infrastructures of the Basque Government were obtained for mussel
collection in public domains of the Basque Coast (Law 6/1998; BOPV N. 62, 1/4/1998). Additional
permits were not required because M. galloprovincialis is not an endangered or protected
species. Mussels were acclimatized for 7 d to laboratory conditions (18?1?C; 12L:12D cycle),
maintained unfed in filtered (0.2 ?m) seawater (dissolved oxygen: 7.6?8.3 mg/l; pH 7.8?8;
After acclimatization, mussels were divided in 4 experimental groups (in 5 l seawater tanks
with constant aeration, n = 20) and fed ad libitum for a week using four different microalgae
diets commonly used as food for mussels in laboratory experimentation: (a) I. galbana, (b) T.
chuii, (c) a mixture of I. galbana and T. chuii, and (d) a commercial food (Microalgae
Composed Diet, Acuinuga SL, A Coru?a, Spain). I. galbana is a brown free-living biflagellate
marine microalga ( 4 ?m ?; Fig 1) (Table 1). T. chuii is a green free-living tetraflagellate
marine microalga ( 10 ?m ?; Fig 1) (Table 1). The used commercial food (Fig 1) is based on a
mixture of 4 microalgae Isochrysis sp. (25%), Tetraselmis sp. (25%), Thalassiosira sp. (25%) and
Nannochloropsis sp. (25%). Following the manufacturer recommendations the commercial
food was stored at -40?C before use. Once opened it was stored at <6?C for 1 week during the
Strains of I. galbana (T. ISO clone) and T. chuii were grown in previously cleaned 30 L
volume methacrylate reactors with natural filtered seawater. Monocultures were maintained
under constant white light exposure (two lamps of 36 W per reactor), room temperature
(T = 17?C) and filtered air flow (0.2 ?m filters). Microalgae culture density was checked daily
using a Beckman Coulter Counter Z2 particle size analyser, and diluted as needed in seawater
enriched with F/2 medium (Easyalgae Fitoplancton Marino SL, Ca?diz, Spain) to keep an
average concentration (cell/mL) of 7.8?1.4?106 for I. galbana and 11.4?3.4?105 for T. chuii. Water
Fig 1. (A-D) Appearance at the light-microscope of the various dietary food types (unstained smears). (A) Isochrysis galbana; (B) Tetraselmis chuii; (C) mixture of I.
galbana and T. chuii; and (D) commercial food. (E) Autofluorescence signal in frozen unstained smear of I. galbana (small particles) and T. chuii (large particles). Scale
bar: 2 ?m.
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and food from mussel tanks (total volume: 3 l seawater+food per tank) were changed every
day: (a) 3 l/d of I. galbana culture; (b) 3 l/d of T. chuii culture; and (c) 1.5 L/d of I. galbana
culture and 1.5 l/d of T. chuii culture for the mixture diet. The commercial food (2?109 particles/
mL; Microalgae Composed Diet) was diluted in seawater in order to provide a concentration
of 5?106 particles/ml in 3 l seawater for daily changes.
Immediately after the acclimatization period and after 5 min, 2 h and 1 wk feeding with I.
galbana and T. chuii and their mixture, the digestive gland was dissected from several mussels,
snap frozen in liquid nitrogen and stored at -80?C until further analysis. The autofluorescence
of cryotome sections (8 ?m) of these digestive glands was examined under the Nikon Eclipse
Ni-Series fluorescence microscope using a 485 nm excitation filter and a 645 nm emission
filter to visualize algal chlorophyll [
]. Schmorl?s staining was applied to visualize lipofuscin in
the same cryotome sections [
After a week of experimental exposures, gills and digestive gland of five mussels were
dissected, frozen in liquid nitrogen and stored at -80?C for biochemical and histochemical
analyses. Mantle and digestive gland of 10 mussels were dissected, fixed in formaldehyde (4% in
seawater) at 4?C and embedded in paraffin for histological analyses. No mortality was
observed during experimental exposures. Gonad histology was examined to provide
supporting data of mussel general condition [
]; upon microscopic examination of mantle tissue
sections all the individuals in all the treatments were found to be at a comparable gametogenic
stage (Gonad Index = 1.25 ? 0.21).
Total lipid content was determined in the digestive gland using a chloroform extraction
]. Briefly, about 50 mg of the digestive gland tissue was homogenized in
chloroform/methanol mixture (2:1 v:v) using tissue: solvent proportion of 1:20 w/v. Samples were
sonicated for 1 min (output 69 W, Sonicator 3000, Misonix, Farmingdale, NY, USA),
incubated overnight at 4?C and centrifuged for 5 min at 13000?g. The supernatant was transferred
in a new tube, mixed with ultrapure water (0.25 volumes of the supernatant), vortexed for 2
min and centrifuged for 5 min at 13000?g. The lower phase (chloroform) was transferred into
a pre-weighed microcentrifuge tube and allowed to evaporate to determine the dry mass of
extracted lipids. For determination of carbohydrates, the digestive gland tissue was powdered
under liquid nitrogen and homogenized with five volumes of ice-cold 0.6 M perchloric acid
(PCA) with 150 mM ethylenediaminetetraacetic acid (EDTA) . An aliquot of the
homogenate was reserved for glycogen determination, and the remaining homogenate was centrifuged
to remove precipitated protein and neutralized with 5 M potassium hydroxide to pH 7.2?7.5.
Precipitated potassium perchloride was removed by a second centrifugation and extracts were
stored at ?80?C. Carbohydrates were measured in neutralized PCA extracts using a standard
NADPH-linked spectrophotometric test . Briefly, assay conditions were as follows: 38.5
mM triethanolamine buffer, pH 7.6, 0.04 mM NADP+, 7 mM MgCl2 6H2O, 0.462 U/ml
glucose-6-phosphate dehydrogenase, 1.8 U /ml hexokinase. Glycogen concentration was
measured in PCA extracts after enzymatic hydrolysis of glycogen to D-glucose by glucoamylase
] and determined by the difference in the D-glucose levels in the tissue extract before and
after glucoamylase treatment. Concentrations of glycogen, lipids and proteins were expressed
in mg/g wet tissue mass.
For total protein content analysis, digestive gland was homogenized in ice-cold
homogenization buffer (100 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM egtazic acid (EGTA),
1% Triton-X 100, 10% glycerol, 0.1% sodium dodecylsulfate, 0.5% deoxycholate, 0.5 ?g
leupeptin/ml,0.7 ?g pepstatin/ml, 40 ?g phenylmethylsulfonyl fluoride (PMSF) /ml and 0.5 ?g /ml
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aprotinin) using Kontes Duall tissue grinders (Fisher Scientific, Suwanee, GA, USA).
Homogenates were sonicated 3?10 sec each (output 69 W, Sonicator 3000, Misonix), with cooling on
ice between sonications, centrifuged for 10 min at 20000 g and 4?C, and supernatants were
used for protein determination. Protein content was measured using the Bio-Rad Protein
Assay kit according to the manufacturer?s protocol (Bio-Rad Laboratories, Hercules, CA,
Activities of the pyruvate kinase (PK; EC 220.127.116.11), phosphoenolpyruvate carboxykinase
(PEPCK; EC 18.104.22.168) and cytochrome c oxidase (COX; EC 22.214.171.124) were determined in the
gills. The tissues were homogenized in enzyme-specific homogenization buffer using
handheld Kontes Duall tissue grinders (Fisher Scientific, Suwanee, GA, USA). Homogenates were
sonicated 3?10 sec each (output 7, Sonic Dismembrator Model 100, Fisher Scientific, Suwanee,
GA) to ensure complete release of the enzymes, with cooling on ice (1 min) between
sonications and centrifuged at 16000?g and 4?C for 25 min. The supernatant was collected and used
for enzyme determination. Enzyme extracts were stored at ?80?C for less than two weeks
before activity assays. For determination of enzyme activities, enzyme extracts were thawed on
ice and immediately analyzed by standard spectrophotometric techniques as described
] using a UV?Vis spectrophotometer (VARIAN Cary 50 Bio, Cary NC,
USA). The temperature of the reaction mixture was controlled at 20?0.1?C using a
water-jacketed cuvette holder. Briefly, isolation and assay conditions for the studied enzymes were as
follows: (a) PK: homogenization buffer: 10 mM Tris?HCl buffer (pH 7.2), 5 mM EDTA, 1 mM
dithiotreitol (DTT), 0.1 mM phenylmethylsulfonyl (PMSF); assay: 50 mM Tris-HCl (pH7.2),
50 mM KCl, 5 mM MgSO4, 1 mM ADP, 0.2 mg/ml NADH, 5.5 U/ml LDH, 0.5 mM
phosphoenolpyruvate (PEP); acquisition wavelength: 340 nm; (b) PEPCK: homogenization buffer: 10
mM Tris?HCl buffer (pH 7.2), 5 mM EDTA, 1 mM DTT, 0.1 mM PMSF; assay: 100 mM
4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2), 2.3 mM MnCl2, 0.5 mM
Inosine-5?- diphosphate trisodium salt (IDP), 5mg/ml KHCO3, 0.2 g/ml NADH, 10 U/ml
malate dehydrogenase (MDH), 15 mM PEP; acquisition wavelength: 550 nm; (c) COX:
homogenization buffer: 25 mM potassium phosphate, pH 7.2, 10 ?g/ml PMSF, 2 ?g/ml
aprotinin; assay: 20 mM potassium phosphate, pH 7.0, 16 ?M reduced cytochrome c(II), 0.45 mM
ndodecyl-b-d-maltoside, 2 ?g/ml antimycin A; acquisition wavelength: 550 nm. Protein
concentration was measured as above described for the digestive gland.
Protein carbonyl groups (CO) were measured spectrophotometrically [
gland was ground under liquid nitrogen and homogenized in buffer containing 50 mM
HEPES, 125 mM KCl, 1.1 EDTA and 0.6 mM MgSO4 (pH 7.4) and protease inhibitors
[leupeptin (0.5 ?g/ml), pepstatin (0.7 ?g/ml), phenylmethylsulfonyl fluoride (40 ?g/ml) and
aprotinin (0.5 ?g/ml)]. Samples were centrifuged at 100000?g for 15 min, supernatant was collected
and incubated at room temperature with 10 mM 2,4-dinitrophenylhydrazine (DNP) in 2 M
HCl. The blanks were incubated with HCl without DNP. After incubation, proteins were
precipitated by adding 100% trichloracetic acid and centrifuged at 11000?g for 10 min. The pellet
was washed with ethanol ethylacetate (1:1 v:v) and resuspended in 6 M guanidine
hydrochloride in 20 mM in KH2PO4 (pH 2.5) until dissolved. The absorbance was measured at 360 nm
on a spectrophotometer (VARIAN Cary 50 Bio, Cary NC, USA) using guanidine HCl solution
as reference. The amount of carbonyls was estimated as a difference in absorbance between
samples and blanks using a molar extinction coefficient of carbonyls ? = 22000 1/(cm?M).
Protein content of the samples was determined using BSA standard prepared in 6 mol/l
guanidine HCL and 20 mmol /L KH2PO4 (pH 2.4). Carbonyl content was normalized to the protein
concentration in the samples.
Protein conjugates of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were
measured as biomarkers of lipid peroxidation using enzyme-linked immunosorbent assay (MDA
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OxiSelect MDA adduct ELISA Kit and HNE OxiSelect HNE-His adduct ELISA Kit,
respectively) according to the manufacturers? protocols (Cell Biolabs, Inc., CA, USA). About 200?
300 mg of digestive gland were homogenized in ice cold phosphate-buffered saline (PBS) (1:5
w:v) with protease inhibitors (50 ?g/l aprotinin and 40 ?M phenylmethylsulfonyl fluoride)
using Kontes Duall tissue grinders (Fisher Scientific, Suwanee, GA, USA). Samples were
centrifuged at 15000?g for 10 min at 4?C. Protein concentration was measured in the supernatant
using the Bio-Rad Protein Assay kit according to the manufacturer?s protocol (Bio-Rad
Laboratories, Hercules, CA, USA). Supernatants were diluted with PBS to a final concentration of 1
Histological and histochemical analyses
lipids) with respect to the digestive epithelium volume (VvNL) was calculated by applying a
stereological procedure [
]. VvNL is expressed as ?m3/ ?m .
Lipofuscin (LPF) accumulation was determined in digestive gland cryostat sections (8 ?m
thick) fixed for 15 min in Baker buffer at 4?C. The sections were rinsed in distilled water and
stained using Schmorl?s reaction [
]. Five measurements using a 400? magnification were
made in each section using image analysis (Sevisan S.L., Spain). The mean value of LPF volume
density (VvLPF = VL/VC) was determined for each mussel digestive gland (N = 5 per
Statistical analyses were made using SPSS v 22.0 software (SPSS INC., Chicago, Illinois).
Parameters were tested for normality (Kolmogorov-Smirnov?s test) and homogeneity
(Levene?s test). For the traits that had normal distribution and homogeneous variances (COX, PK,
PEPCK, PK/PEPCK, MDA, HNE, VvNL, VvLPF VvBAS, MLR/MET and CTD), one-way
ANOVA and Duncan?s post-hoc tests were used to test for the effects of the diet type and
conduct the pairwise comparisons of group means, respectively. For LP, non-parametric statistics
(Mann-Whitney?s U-test) was used. The Z-score test was used when the sample size was too
small (N = 4) for reliable Duncan?s or Mann-Whitney?s U-test including the following traits:
total lipid content, glycogen, total protein content, VvLYS, S/VLYS, and NvLYS. Significance for
all statistical tests was established at p<0.05.
Microalgae distribution and fate in the midgut
After 7 days of acclimatization without food, some brownish granules were observed in the
lumen and in the epithelium of digestive alveoli (Fig 2A). These granules exhibited weak
fluorescence (Fig 2B) and were identified as LPFs (Fig 3A and 3B), likely related to residual bodies
of digestive cells.
Similar fluorescent LPF-like granules were observed after feeding mussels with I. galbana
for 5 min (Figs 2C, 2D, 3C and 3D). Microalgae were found in the lumen of the stomach as
well as in primary and secondary digestive ducts (Fig 2C). These microalgae presented a strong
fluorescence (Fig 2D). In contrast, no microalgae were found, nor fluorescence detected in the
digestive alveoli (Fig 2C and 2D). Although microalgae pigments also stained with the
Schmorl?s method, they were easily distinguishable from the mussels? LPFs because of the
different morphology and much higher staining intensity of the microalgae (Fig 3C and 3D).
After 2hr, microalgae-like bodies were observed within the epithelium of digestive alveoli (Fig
2E), which exhibited a remarkable fluorescence intensity (Fig 2F). After 1 week of feeding with
I. galbana, abundant dark brown bodies were found in the epithelium of digestive alveoli
together with yellowish corpuscles resembling microalgae (Fig 2G). Schmorl-positive materials
(both LPFs and microalgae, which were indistinguishable due to the high intensity of the
Schmorl?s reaction) were extremely abundant (Fig 3F and 3G) and fluorescence intensity
increased throughout the epithelium, although some small patchy areas with apparent LPFs
appeared dark (Fig 2H).
After feeding mussels ad libitum with T. chuii for 5 min, Schmorl-positive brownish bodies
with background weak fluoresce were found (Figs 2I, 2J, 3H and 3I), similar to those found
after 7 days of starvation during acclimatization (Fig 2A, 2B, 2I and 2J). In contrast, after 2 h of
feeding, abundant microalgae and their large fragments were found in the stomach but not in
the digestive alveoli (Fig 2K). These microalgae exhibited intense fluorescence (Fig 2L) and
were highly reactive after Schmorl?s staining (Fig 3J and 3K). No change was observed in the
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Fig 2. Cryostat sections (8 ?m) of unstained fresh tissue of mussels: (A, C, E, G, I, K, M, O, Q, S, U, W) Before feeding and after ad
libitum feeding for 5 min, 2 h and 1 week with I. galbana, T. chuii and I. galbana + T. chuii; (B, D, F, H, J, L, N, P, R, T, V, X) The same
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tissue section fields examined at the fluorescence microscope with 485 nm excitation filter and 645 nm emission filter. B, D and F: 12%
light intensity; H: 6% light intensity. J, L, N, P, R, T, V and Y: 3% light intensity. Scale bar: 20 ?m. I, I. galbana-like body; T, T. chuii;
AE, alveolus epithelium.
reactivity of the alveolus epithelium after Schmorl?s staining (Fig 3J and 3K). After 1 week,
highly fluorescent microalgae and their fragments were found in the lumen of alveoli, but not
in the epithelium (Fig 2M and 2N). In contrast, the amount and staining intensity of LPFs in
the epithelium of digestive alveoli increased (Fig 3L and 3M).
In mussels fed with the mixture of both microalgae species (I+T) for 5 min, a compact
highly fluorescent mass was found in the stomach lumen in which some bodies resembling I.
galbana could be identified (Fig 2O and 2P). After 2 h, both microalgae species were found in
the lumen of the stomach (Fig 2Q and 2R) and of the alveoli (Fig 2S and 2T). After 1 week, the
stomach lumen was full of microalgae and microalgae fragments (Fig 2W and 2X), with a
strong fluorescence (Fig 2V and 2X). However the appearance of digestive alveoli was diverse.
Some alveoli with empty lumen presented high fluorescence intensity in their epithelium (Fig
2V) whilst other had the lumen full of T. chuii and fragments, and exhibited low fluorescence
within the epithelium (Fig 2U and 2V). Overall, the LPF content increased greatly in the
epithelium of the digestive alveoli after 1 week of feeding with the algal mixture (Fig 3R).
Food type influence on biomarkers
While the total protein levels were similar in all diets, the total lipid content was higher and the
glycogen levels lower in I. galbana than in the other diets (Table 2). Likewise, the total protein
levels were similar in all experimental mussels irrespective of the diet, while the total lipid
content was lower and the glycogen levels higher in the digestive gland of mussels fed commercial
food compared with other experimental groups (Table 2).
Most studied biomarker values differed between the groups of mussels fed with the different
diets (S1 Table). PK activity was lower in mussels fed commercial food than in any other
experimental group (Fig 4A; S1 Table) whereas PEPCK was much higher (Fig 4B), thus
resulting in a low PK/PEPCK ratio (Fig 4C). The response profiles of MDA and HNE were similar,
with higher values in mussels fed T. chuii and commercial food, especially in MDA (Fig 4D
and 4E; S1 Table). The CO values and COX activity were not significantly different between
the treatments (S1 Fig; S1 Table).
LP could not be determined in mussels fed I. galbana alone or in mixture with T. chuii
because histochemical hexosaminidase activity in digestive alveoli was not clearly
discriminated from the background brownish coloration caused by LPFs and microalgae (Hex in Fig
5). LP values between 15 and 20 min were recorded in mussels fed T. chuii and commercial
food (Fig 4F), although in the former the measurements were difficult due to the presence of
extensive brownish bodies in the digestive cells. Similarly, lysosomal structural changes (VvLYS
and NvLYS) were difficult to measure in mussels fed I. galbana due to the massive amount of
microalgae within the epithelium of digestive alveoli (?-Gus in Fig 5. With this caveat, VvLYS
was higher in mussels fed I. galbana and I+T than in those fed T. chuii or commercial food
(Fig 4G; S1 Table). In contrast, S/VLYS and NvLYS values did not differ among experimental
groups, according to 1-way ANOVA (S1 Table). VvNL was higher in mussels fed I. galbana and
I+T than in mussels fed T. chuii or commercial food, with the lowest values being in the latter
group (Fig 4J; NL in Fig 5; S1 Table). VvLPF was the highest in mussels fed I. galbana and the
lowest in the mussels fed commercial food, with intermediate values in the mussels fed T. chuii
and the mixture of microalgae (LPF in Fig 5; Fig 4K; S1 Table).
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Fig 3. Histochemistry of lipofuscins in digestive gland of mussels, before feeding and after feeding ad libitum with I. galbana, T.
chuii and I. galbana + T. chuii for 5 min, 2 h and 1 wk. Scale bar is 30 ?m in A, C, F, H, Q, L, O);. 20 ?m in B, D, E, G, I, J, K, M, N, P,
S); and 50 ?m in S. I, I. galbana-like body; T, T. chuii; St, stomach; L, lipofuscin.
12 / 25
Estimated as the average between I. galbana and T. chuii (1:1)
Estimates, according to the product label, subject to some variability among batches
VvBAS was lower in mussels fed I. galbana than in other experimental groups and the
highest in mussels fed I+T and commercial food (Fig 4L; T&E in Fig 5; S1 Table). The lowest MLR/
MET values were found in the mussels fed I. galbana and the highest in those fed commercial
food (Fig 4M; T&E in Fig 5; S1 Table). CTD ratio was higher in mussels fed commercial food
than in the other experimental groups (Fig 4N; S1 Table).
Microalgae distribution and fate in the midgut
Microalgae species used as a food source for bivalves can differ in cell size and morphology,
digestibility, biochemical composition and toxicity. Some microalgae may have a high
nutritional value and be readily ingested by mussels, yet this does not necessarily imply that they
will be subject to digestion [
]. For instance, after testing ten species of microalgae, only two
species (Isochrysis and Pavlova) were digested by winged pearl oyster larvae [
After 5 min of feeding, strongly fluorescent I. galbana reached the lumen of the stomach
and digestive duct of the mussels and were inside the digestive cells after 2 h of feeding or later.
This finding agrees with the reported length of the digestion cycle (~ 4 h) in the intertidal
mussels and the findings that I. galbana is internalized (phagocytosed) in digestive cells for
intracellular digestion [
]. Similarly, digestion of Isochrysis sp. by giant clam veliger larvae was
observed 2 h after the start of feeding [
]. With the exception of a previous study [
massive presence of microalgae-like spherical bodies in mussel digestive cells has not been
previously reported in laboratory experiments which used I. galbana as food [
] even though
several of these studies were based on microscopic observations of digestive gland tissue
sections. Therefore, further research is needed in order to understand the mechanism through
which small and relatively easily digestible microalgae such as I. galbana are digested in
Unlike I. galbana, T. chuii took 2 h to reach the stomach and was never found within
digestive cells. Similarly, digestion of Tetraselmis sp. by giant clam veligers was only observed at
time periods exceeding 4?8 h after feeding [
]. Although T. chuii reached the lumen of
digestive alveoli of the mussels and the digestive cells were rich in lipofuscins after 1 week of
feeding, the epithelium was weakly fluorescent. Fluorescence intensity decays as the degree of
lysis and digestion of phytoplankton cells increases [
]. Therefore, T. chuii digestion appears
to be mainly extracellular and subject to extended gut retention times. Gut retention time (and
associated absorption efficiency; [
]) are determined by the amount and quality, and most
13 / 25
Fig 4. Biomarkers recorded in mussels fed ad libitum for 1 week with 4 different diets (I. galbana (I); T. chuii (T); mixture of I. galbana and
T. chuii (I+T); and commercial food (CF)): pyruvate kinase (PK) (A), and phosphoenolpyruvate carboxykinase (PEPCK) (B) activities, and PK/
PEPCK ratio (C) in gills; malondialdehyde (MDA)-protein conjugates (D), 4-hydroxynonemal (HNE)-protein conjugates (E), labilization period
(LP) of the lysosomal membrane (F), lysosomal volume density (VvLYS) (G), surface-to-volume ratio (S/VLYS) (H) and numerical density (NvLYS)
(I), volume density of neutral lipids (VvNL) (J) and lipofuscins (VvLPF) (K), volume density of basophilic cells (VvBAS) (L),
mean-luminal-radiusto-mean-epithelial-thickness (MLR/MET) (M) and connective-to-digestive-tissue (CTD) ratio (N) in digestive gland. Intervals indicate standard
error. Groups labelled with a different letter are significantly different (p<0.05) from each other according to the Duncan?s test performed after
one-way ANOVAs except for F-I. (F-I) Different letters indicate significant differences (p<0.05) among diets according to the Mann-Whitney?s
U-test for LP and the Z-score test for CO, VvLYS, S/VLYS and NvLYS. ?, no reliable measurement.
importantly, by the digestibility of the ingested food [
]. In adult oysters and mussels
fed Tetraselmis, the gut retention time can go beyond 10 h . Similarly, mussels have more
difficulties in absorbing Tetraselmis, compared with other microalgae, and the absorption
efficiency of Tetraselmis is half of that recorded for Isochrysis [
]. The large cell size can hamper
The presence of refractory cell walls is another potential cause for indigestibility as shown
for chlorophytes in bivalves [
]. Cell walls can contain highly refractory components
resistant to enzymatic attack and strong acid degradation, so that there might be little advantage in
a more prolonged retention of such cells in the gut [
]. The digestibility of the cell
wall of Tetraselmis appears low in mussels, reflected in low absorption efficiency [
Tetraselmis cells are not easily digested due to their thick cellulose-rich cell wall, which renders
intracellular starch granules and other components unavailable to the gut digestive enzymes
]. Cellulase is a common molluscan enzyme; however, hydrolysis of structural cellulose is
generally low in bivalves [
]. Moreover, the cell wall of Tetraselmis is made of a pectin-like
material, with galactose, galacturonic acid and unusual 2-keto sugar acids as major
components, which make the cells walls acidic and difficult to degrade [
]. As a result, Tetraselmis
is known to be less nutritious that I. galbana in a variety of bivalve species [
It is worth noting that absorption in bivalves? gut is not intestinal but depends on
endocytosis and phagocytosis in digestive cells and further intracellular food digestion and nutrient
delivery to haemocytes. Whilst the entire cells of I. galbana appeared taken into the digestive
diverticula, in the case of T. chuii only food materials derived from extracellular digestion
would be taken up by digestive cells (and lead to residual products of digestion such as
lipofuscins). Interestingly, the presence of I. galbana seemed to facilitate the distribution of T. chuii
towards digestive alveoli, reflected in the intermediate distribution profile of the algal cells in
mussels fed with the mixture of I. galbana and T. chuii compared with the single-species diets.
In summary, different microalgae show different distribution and fate in mussel digestive
gland. Whereas small microalgae such as I. galbana readily reach digestive alveoli and are
intracellularly digested (albeit the extracellular pre-digestion cannot be discarded), large and
hardly degradable T. chuii are retained in the stomach and digestive ducts. As a result of the
presence of microalgae in the gut and digestive gland epithelium, the enzyme activities and
metabolites (e.g. pigments and lipofuscins) of the microalgae or resulting from the mussel
response to the microalgae can influence the determination of biochemical biomarkers.
Furthermore, due to the long retention times and extracellular digestion of large microalgae, the
algae-gut interactions might affect the morphology and function of the digestive cell lysosomes
and thus influence lysosomal and tissue-level biomarkers in the digestive gland epithelium.
Food type influence on biomarkers
The nutritional condition of mussels varied significantly depending on the diet. Lower total
lipid content in the digestive gland of mussels fed commercial food than in those fed live
microalgae reflects the relative lipid content of the diet. However, the glycogen levels in the
15 / 25
Fig 5. Micrographs of digestive gland of mussels fed ad libitum for 1 week with 4 different microalgae diets (I. galbana; T. chuii;
mixture of I. galbana and T. chuii (I+T); and commercial food (CF)): hexosaminidase (Hex) and ?-glucuronidase (?-gus) enzyme
16 / 25
histochemistry, Oil Red O (neutral lipids: NL) and Schmorl?s (lipofuscins: LPF) histochemistry and toluidine-eosine staining (T&E)
topographical staining. Scale bar: 20 ?m.
digestive gland of mussels fed T. chuii were much lower despite the similar carbohydrate levels
in the commercial food and in T. chuii, possibly reflecting the lower digestibility and
absorption efficiency of T. chuii. A large part of the carbohydrates determined in T. chuii would
correspond to cellulose and pectin-like material [
], which remained in the gut lumen and
did not contribute to the carbohydrates found in the digestive gland. Overall, the commercial
food (poorest diet) and I. galbana (richest diet) represented the two extreme nutritional
conditions, as envisaged in the basic biochemical components measured in the digestive gland of
mussels. The low digestibility of freeze-dried microalgae used in commercial food has been
shown to cause reduced growth rates of bivalve seed in comparison with fresh microalgae diets
. Accordingly, the most striking differences in biomarker values were found when
commercial food and I. galbana were compared, with the intermediate values in the mussels fed T.
chuii and the I. galbana+T. chuii mixture. This finding is consistent with the earlier reports
that the nutritive condition can strongly affect the biomarker values in mussels [
], albeit in
this latter study the quantity rather than the quality of the food was manipulated.
Overall, differences between the groups of mussels fed different diets were found for most
biomarkers investigated in our present study. Thus, PK/PEPCK was much lower in mussels
fed commercial food than in those fed live microalgae, as the result of low PK and high PEPCK
activities, indicating decreased aerobic scope and increased gluconeogenesis [
peroxidation (indicated by high MDA and HNE values; [
] was enhanced in mussels fed
T. chuii or commercial food. Likewise, intracellular digestion was reduced (low VvLYS and
NvLYS and high VvBAS, MLR/MET and CTD ratio; [
]) and the levels of neutral lipids
(indicative of nutritional status; ) and lipofuscins (residual product of lipid digestion or
]) were low in mussels fed the commercial food. This profile might reflect lower
nutritional status in mussels fed commercial food and to a lesser extent, in mussels fed T.
chuii, which is known to affect biomarkers and biomarker responsiveness [
Furthermore, the presence of the food particles, which may vary depending on the food
type and regime, may interfere with the measurement of the biomarkers. For instance, LP
could not be determined in mussels fed I. galbana alone or in mixture with T. chuii because
the hexosaminidase activity used to visualize lysosomes in digestive cells could not be easily
discriminated against the background of brownish coloration of the lipofuscins and
microalgae pigments. Likewise, VvLYS and NvLYS could not be reliably measured in mussels fed I.
galbana due to the presence of massive amounts of microalgae within digestive cells, which also
hampered any distinction between microalgae and lipofuscins. More subtly, biochemical
determinations (e.g. MDA and HNE in digestive gland) might include the contamination with
the algal-derived products potentially biasing the assessment of these biomarkers in the mussel
digestive gland tissue. Future studies are needed to determine alternative experimental and
analytical approaches to mitigate or reduce the diet-induced bias in biomarkers and their
assessment. Furthermore, the effects of the nutritional condition and the diet must be taken
into account in the biomarker assessment, as the potential effects of the nutrition are likely to
be pervasive and not limited to a single tissue type.
According to the present study, Best Available Practices for biomarker-based toxicological
experiments should include the appropriate selection and reporting of the food type and
feeding regime to achieve reliable and comparable experimental data on the biological effects of
17 / 25
pollutants. Commercial food based on frozen or freeze-dried diets might not be the best option
for feeding during toxicological experiments, similar to what was earlier shown for aquaculture
]. Live commercial phytoplankton might be a viable alternative, yet the
dietary microalgae should be selected on the basis of the suitable dimension (size, volume,
weight) of algal cells, high digestibility and balanced nutritional value . Furthermore,
different live microalgae affect biomarkers in different ways. T. chuii that has low digestibility and
long gut retention times [
] appears to influence nutritional status, oxidative stress
and digestion processes in mussels. Alternatively, the massive presence of I. galbana within
digestive cells may hamper the measurement of fluorescent-based cytochemical biomarkers
and may bias biochemical biomarkers due to the high abundance of the algae in the mussel
tissue. Interestingly, at low dietary cell concentrations of I. galbana (2?104 cells/mL) the
occurrence of microalgae within digestive cells is negligible [
]; however, rations over 2?104 cells/
mL are recommended and commonly used in physiological experiments [
research is needed to optimize dietary food type, composition, regime and rations for
toxicological experimentation. Meanwhile, it is important that research papers include a detailed
description of the food type and feeding conditions to aid in comparison and interpretation of
the biological responses elicited by pollutants in mussels.
S1 Fig. Cytochrome-c-oxidase (COX) activity in gills (A) and protein carbonyl groups
(CO) in digestive gland (B); as recorded in mussels fed ad libitum for 1 week with 4 different
diets (I. galbana (I); T. chuii (T); mixture of I. galbana and T. chuii (I+T); and commercial
S1 Table. The effects of food type (d.f. = 3) on biomarkers in mussels fed ad libitum for 1
week with 4 different microalgae diets (I. galbana; T.chuii; I. galbana + T. chuii mixture;
and commercial food).
Our special thanks to the undergraduate visiting student Merle U?cker for her invaluable
assistance during experimentation.
Conceptualization: Esther Blanco-Rayo?n, Ionan Marigo?mez, Urtzi Izagirre.
Data curation: Esther Blanco-Rayo?n, Urtzi Izagirre.
Formal analysis: Esther Blanco-Rayo?n, Urtzi Izagirre.
Funding acquisition: Ionan Marigo?mez.
Investigation: Esther Blanco-Rayo?n, Anna V. Ivanina, Ionan Marigo?mez.
Methodology: Esther Blanco-Rayo?n, Anna V. Ivanina, Inna M. Sokolova, Ionan Marigo?mez,
Project administration: Urtzi Izagirre.
Resources: Anna V. Ivanina.
Supervision: Anna V. Ivanina, Inna M. Sokolova, Ionan Marigo?mez, Urtzi Izagirre.
18 / 25
Validation: Inna M. Sokolova.
Writing ? original draft: Esther Blanco-Rayo?n, Ionan Marigo?mez. Writing ? review & editing: Esther Blanco-Rayo?n, Anna V. Ivanina, Inna M. Sokolova, Ionan Marigo?mez, Urtzi Izagirre.
19 / 25
galloprovincialis. Chemosphere. 2017; 169: 493?502. https://doi.org/10.1016/j.chemosphere.2016.
11.093 PMID: 27894055
20 / 25
21 / 25
22 / 25
23 / 25
98. Bergmeyer HU. Methods of enzymatic analysis. Vol VI. Metabolites 1: Carbohydrates. Vol. VIII.
Metabolites 3: Lipids, Amino Acids and Related Compounds. VCH Verlagsgesellschaft, Weinheim;
24 / 25
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