Shotgun proteomics analysis discards alkali labile phosphate (ALP) as

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Shotgun proteomics analysis discards alkali labile phosphate (ALP) as a reliable method to assess vitellogenin levels in Mytilus galloprovincialis Paula Sanchez-Marin, Laura E. Fernández-González, Leonardo Mantilla-Aldana, Angel P. Diz, and Ricardo Beiras Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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Environmental Science & Technology

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Shotgun proteomics analysis discards alkali

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labile phosphate (ALP) as a reliable method to

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assess vitellogenin levels in Mytilus

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galloprovincialis

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Paula Sánchez-Marín1,2,*, Laura E. Fernández-González1,2,3, Leonardo Mantilla-

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Aldana1,2, Angel P. Diz1,3,# and Ricardo Beiras1,2, #.

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1

Toralla Marine Science Station (ECIMAT), University of Vigo, Illa de Toralla, E-

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36331, Vigo, Galicia, Spain.

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2

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of Vigo, E-36200, Vigo, Galicia, Spain.

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University of Vigo, E-36200, Vigo, Galicia, Spain.

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#

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*Corresponding author phone and e-mail: (+34)986818780; [email protected].

Department of Ecology and Animal Biology, Faculty of Marine Sciences, University

Department of Biochemistry, Genetics and Immunology, Faculty of Biology,

Authors with equal contribution.

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1 ACS Paragon Plus Environment


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ABSTRACT

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Vitellogenin, the egg yolk precursor, is a well known biomarker of endocrine disruption

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in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last

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10 years for the same purpose, despite the limited knowledge of invertebrate

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endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect

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technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most

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abundant phosphorylated protein in the analyzed tissue. In this study, we applied

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shotgun proteomics for the identification and quantification of vitellogenin in marine

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mussel gonads and compared the results with those obtained with the ALP method. The

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proteomic analysis revealed that vitellogenin is only detected in female gonads with

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expression levels that are rather variable among female mussels at different stages of

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gonad development. ALP analysis, on the contrary, detected similar amounts of

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phosphorylated proteins regardless sex or gonad development stage. These results show

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evidence that the ALP method is not providing reliable information about Vtg levels, at

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least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine

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mussels and careful verification of the adequacy of the procedure should be done before

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ALP is further assumed as a proxy of Vtg in other bivalve mollusks.

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KEYWORDS

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Mytilus, endocrine disruption, biomarker, proteomics, mass spectrometry analysis,

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label-free quantification

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TOC/ABSTRACT ART

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INTRODUCTION

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In the marine environment, the blue mussel (including species of the Mytilus edulis

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complex) is widely used as a sentinel species in pollution biomonitoring because it is

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sessile, ubiquitous, filters high volumes of water and accumulates pollutants in its

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tissues. Its use in pollution monitoring studies includes not only the measurement of the

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bioaccumulated pollutants,1-2 but also the analysis of several biochemical biomarkers of

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exposure or effects, such as different antioxidant enzymatic activities,3 or physiological

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energetic endpoints.4 More recently, a biomarker of endocrine disruption has also been

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added to this list: this is alkali labile phosphate (ALP), an easy and inexpensive

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alternative for the indirect quantification of vitellogenin (Vtg), the egg-yolk precursor

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(reviewed in5-6). In oviparous vertebrates, Vtg is synthesized in the liver in response to

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estrogens in maturing females, from where it is transported to the gonads through blood.

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Vtg is normally undetectable in the plasma of males and juveniles, which makes its

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anomalous presence in males an excellent biomarker of estrogenicity and feminization

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in fishes.7-9 Vtg is usually measured in fish serum using an enzyme-linked

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immunosorbent assay (ELISA), which has the limitation of being species specific.

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When specific antibodies are not available for the test species, the ALP method has

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been proved to be useful for the estimation of Vtg levels in fish serum,10 under the

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assumption that Vtg is the most abundant phosphorylated protein in this fluid.11

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The ALP assay was also modified for its use in mollusks, and applied in exposure

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studies that showed increases in ALP levels in females of freshwater mussels and clams

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that were injected 17β-estradiol or estrogen mimics such as nonylphenol. 12-13 Since

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then, the ALP assay has been widely applied in several mollusk species, including blue

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mussels.5-6 Concerning marine mussels, different studies using ALP as a proxy for

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measuring Vtg levels have shown contrasting results. One study showed increases in 4 ACS Paragon Plus Environment

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ALP levels in male and female mussels upon exposure to nonylphenol,14 while other

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studies failed in detecting changes in ALP upon exposure to a related chemical,

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bisphenol A.15-16 Other studies with the Mytilus family have used ALP in the evaluation

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of the effects of xenobiotics with different modes of action, such as hydrocarbons or

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pharmaceuticals. In these studies, increases or decreases in ALP as a response to those

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chemicals were also observed, and the influence of sex in the response was very

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variable, being in some cases only male or female specific, while in other cases both

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sexes were affected. 17-19 Surprisingly, no attempt has been made to relate ALP levels

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with exposure to natural or synthetic estrogens in marine mussels, so it is not confirmed

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if changes in ALP levels can be attributed to estrogenic or androgenic effects of

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contaminants. An alternative technique to study Vtg induction showed that Vtg mRNA

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expression increased several orders of magnitude in both male and female mussels upon

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exposure to natural and synthetic estrogens only when the mussels were at early

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gametogenesis stage.20 In situ hybridization has shown that Vtg is only detected in the

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female gonad, suggesting that this is the primary site of both synthesis and

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accumulation. 21 However, since ALP can be measured in any tissue, ALP levels have

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been reported in haemolymph, 22 digestive gland 14 and both unexposed male and

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female gonads 23 in Mytilus. Recent studies have shown that the most abundant

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phosphorylated protein present in mussel haemolymph –and reported in ALP

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measurements– was in fact extrapalial protein precursor, and not Vtg.24 Therefore, the

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usefulness of ALP for the detection of Vtg in mollusks has been questioned, and more

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reliable methods are needed before this protein can be used as a biomarker of endocrine

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disruption in marine mussels.

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The present study tested whether results from ALP method are a good proxy for Vtg

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levels in mussel gonads, the primary site of Vtg production and accumulation, using a 5 ACS Paragon Plus Environment


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shotgun label-free proteomics approach. To this aim, several male and female mussel

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gonads were analyzed for Vtg protein identification and quantification, and results

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discussed in terms of the suitability of this protein as a valid biomarker for the detection

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of effects caused by estrogenic endocrine disruptors in marine mussels.

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EXPERIMENTAL

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Sampling and histological analysis

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Mussels (Mytilus galloprovincialis) between 40 and 50 mm length were collected in

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unpolluted areas in the Galician Rías (NW Iberian Peninsula) in two different seasons,

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representing different stages of the gametogenesis cycle: December 2014, when mussels

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are mainly in the early gametogenesis stage, and in the pre-spawning season, April

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2015, when most mussels are fully mature.25

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Once in the laboratory, mussels were opened with a scalpel and the mantle-gonad was

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excised and split in three (December) or two (April) fragments. Two of the gonad

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fragments were introduced in microtubes, quick-frozen in liquid nitrogen and conserved

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at -80 ºC until further processing for ALP and proteomic analysis. The third gonad

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fragment was used for determination of sex and gonad gametogenesis stage after

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standard histological processing using haematoxylin-eosin stain. For individuals

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collected in April, sex and gametogenesis stage were determined by direct observation

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(size of the gonad, color and texture) and confirmed by observation of a gonad smear

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under the microscope.

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ALP analysis


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ALP extraction was performed following a method modified from Gagné et al. 26 Tissue

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samples were homogenized on ice with micropestles in 1:5 volume of cold (4 ºC)

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homogenization buffer (25 mM Hepes, 125 mM NaCl, 1 mM dithiothreitol and 1 mM

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EDTA; pH 7.4), centrifuged (12,000 g, 30 min, 2 ºC) and the supernatant collected. An

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aliquot was taken for total protein determination using the Bradford method27 adapted to

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microplate, using bovine serum albumin (BSA) as standard. Another aliquot was

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adjusted to 35% acetone, let on ice during 10 min, and centrifuged (10,000 g, 5 min, 2

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ºC). The pellet was dissolved in 1 M NaOH at 60 ºC during 30 min to hydrolyze the

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phosphate groups. Before the analysis of inorganic phosphate, a last step was

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introduced as a modification of the Gagné et al. procedure in order to eliminate turbidity

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interferences (see text S1 in supporting information). Trichloroacetic acid (TCA) 6.1 N

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was added to the samples, in a dose of 25 µL per 100 µL of sample, and samples were

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centrifuged (12,000 g, 15 min). Analysis of released inorganic phosphate was

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performed in the supernatant. This last step prevents the formation of turbidity that

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interferes with the spectrophotometric analysis of phosphate if TCA is directly added to

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the sample in the spectrophotometric well (see text S1 and Figure S1).

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Measurement of phosphate was performed using a spectrophotometric method. In

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preliminary tests (see text S1), the phosphomolybdenum method 28 was used when the

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concentration of phosphate in the sample was between 5-200 mg/L and for lower

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concentrations, between 0.2 - 2 mg/L PO43- , the malachite green method29 was used

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instead. The malachite green method was the preferred method afterwards due to the

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low phosphate concentration of most samples.

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Phosvitin (from egg yolk, Sigma) (8-10% P) was used as internal standard for quality

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control. The recovery of PO43- from phosvitin was 96% assuming a P content of 9%.

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Details on the preliminary tests, quality control and comparison with the results 7 ACS Paragon Plus Environment


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obtained with the original method –without turbidity removal– are given in the

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supporting information (Text S1).

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Proteomic analysis

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Protein extraction and quantification. Protein extraction was performed as previously

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described,30 by sonication on ice, in 1:5 vol of lysis buffer (7 M urea, 2 M thiourea and

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4% CHAPS), followed by centrifugation (20,000 g, 30 min, 4 ºC) and supernatant

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storing at -80ºC until electrophoresis. Total protein concentration was measured with

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the Bradford method adapted to microplate, using a similar matrix (10% lysis buffer) in

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both BSA standards and samples.

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1-D gel electrophoresis. In a first analysis, 20 µg of protein from a mature male and a

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mature female gonad were separated according to their molecular weight by 12% SDS-

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PAGE electrophoresis, and stained using OrioleTM fluorescent gel stain (Bio-Rad

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Laboratories, USA). 1-D gels were visualized on a blue-light DarkReader (Clare

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Chemical Research, CO, USA). EZ-Run Protein Marker (Fisher BioReagents, NJ, USA)

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was used as molecular weight marker. The gel was fractionated in 7 sections according

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to the protein's molecular weight (MW) (>116 kDa, 116-66.2 kDa, 66.2-45 kDa, 45-35

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kDa, 35-25 kDa, 25-18.4 kDa, and 116 kDa fraction,

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we proceed to Vtg quantification using a label-free MS analysis approach in different

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samples focused only on the highest MW gel fraction.

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Tryptic digestion. Gel sections were cut in small pieces, washed with ultrapure water

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and acetonitrile, and proteins reduced by treatment with 10 mM dithiothreitol and

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alkylated with 100 mM iodoacetamide. Samples were then dehydrated in acetonitrile,


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rehydrated in 25mM ammonium bicarbonate solution followed by addition of

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acetonitrile, and dried at room temperature. Forty µl of sequencing-grade modified

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trypsin (12.5 ng µl-1; Promega, Madison, WI) in 25 mM ammonium bicarbonate

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solution was added to each sample, kept on ice during 45 min, followed by an

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incubation at 37 ºC overnight. Peptides were recovered and transferred to a clean

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microtube using 0.1% formic acid/50% acetonitrile and dried under speed-vacuum

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(Concentrator plus, Eppendorf, Hamburg, Germany).

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Mass spectrometry (LC-MS/MS) analysis. Tryptic digests were acidified with 0.5%

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formic acid and analysed in a LTQ-Orbitrap Elite mass spectrometer coupled to a

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Proxeon EASY-nLC 1000 UHPLC system (Thermo Fisher, San Jose, CA). For protein

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identification, MS/MS spectra were searched using PEAKS Studio v7.0 software

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(Bioinformatics Solutions Inc., Waterloo, Canada) against two customized protein

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databases: one containing 411,509 non-redundant protein sequences of mollusks

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retrieved from NCBI in 11th May of 2016, and another one resulting from the six-frame

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translation of 151,320 sequences from available M. galloprovincialis transcriptomes

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(RNA-seq data) from different tissues, including the mantle-gonad. 31 This database

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contains two sequences that were annotated as vitellogenin by sequence homology with

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other species (CL1077.Contig3_All_5 and Unigene21_All_6). The second one

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(Unigene21_All_6), hereafter referred to as "the M. galloprovincialis Vtg sequence",

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was used for the identification and quantification of Vtg and included also in the

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database retrieved from NCBI. A list of common contaminants was also added to both

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databases, and a decoy sequence database was included in the analysis to calculate the

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false discovery rate (FDR). All cysteines were considered as fully

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carbamidomethylated, while methionine oxidation was searched as a variable

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modification. Positive protein identifications were only accepted when the number of 9 ACS Paragon Plus Environment


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matched peptide sequences > 2, unique peptides > 1, peptide spectrum matches (PSMs)

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FDR < 0.1%, and protein identification PEAKS score > 20.32

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Label-free quantification of vitellogenin. The relative abundance of Vtg in the different

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samples was estimated using the normalized spectral abundance factor (NSAF),33 using

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the spectral counts derived from protein identifications against the customized protein

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database generated from RNA-seq data (see above). This was done using PatternLab for

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proteomics 4.0.34 Positive protein identifications were only accepted when the number

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of matched peptide sequences > 2, unique peptides > 1, PSMs FDR < 3%, peptide FDR

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< 2% and protein FDR < 1 %. Calculation of fold change was performed by dividing the

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mean NSAF value of Vtg in each treatment by the mean NSAF value of Vtg in the

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treatment with the lowest value.

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Statistics

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Data normality and homoscedasticity was tested by the Shapiro-Wilk and Levene tests

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respectively before carrying out an analysis of variance (ANOVA) followed by the post

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hoc Tukey HSD when multiple treatments were compared. Statistical analysis were

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performed with IBM SPSS Statistics 23.

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RESULTS AND DISCUSSION

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Gametogenesis stage

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The individuals collected in April, hereafter referred to as "mature", presented very

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thick, uniform gonads with no signs of spawning. The gonads were white or cream-

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colored in the case of males and sperm poured easily when the gonads were lightly

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pressed with a scalpel. Female gonads were orange-colored and with a granular texture.


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The smear observation revealed spherical full grown oocytes in the case of females and

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mobile sperm in the case of males.

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Individuals collected in December, hereafter referred to as "immature", presented very

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thin mantle tissues that were almost transparent. Histological observations revealed the

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presence of very few follicles embedded in the connective tissue, and presented germ

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cells at different stages of development. Gametogenesis stage was classified in the

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histological stage I or II according to classification given in Martinez-Castro and

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Vazquez.35 Histological sections of selected specimens (representative of these

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gametogenesis stages) are shown in the supporting information (Table S2).

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ALP measurements

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Results of ALP analysis are presented in Table 1. ALP data normalized by total protein

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was quite similar for all mussel groups, regardless sex or gametogenesis stage, with the

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only exception of mature females, which showed significantly lower ALP levels than

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males. These values are not expected under the assumptions that PO43- released in ALP

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measurements would come mostly from Vtg proteins, since Vtg proteins are supposed

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to be mainly, if not only, synthesized in female tissues, and should be higher in mature

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individuals.36 Other studies have also found similar or even higher ALP values in males

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compared to females samples of different bivalve mollusks,37-39 including blue

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mussels15, 18, under control conditions. It has been proposed that these unexpected

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results are explained by over-normalization due to the higher protein content of females

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at advanced gametogenesis stages, and normalization by tissue weight was proposed to

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solve this problem.36 ALP values normalized by tissue weight are indeed around twice

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for mature females compared to mature males (Table 1), and higher for mature than for

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immature individuals, but still there are not differences between males and females

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when mussels are not mature. Despite this normalization gives values that are more 11 ACS Paragon Plus Environment


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similar to the expected if variation in ALP data would be mainly explained by

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differences in Vtg levels, it is important to note that they follow exactly the same

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pattern as total protein contents in mantle tissue, as confirmed by a statistically

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significant high correlation coefficient among both variables (ALP normalized by tissue

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weight vs total protein; r = 0.954; p = 0.046). It seems that the higher the amount of

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protein extracted, the higher the amount of PO43- extracted from those proteins. This

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phosphate may come from any phosphorylated proteins in the sample, indicating that

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the basal levels of phosphorylation in gonads might be similar or higher than those of

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Vtg, which compromises the use of ALP for Vtg determination in these tissues.

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Other studies have shown higher ALP values (normalized by total protein) in females

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than in males of blue mussels17 and other bivalves,13 as well as covariations between

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ALP levels and gametogenesis cycle.40,12 These results are not concordant with others

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provided in different studies,15, 18, 37-39 nor with those from the present study. This

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important discrepancy among studies provide evidence that reported ALP levels show

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low reproducibility due to high technical variation. In the present study, the

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methodology was carefully examined to assure that ALP values are correct and not

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overestimated due to sample turbidity (Text S1).

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Identification of vitellogenin by shotgun proteomics

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The protein sequence of M. galloprovincialis vitellogenin, consists on a 2828 amino

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acids length sequence and presents homology with other vitellogenins described so far

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for mollusks. It should be noted, however, that the last ~200 amino acids are not present

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in other Vtg sequences, neither detected in LC-MS/MS analysis (see below), indicating

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that they probably correspond to a 3' UTR (untranslated region). An NCBI-BLASTp

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search41 reports the highest alignment scores (> 800) and lowest E-values (< 10-141 )

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with Vtg sequences described for other marine bivalves, such as Pecten maximus, 12 ACS Paragon Plus Environment

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Saccostrea gigas and Mizuhopecten yessoensis, with >55% of coverage and 30 to 40%

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identity for complete sequences. An almost perfect alignment (99% identity) was also

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found with the partial sequence of M. galloprovincialis Vtg available at NCBI

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(accession number AHB63226.1), that only covers a 4% of the total protein length. It

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also presents 94% identity with a Vtg sequence of 2593 amino acids length obtained

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from a first release of the M. galloprovincialis genome.42 The alignment of the complete

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M. galloprovincialis Vtg sequence with that of P. maximus (the one showing the lowest

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E-value) is presented in Figure 1.

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LC-MS/MS analysis of proteins extracted from the gonad of a mature female detected

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Vtg with a coverage of 66% (280 peptides -all unique-, including post-translational

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modifications, and 3100 peptide spectrum matches) (see Fig. 1 and supporting file 1).

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Vtg was detected mainly in the >116 kDa fraction of the gel, which is in agreement with

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the expected molecular weight of the protein, 326 kDa. On the contrary, none of the

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peptides detected in the male gonad correspond to the M. galloprovincialis Vtg

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sequence, neither against any other vitellogenin or vitelin-like sequence when the

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analysis was repeated against the NCBI database for mollusks.

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Subsequent proteomic analysis were only performed in the >116 KDa fraction of the

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electrophoresis gel.

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Label-free quantification of vitellogenin by shotgun proteomics

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Results of the label-free LC-MS/MS quantification of Vtg are represented in Table 2.

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We did not find any evidence of Vtg in male gonads. On the contrary, we provide

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evidence of the presence of Vtg in female gonads in an amount that is largely dependent

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on the gametogenesis stage of mussel. Mature females presented 8.5 times more Vtg

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than immature females (in stage II of gonad development). Immature females in stage I 13 ACS Paragon Plus Environment


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of gonad development (which do not present vitellogenic oocytes)35 did not present

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enough amount of Vtg to be detected by the methodology used in this study, despite

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there is evidence of its synthesis in adipogranular and follicle cells of female gonads at

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early stages of development. 21 According to mean NSAF values, Vtg represents around

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0.9% of the detected gonad proteins in the >116 KDa fraction for females in early

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gametogenesis, and around 7.5% for mature females.

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The fact that Vtg is only detected in female gonads at advanced stages of gametogenesis

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is in concordance with a study using in situ hybridization of Vtg in Crassostrea gigas,

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that shows the presence of this protein only in follicle cells of females in late

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vitellogenesis or ripe state.43 In contrast, similar contents of Vtg were detected by HPLC

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in males and females of the species Saccostrea glomerata, both unexposed and after

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exposure to xenoestrogens.44-45 The technique used in the present study is highly

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sensitive.46 In fact, from 108 to 242 proteins were detected only in the >116 KDa

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fraction of female gonad samples, disregarding gametogenesis stage, and from 141 to

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223 in male samples. Despite this, we were unable to detect Vtg neither in males nor in

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females at the very early stage of gametogenesis (stage I, see Table S2 in supporting

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information). Therefore, Vtg is either absent or expressed at extremely low levels in the

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gonads of male individuals that were not exposed to estrogens. It would be interesting

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in the future to test if Vtg synthesis is induced upon exposure to estrogens, and in case

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this is confirmed in males, it would enable the use of Vtg as a valid biomarker for

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endocrine disruption in this species. In fact, it has been already shown that Vtg mRNA

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expression was induced both in males and females of the closely related species M.

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edulis upon exposure to 17β-estradiol and synthetic estrogens.20 It is still unknown

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whether or not this mRNA is effectively translated to proteins in males. A partial

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sequence of Vtg was also found in an unpublished RNA-seq project from M. edulis and 14 ACS Paragon Plus Environment

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M. galloprovincialis mature male gonads (Romero et al., manuscript in preparation), but

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the Vtg protein was not found within highly expressed proteins in M. edulis sperm.47

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Comparison of the indirect method (ALP) with direct Vtg quantification

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The disagreement among the indirect ALP method and direct Vtg quantification by

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shotgun proteomics is evident when comparing Fig. 2a and Fig. 2b. Since Vtg was so

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low in females in stage I of gametogenesis that it could not be detected by LC-MS/MS,

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only females in stage II were chosen for this comparison, and therefore ALP values

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change slightly compared to those presented in Table 1. In any case, there were not

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significant differences in ALP values of females in stage I (7 ± 3 mg PO43- / g of

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protein; n = 4) or II (4 ± 1 mg PO43- / g of protein; n = 6).

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The results of both methods are in high disagreement since ALP levels were as high in

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males as in females, despite Vtg being undetectable in males by LC-MS/MS. Regarding

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females, mature individuals presented 8.5 fold more Vtg than immature ones, while

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ALP values were again similar for both gametogenesis stages. ALP normalization by

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tissue weight does not represent a significant improvement in the results, since high

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ALP levels are still observed in mature males, that exceed those observed in immature

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females, despite LC-MS/MS analysis shows the opposite trend (Figure S3, supporting

329

information). This clearly indicates that ALP is not suitable as a proxy of Vtg in mussel

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gonads.

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In fish plasma, significant correlations have been found among ALP levels and Vtg

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measured by direct techniques such as enzyme immunoassays,10, 48 but similar

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correlations have not yet been shown for mollusks, despite this has been frequently

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assumed. On the contrary, no correlation was observed between ALP levels and yolk

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protein levels (measured by ELISA) in haemolymph of the freshwater mussel Unio



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tumidus, and, as occurred for blue mussels, ALP levels did not present differences

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depending on the sex, while ELISA detected higher yolk protein levels in females.49 It

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seems clear that although ALP was a suitable technique for fish plasma, where Vtg is

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the most abundant phosphorylated protein,48, 50 this seems far from being the case for

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mollusk tissues. Indeed, the ALP method was shown to be unsuitable for the

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measurement of Vtg-like proteins in gastropods, masked by the background level of

342

phosphorylation of other proteins that fractionate in acetone.51 The origin of ALP in M.

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galloprovincialis haemolymph was revealed to be extrapallial protein precursor, and not

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Vtg,24 and the present results reveal that ALP in M. galloprovincialis gonads may well

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come from different proteins, also extracted with acetone fractionation, with a

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phosphorylation level that might be higher or similar to that of Vtg, as evidenced by the

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high ALP values observed in males, and no evidence of Vtg expression on the basis of

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LC-MS/MS analysis. Protein phosphorylation is indeed a common process initiated by

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numerous factors,52 so this result is not surprising at all.

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Not only there might be another phosphorylated proteins in mollusks gonads, but also, it

351

is reasonable to question if mollusks' Vtgs are also highly phosphorylated as those of

352

vertebrates,49 which is the main basis for the application of the ALP methodology.

353

Vertebrate Vtgs are highly phosphorylated especially because of the presence of high

354

serine contents in the phosvitin moiety, but invertebrate Vtgs seem to lack this phosvitin

355

domain.53 This is the case for the oysters Saccostrea glomerata and Crassostrea gigas,54

356

although their serine contents (12-14%) are comparable to that of vertebrate Vtgs.53-54

357

According to Gagné,36 Vtg from Mytilus edulis has about 14.8% of potential

358

phosphorylation sites (serine and threonine) , slightly higher than the mean serine and

359

threonine contents in eukariotic proteins (12.9%).53 Similarly, the M. galloprovincialis

360

Vtg sequence has 9.8% serine and 3.7% threonine in its composition, and the final 16 ACS Paragon Plus Environment

Page 17 of 34


361

degree of phosphorylation of the protein –depending on the level of phosphorylation

362

that is actually achieved during post-translational modifications– is predicted to be 11%

363

on the basis of amino acid sequence. 55-56

364

The present results clearly showed that ALP was not suitable as a proxy for Vtg in this

365

species, probably due to a high ratio of background to Vtg phosphorylation. It is highly

366

probable that this is the case for other bivalves too, that may show a similar proteomic

367

composition in their gonads, so careful verification of the adequacy of the procedure

368

should be done before ALP is further assumed as a proxy of Vtg in bivalve mollusks.

369

As already noted by Morthorst et al.,49 in agreement with this study, the assumption that

370

ALP measurements reflected yolk protein levels in bivalves has been the foundation for

371

more than 50 publications, whose conclusions should be carefully revisited.

372

Environmental implications

373

There is a wide variety of chemicals with confirmed or suspected endocrine disruptor

374

ability,57 that are spilled in the environment and can reach coastal areas through

375

effluents of wastewater treatment plants, runoff or deposition. Deleterious effects of

376

xenoestrogens have been reported in recent years in European estuaries, and bivalve

377

molluscs exhibiting intersex, i.e. the simultaneous presence of both testicular and

378

ovarian tissue in the male gonad, are increasing in number and sites affected.58-59 Due to

379

the difficulty in analyzing and monitoring this myriad of chemicals, it is therefore

380

desirable to count with an early-warning biomarker of endocrine disruption in marine

381

biota.

382

Concerning mollusks, very little is known about their endocrinology, and despite it has

383

been frequently assumed that reproductive hormones play similar roles as those

384

described for vertebrates, the topic is under debate,60-61 and it has not been



Page 18 of 34

385

unequivocally demonstrated that vertebrate-like hormones such as estrogens play any

386

role in reproduction in mollusks, and it has been also proposed that these processes are

387

regulated by other mechanisms, such as by neuropeptides.62 Some recent studies

388

showed evidence supporting the theory that the estrogen receptor (ER) plays a role in

389

mollusks' reproduction and that ER and Vtg mRNA expression are induced in response

390

to estrogens.20, 54, 63-65 Also, direct Vtg analysis by HPLC showed that its synthesis is

391

induced in oysters in response to estrogens.44-45 More studies are needed to better

392

understand the mechanism of estrogen signaling in mollusks' endocrinology and its role

393

in Vtg induction. In particular, the analysis of Vtg using robust techniques (such as LC-

394

MS/MS) will be very useful in this research process, especially to confirm if the

395

upregulation of Vtg mRNA is reflected at the protein level.66

396

Also, Vtg analysis will be of interest in the quantitative determination of gametogenesis

397

stage in females, or for other applications, given the other non-reproductive functions

398

attributed to Vtg.67-68 If less expensive techniques are preferred for routine analysis of

399

Vtg in marine mussels, the use of other techniques such as immunoassays should be

400

explored, given that ALP is not a reliable alternative.

401 402

Supporting information

403

Details on the standardization and quality control of ALP analysis, including

404

comparison with the results obtained with the original method (without turbidity

405

removal) and full absorbance spectra. A table showing histological sections of gonads

406

representative of stage I and II of male and females mussels. An image file (supporting

407

file 1) obtained from PEAKS Studio v7.0 software showing the coverage detected by

408

LC-MS/MS of the Vtg sequence in a female gonad including PTMs.


Page 19 of 34


409

410

ACKNOWLEDGEMENTS

411

LC-MS/MS analysis were performed in the CACTI and histological analysis were

412

performed in the ECIMAT, both centers belonging to University of Vigo, by Manuel

413

Marcos (LC-MS/MS) and Rosana Rodriguez (histology). Carlos Canchaya provided us

414

with a Vtg sequence obtained from the M. galloprovincialis genome project. Mónica

415

Rodriguez-Romero is also acknowledged for technical assistance. This study was

416

funded by the Spanish Government through projects CTM2013-48194-C3-3-R and

417

PCIN-2015-187-C03-03, and by "Consellería de Educación e Ordenación Universitaria"

418

Xunta de Galicia (Galician Regional Government), with cofunding from the European

419

Regional Development Fund (ERDF). P.S.-M. was supported by the People Programme

420

(Marie Curie Actions) of the European Union's Seventh Framework Programme

421

(FP7/2007-2013) under REA grant agreement n° 600391 and A.P.D. was supported by

422

the Spanish “Ministerio de Economía y Competitividad” (code AGL2014-52062-R),

423

Fondos Feder and Xunta de Galicia (“Grupos de Referencia Competitiva” ED431C

424

2016-037)".



Page 20 of 34

425

TABLES

426

Table 1. Levels of ALP measured in M. galloprovincialis gonad tissues, normalized

427

by protein or by tissue weight, and total protein extracted per gram of tissue. Mussel's group

ALP (µg PO43- / mg protein)

ALP (µg PO43- / g tissue)

Total protein (mg / g tissue)

Mature females

2.9 ± 0.8 a

221 ± 51 a

78 ± 7

Mature males

6.5 ± 2.9 b

122 ± 53 b

19 ± 2

Immature females

5.2 ± 2.6 ab

52 ± 29 c

11 ± 5

Immature males

5.5 ± 1.9 b

43 ± 14 c

8±2

428

Mean ± SD is represented (n = 10).

429

Values sharing the same superscript letter do not present significant differences among

430

them (HSD Tukey, α = 0.05).

431


Page 21 of 34


432

Table 2. Proteins detected, Vtg spectral counts and NSAF values obtained from

433

LC-MS/MS analysis in the > 116 kDa fraction of proteins extracted from M.

434

galloprovincialis gonad tissues (three biological replicates per treatment).

435

Mussel's group

Nº Proteins detected

Vtg spectral counts

Vtg NSAF

Mature females

108 242 177

434 1182 801

0.0708 0.0781 0.0749

Mature males

162 141 195

0 0 0

n.a. n.a. n.a.

Immature females (stage II)

232 139 111

143 79 4

0.0141 0.0113 0.0006

Immature males

159 211 223

0 0 0

n.a. n.a. n.a.

n.a. = not applicable due to zero values in spectral counts.

436



Page 22 of 34

437

FIGURE CAPTIONS

438

Figure 1. Alignment of the M. galloprovincialis vitellogenin sequence (obtained from

439

the RNA_seq sequence Unigene21_All_6 )31 with that showing the best identity (lowest

440

E-value) in NCBI-Blast search, Pecten maximus (GenBank: CAQ06469.2). Red lines

441

represent the peptide coverage detected by LC-MS/MS in a M. galloprovincialis mature

442

female gonad sample.

443

444

Figure 2. Comparison of (a) indirect quantification of Vtg using ALP and (b) direct

445

quantification using LC-MS/MS in gonads of Mytilus galloprovincialis females (black

446

bars) or males (white bars) in different stages of gametogenesis: stage II of

447

gametogenesis (Immature) and fully mature individuals (Mature). The absent bars

448

corresponding to males in figure (b) correspond to 0 values (undetected Vtg).

449


Page 23 of 34


450

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Figure 1. Alignment of the M. galloprovincialis vitellogenin sequence (obtained from the RNA_seq sequence Unigene21_All_6 31) with that showing the best identity (lowest E-value) in NCBI-Blast search, Pecten maximus (GenBank: CAQ06469.2). Red lines represent the peptide coverage detected by LC-MS/MS in a M. galloprovincialis mature female gonad sample. 664x1436mm (72 x 72 DPI)

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Figure 2. Comparison of (a) indirect quantification of Vtg using ALP and (b) direct quantification using LCMS/MS in gonads of Mytilus galloprovincialis females (black bars) or males (white bars) in different stages of gametogenesis: stage II of gametogenesis (Immature) and fully mature individuals (Mature). The absent bars corresponding to males in figure (b) correspond to 0 values (undetected Vtg). 331x209mm (150 x 150 DPI)

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Shotgun proteomics analysis discards alkali labile phosphate (ALP) as

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