Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis

Accepted Manuscript Title: Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis Author: M. Roussel A Villay F Delbac P Michaud C Laroche D Roriz H El Alaoui M Diogon PII: DOI: Reference:

S0144-8617(15)00651-7 http://dx.doi.org/doi:10.1016/j.carbpol.2015.07.022 CARP 10124

To appear in: Received date: Revised date: Accepted date:

22-12-2014 30-6-2015 2-7-2015

Please cite this article as: Roussel, M., Villay, A., Delbac, F., Michaud, P., Laroche, C., Roriz, D., Alaoui, H. E., and Diogon, M.,Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.07.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Highlights (for review)

► Sulphated polysaccharides were extracted from Porphyridium sp. ► Two polysaccharides decreased the growth of Encephalitozoon cuniculi in vitro. ► Honeybees were infected by the microsporidia Nosema ceranae. ► The polysaccharide from P. marinum decreased the infected-honeybee mortality. ► The polysaccharide from P. marinum reduced the parasite

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*Response to Reviewers

_____________________________________________________________________________________ LABORATOIRE MICROORGANISMES : GENOME ET ENVIRONNEMENT UMR CNRS 6023

Dr Hicham El Alaoui

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Laboratoire Microorganismes : Génome et Environnement - UMR CNRS 6023 Université Blaise Pascal, Bâtiment Biologie A 24 Avenue des Landais BP 80026 63171 AUBIERE Cedex – France Tel : 33.4.73.40.74.31 e-mail : [email protected]

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Aubière, june 18th, 2015

Dear Editor and editorial team,

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The reviewers did nice comments to improve the quality of our manuscript, and we thank them for their help. We have addressed below each of the issues raised by the reviewers in a point-by-point reply. We hope that changes did in our amended manuscript will answer to the

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reviewer queries. All authors agree with this new version of our manuscript. Here are our answers to all comments: the reviewer comments are indicated in black and our

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responses in red :

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Reviewer #1: The manuscript presented a very interesting study which could potentially have big impact on a critic issue affecting the ecology of western honeybees. Indeed, recently, the number of bee colonies has decreased significantly due to unknown reason. One of the possibilities is due to nosemosis, which is caused by gut parasites Nosema species. Since fumagillin, the only one for nosemosis treatment is no longer licensed in some EU countries, there's a urgent need to search for new alternatives. In the current study, the authors isolated polysaccharides from algae, which were further characterized. Moreover, the effects of the polysaccharide on the mortality of honeybee infected with Nosema were further investigated. In addition, the experiments were well designed and executed. The conclusions made here were well supported by the data. The manuscript was well organized. Minor revision! 1. Please pay attention to the following sentence. It's not a good idea to include the latter part in abstract, namely " because fumagillin, the only product available for nosemosis treatment, is no longer licensed in several European countries".

This part of the sentence has been removed (line 29).

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3. Highlights should be rewritten so that it can get readers interested. It did not reflect the essence of the manuscript. They have been rewritten.

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Reviewer #2: Manuscript Number: CARBPOL-D-14-03195 The manuscript entitled as "Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis" is well written and well organized consecutively. The authors were evaluated 10 sulphated polysaccharides from algae for antimicrosporidian activity and they found as algal sulphated polysaccharides could be used to improve the survival of N. ceranae-infected honeybees and reduce the parasite load. There is very few research works on antimicrosporidian activity using polysaccharides, hence this work also possess significant novelty and it would more appropriate to Carbohydrate polymers journal. There is few corrections are needed to be done before the publication and the corrections and clarifications as follow:

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1. In methodology section, extraction of polysaccharides from microalgae culture media: How can you consider the whole extract as secreted polysaccharide and have you done any column chromatography or further purification methods. Cell free extract may contain a mixture of polymers, so authors should make it clear their detailed extraction procedures to the broad readership.

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Exopolysaccharides from red microalgae used in this study have been extracted from cell free broth by dialysis (removing of salts and low molecular weights compounds) as described in material and methods section of the revised manuscript. No chromatographic procedure of purification has been applied to separate macromolecules from a putative mixture of biopolymers because the polydispersity and the molecular homogeneity have been previously published by authors working with similar processes applied to the same red microalga (Geresh et al., 2002; Patel et al., 2013, Chen et al., 2009 as examples). - Geresh S., Adin I., Yarmolinsky E., Karpasas M. (2002). Characterization of the extracellular polysaccharide of porphyridium sp.: molecular weight determination and rheological properties. Carbohydrate Polymers 50: 183-189. - Patel K.A., Laroche C., Marcati A., Ursu V., Jubeau S., Marchal L., Petit E., Djelveh G., Michaud P. (2013). Separation and fractionation of exopolysaccharides from Porphyridium cruentum. Bioresource Technology 145:345-350. - Chen B., You W., Huang J.Yu Y., Chen W. (2009). Isolation and antioxidant properties of the extracellular polysaccharide from Rhodella reticulata. World Journal of Microbiology and Biotechnology 26(5): 833-840. 2. Is it partially purified or pure polysaccharides? As suggested by the reviewer “purified polysaccharide” as been replaced by “partially purified polysaccharide” in the revised manuscript (lines 142-143 and 275-276). 3. Why authors didn't conduct any chemical characterization like FTIR and also more reliable one as NMR for microalgae polysaccharides. Since, the number of polysaccharides is high around 10, but authors can exploit the structure of an effective polysaccharide because this will make more sensible to publish this work in Carbohydrate polymers journal.

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Authors didn’t conduct any chemical characterization (FTIR or 1H and 13C NMR) because these analyses have been previously published by other authors in literature and some structures are already available.

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Polysaccharide of Porphyridium species: -Gloaguen V., Ruiz G., Morvan H., Mouradi-Givernaud A., Maes E., Krausz P., Strecker G. (2004). The extracellular polysaccharide of Porphyridium sp.: an NMR study of lithium resistant oligosaccharidic fragments. Carbohydrate Research. 339 : 97-103 -Geresh S., Arad S., Levy-Ontman O., Zhang W., Tekoah Y., Glaser R. (2009). Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp. Carbohydrate Research. 344(3) : 343-9.

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Polysaccharide of Rhodella species: -Capek P., Matulova M., Combourieu B. (2008). The extracellular proteoglycan produced by Rhodella grisea. International Journal of Biological Macromolecules. 43: 390-393.

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Polysaccharide of Arthrospira -Trabelsi L., M’Sakni N.H., Ben Ouada H., Bacha H., Roudesli S. (2009). Partial characterization of polysaccharides produced by cyanobacterium Arthrospira platensis. Biotechnology and Bioprocess Engineering. 14: 27-31.

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Polysaccharide from Ulva: -Lahaye M., Alvarez-Cabal E., Kuhlenkamp R., Quemener B., Lognon V., Dion P. (1999). Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta). Journal of Applied Phycology. 11: 1–7.

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Polysaccharide from Halymenia: -Fenoradosoa T.A., Delattre C., Laroche C., Wadouachi A., Dulong V., Picton L., Andriamadio P., Michaud P. (2009). Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts. International Journal of Biological Macromolecules. 45 : 140–145.

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Carrageenans -Van de Velde F., Knutsen S.H., Usov A.I., Rollema H.S., Cerezo A.S. (2002). 1H and 13C high resolution NMR spectroscopy of carrageenans: application in research and industry. Trends in Food Science and Technology 13: 73-92. Looking forward to hearing from you, Yours sincerely,

Dr Hicham El Alaoui

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*Manuscript Click here to view linked References

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Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to

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control honeybee nosemosis

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Roussel M a,b, Villay A c,d, Delbac F a,b, Michaud P c,d, Laroche C c,d, Roriz D a,b, El Alaoui H a,b,*,

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Diogon M a,b,*

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Environnement », BP 10448, F-63000 Clermont-Ferrand, France.

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Ferrand, France.

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Clermont Université, Université Blaise Pascal, Laboratoire « Microorganismes : Génome et

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CNRS, UMR 6023, LMGE, F-63171 AUBIERE, France.

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Clermont Université, Université Blaise Pascal, Institut Pascal, BP 10448, F-63000 Clermont-

CNRS, UMR 6602, IP, F-63171 Aubière, FRANCE

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*

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LMGE, UMR CNRS 6023, Université Blaise Pascal, 24 Avenue des Landais 63177 Aubière

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Cedex, France. Tel.: +33 4 73 40 78 68, Fax: +33 4 73 40 76 70.

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E-mail

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[email protected],

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[email protected], [email protected], hicham.el_alaoui@univ-

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bpclermont.fr, [email protected]

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Corresponding authors: Hicham El Alaoui and Marie Diogon. Interactions Hôtes-Parasites,

addresses:

[email protected],

[email protected],

[email protected],

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Abstract

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Nosemosis is one of the most common and widespread diseases of adult honeybees. The causative

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agents, Nosema apis and Nosema ceranae, belong to microsporidia some obligate intracellular

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eukaryotic parasites. In this study, 10 sulphated polysaccharides from algae were evaluated for their

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antimicrosporidian activity. They were first shown to inhibit the in vitro growth of the mammal

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microsporidian model, Encephalitozoon cuniculi. The most efficient polysaccharides were then

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tested for their ability to inhibit the growth of Nosema ceranae in experimentally-infected adult

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honeybees. Two polysaccharides extracted from Porphyridium spp. did not show any toxicity in

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honeybees and one of them allowed a decrease of both parasite load and mortality rate due to N.

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ceranae infection. A decrease in parasite abundance but not in mortality rate was also observed with

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an iota carrageenan. Our results are promising and suggest that algal sulphated polysaccharides

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could be used to prevent and/or control bee nosemosis.

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Keywords: Apis mellifera, honeybee, Nosema ceranae, polysaccharides, antimicrosporidian

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activity, algae

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1.

Introduction

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The western honeybee Apis mellifera is widely recognized as a beneficial insect of agronomic and

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environmental importance (Gallai, Salles, Settele, & Vaissière, 2009). However, in the last decades,

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bee colony losses have been reported worldwide at alarming rates. While the causes remain unclear,

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they are likely to be multifactorial including pathogens and pests, invasive species, climate change,

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decrease in food resources, fragmentation of habitat and pesticides (Vanengelsdorp & Meixner,

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2010). Nosemosis is one of the most common diseases of the european honeybee and was long-time

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attributed exclusively to the gut parasite Nosema apis. A second species named Nosema ceranae

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was more recently described and is now the predominant microsporidian species in Apis mellifera.

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Interestingly, N. ceranae has been shown to have more negative impacts on honey bees than N. apis

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(Williams, Shutler, Burgher-MacLellan, & Rogers, 2014) and some studies provided evidence of a

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role of N. ceranae in colony losses alone (Higes et al., 2008), or in combination with other

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stressors such as pesticides (Aufauvre et al., 2012; Vidau et al., 2011), viruses (Bromenshenk et al.,

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2010). It has also been demonstrated that infection of honeybee colonies by Nosema may decrease

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the efficacy of acaricide treatment used to fight the mite Varroa destructor (Botías et al., 2012).

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Currently, the only treatment to control nosemosis, fumagillin, is forbidden in the majority of EU

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member states due to the absence of maximum residue limit (MRL) determination for honey

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(Reybroeck, Daeseleire, De Brabander, & Herman, 2012). Alternative therapeutic treatments have

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been experimented to control the disease, such as bacteriocin, itraconazole, benzoic and acetic

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acids, thymol, resveratrol or Artemisia extract (Forsgren & Fries, 2005; Liu & Myrick, 1989;

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Maistrello et al., 2008; Pohorecka, 2004; Porrini et al., 2010). However, all these treatments

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resulted in a small reduction in the number of parasites but did not protect from nosemosis. It is

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therefore of high interest to propose new strategies to prevent and control bee nosemosis. One

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strategy to prevent the infection would consist in the blocking of the invasion step by inhibiting the

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adhesion of the microsporidian spore to the host cell using mimetics of extracellular

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glycosaminoglycans (GAGs) located at the host cell surface. Indeed, spores need to be close to host

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cells in order to allow their polar tube to pierce the cell membrane and inject the spore contents.

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Like many pathogens, microsporidia may use specific receptors at the host cell surface for

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adherence that allows a successful infection.

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GAGs are linear and negatively charged polysaccharides ubiquitous in animals bodies and known

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for their multiple biological functions essential for life (Taylor & Gallo, 2006). Many of these

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biological functions and notably those of cellular signaling pathways are conferred by the unique

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chemical structure of GAGs, consisting of repeating disaccharide units that are specific for each

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GAG species. They are composed of uronic acid (L-iduronic acid or D-glucuronic acid) and

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hexosamine (D-glucosamine or D-galactosamine) sugars. Each sugar is differentially sulphated at

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the hydroxyl and/or amino positions. Sulphated GAG species such as chondroitin sulfate (CS),

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heparin, heparan sulfate (HS), dermatan sulfate (DS), and keratan sulfate (KS) bear negative

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charges that vary in density and position within the disaccharide units. The hyaluronic acid (HA) is

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not sulphated and therefore is the GAG with the least net negative charge (Griffin & Hsieh-Wilson,

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2013; Taylor & Gallo, 2006). Many studies have investigated the modulation of some biological

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responses using either native, chemically modified, synthetic or naturals GAG mimetics (Delattre,

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Fenoradosoa, & Michaud, 2011; Yip, Smollich, & Götte, 2006). In this context, some sulphated

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carbohydrates such as carrageenans or GAG mimetic blocks have been clearly identified as playing

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a role in both adhesion and invasion processes that occur between the malaria parasite Plasmodium

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falciparum and human host cells (Adams, Smith, Schwartz-Albiez, & Andrews, 2005; Mathias et

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al., 2013). Using sulphated glycans, Hayman et al. (2005) have suggested that the microsporidian

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Encephalitozoon intestinalis exploits cell surface glycosaminoglycans (GAGs) to detect and bind to

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host cell receptors. So, it appears that sulphated polysaccharides could be potential candidates to

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prevent microsporidian spore adherence to host cells and subsequent infection of these cells. In the

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natural environment, sulphated polysaccharides are abundantly present in marine macroalgae,

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microalgae and cyanobacteria. Their potential as GAG mimetics and/or biological agents with

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anticoagulant, antiviral, antioxidant or antitumor activities has been largely described (Delattre et

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al., 2011; Jiao, Yu, Zhang, & Ewart, 2011; Yip et al., 2006) but these compounds have not been

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studied for their antimicrosporidian activity. The aim of this study was first to evaluate the potential

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of sulphated polysaccharides from macro and microalgae to control the development of

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Encephalitozoon cuniculi a mammal microsporidia maintained in cell culture as the only one in

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vitro system that exists for N. ceranae provides an initial infection rate of approximately

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15–30 % (Gisder, Möckel, Linde, & Genersch, 2011) that is not powerful enough to be used in

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ELISA systematic screens. The oral administration of polysaccharides to N. ceranae-infected adult

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honeybees was then performed in order to test their capacity to control the parasite growth and/or to

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reduce deleterious effects on host.

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109 2.

Material and methods

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2.1

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Microalgae strains and culture conditions

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Polysaccharides were extracted from 6 microalgae strains: Porphyridium purpureum CCAP

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1380/1A (polysaccharide PP); P. marinum CCAP 1380/10 (polysaccharide PM); Rhodella violacea

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LMGEIP 001 (polysaccharide RL); R. violacea CCAP 1388/5 (polysaccharide RC); R. maculata

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CCAP 1388/2 (polysaccharide RM) and the cyanobacteria Arthrospira platensis PCC 8005 (Institut

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Pasteur culture collection) (polysaccharide S). Rhodella violacea LMGEIP 001 was obtained from

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the collection of cultures of microalgae, Clermont Université, Université Blaise Pascal (Villay et al.,

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2013).

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Microalgae were grown photoautotrophically in flasks (50 to 1000 mL) stirred at 110 rpm at 24°C

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for one month (R. violacea CCAP 1388/5; R. maculata, A. platensis), in a 16h-8h light-dark regime

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(150 µmol photons.m-2.s-1) or in 5L cylindrical photobioreactor (P. purpureum, P. marinum, R.

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violacea LMGEIP001) equipped with pH and pO2 probes, and adjustable lighting system composed

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of 55 halogen lamps (Sylvania professional 25, BAB 38°, 12 V, 20 W), allowing irradiances

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between 0 to 2000 µmol photons.m-2.s-1. Cultures were performed with a gas flow of 100 mL.min-1

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Production and extraction of native polysaccharides from macro- and microalgae

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containing 1% CO2. Stirring was set up between 100 and 250 rpm depending on the strain. For the

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culture of Rhodella strains the f/2 medium (Guillard & Ryther, 1962) modified by Villay et al.

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(2013) was used.

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Cultures of A. platensis PCC 8005 were done in a modified Zarrouk media (Cogne, Lehmann,

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Dussap, & Gros, 2003).

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P. purpureum and P. marinum was grown in a modified artificial sea water (for 1 L in milliQ water:

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NaCl 28.13 g, KCl 0.77 g, CaCl2, 2H2O 1.6 g, MgCl2, 6H2O 4.8 g, NaHCO3 0.11 g, MgSO4, 7H2O

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3.5 g) supplemented with Na2EDTA 0.4 g, NaNO3 1.12 g, Na2HPO4 0.2 g, NaH2PO41.04 g, before

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to be adjusted to pH 7.8. After sterilization at 120°C during 20 min, this medium was supplemented

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by 2.5 mL of vitamin solution (composed for 100 mL of cyanocobalamin 4 mg, thiamin HCl 0.1 g,

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biotin 1 mg, inositol 1g, folic acid 1 mg) and 1 mL of trace metal elements (for 100 mL:

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FeCl3,6H2O 0.28 g, CuSO4,5H2O 10 mg, ZnSO4,7H2O 40 mg, CoCl2,6H2O 5 mg, MnCl2,4H2O 40

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mg, (NH4)2Mo7O24,4H2O 37 mg) filtered at 0.2 µm.

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Extraction of exopolysaccharides from microalgae culture media

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Microalgae cultures were centrifuged at 10,000 x g for 30 min at 15°C to eliminate biomass.

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Supernatants were concentrated 2 times by evaporation (Büchi RII) under 72 mbar at 60°C and

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dialysed (10,000 Da) against milliQ water at 4°C during 72 h (9 baths). Finally, products (partially

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purified polysaccharides) were freeze dried and stored at room temperature.

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Extraction of polysaccharides from macroalgae

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Two commercially available iota carrageenans were used: C1 from Rhodia Food (France), and C3

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aquagel from Ikeda Corp. (Japan). A highly sulphated galactan was extracted from the marine

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macroalgae Halymenia durvillei collected in the coastal waters of small island of Madagascar,

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Nosy-be in Indian Ocean (polysaccharide H) (Fenoradosoa et al., 2009) and ulvan (polysaccharide

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U) was obtained from Ulva lactuca (AGULV003 Agrimer) as described previously (Lahaye et al.,

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1999).

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2.2

Characterization of polysaccharides

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Colorimetric assays

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Purity was evaluated at A492 by the phenol-sulphuric acid method as described by Dubois et al.

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(1956). The results were expressed in g.L-1 or mg.g-1 of D-glucose equivalent (GlcEq). Sulfur

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content was determined by the turbidimetric method (Dodgson & Price, 1962). Uronic acids and

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neutral sugar contents of exopolysaccharide extracts were assayed with meta-hydroxyldiphenyl and

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resorcinol as previously described (Blumenkrantz and Asboe-Hansen, 1973; Monsigny et al., 1988).

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Quantification of neutral sugars was done according to the corrective formula described by

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Montreuil et al. (1963). Results were expressed in mg.g-1 of D-glucose equivalent (GlcEq) for

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neutral sugars and in mg.g-1 of D-glucuronic acid equivalent for uronic acids. The protein content

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was estimated by the Bradford method (Bradford, 1976).

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Monosaccharide composition of polysaccharides

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Monosaccharide composition of polysaccharides was evaluated by High Pressure Anion Exchange

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Chromatography (HPAEC) on an ICS 3000 (Dionex, USA) equipped with pulsed amperometric

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detection and AS 50 autosampler. It was assembled with a guard CarboPacTM PA1-column (4 × 50

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mm) and analytical CarboPacTMPA1-column (4 × 250 mm). Before analysis, polysaccharide

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samples were hydrolyzed in 4 M TFA for 8 h at 100°C and neutralized by 2 M NH4OH. Samples (1

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mg.mL-1) were filtered using 0.2 µm membrane filter and injection volume was fixed at 25 µL.

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Before each injection, columns were equilibrated by running during 15 min with 18 mM NaOH.

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Samples were eluted isocratically with 18 mM NaOH for 25 min, followed by a linear gradient

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between 0 to 0.5 M sodium acetate in 200 mM NaOH for 20 min to elute acidic monosaccharides.

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Run was followed by 15 min washing with 200 mM NaOH. The eluent flow rate was kept constant

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at 1 mL.min-1. Columns were thermostated at 25°C. Data were collected and analyzed with Dionex

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Chromeleon 6.80 software (Sunnyvale, USA).

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2.3

Cytotoxicity assays

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Human Foreskin Fibroblast (HFF, American Type Culture Collection) cells were seeded in 96 well

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cell culture microplates (Cellstar®, Greiner Bio-One) at a density of 2 x 104 cells/well in MEM

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medium supplemented with 10% inactivated Fetal Bovine Serum (FBS), 2 mM glutamine, 2.5

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µg.mL-1 amphotericin B, 100 µg.mL-1 penicillin/streptomycin (GIBCO®, Invitrogen) and 100

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µg.mL-1 ampicillin solution (Sigma-Aldrich). Cells were incubated at 37°C in a humidified 5% CO2

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atmosphere for approximately 48 h to reach confluence. The medium was then replaced by fresh

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culture medium contained different concentrations of sulphated polysaccharides (50, 100 and 200

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µg.mL-1). Negative control (cells with culture medium only) and positive control (cells with 20%

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DMSO diluted in the medium) were also added to each 96-well culture plate. Each condition was

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tested in triplicate. After incubation for 96 h at 37°C in a humidified 5% CO2 atmosphere, the

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medium was removed and cells were fixed with 10% trichloroacetic acid in culture medium (v/v)

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and incubated 2 h at 4°C. Cells were washed five times with water and dried at room temperature.

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Then, 0.4% sulforhodamine B (Sigma-Aldrich) in 1% acetic acid (w/v) solution was added and

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incubated 20 min at room temperature. The dye was removed and the plates washed five times with

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1% acetic acid. After the last washing, 10 mM Tris base solution (Sigma-Aldrich) was added and

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incubated at 37°C for 15 min in gentle shaking to dissolve the protein-bound dye for OD

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determination at 550 nm using a microplate reader (Multiskan FC, Thermo-Scientific).

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2.4

Parasite culture and in vitro growth inhibition assays

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After validation of non-cytotoxic effect of sulphated polysaccharides, the anti-microsporidian assay

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was performed using the highest non-cytotoxic concentrations on Encephalitozoon cuniculi-infected

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HFF cells. Host cells were first seeded in 48 well culture plates (Nunclon™ Surface, Nunc) at a

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density of 4 x 104 cells/well in the same culture medium described above. Cells were allowed to

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grow in a humidified 5% CO2 atmosphere at 37°C for approximately 48h.

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Four groups of triplicates wells were created on the plate: (i) uninfected control with medium only

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(ii) infected control with medium only, (iii) infected group with medium containing fumagillin at 1

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µg.mL-1 (Sigma-aldrich), and (iv) infected group with medium containing the diluted sulphated

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polysaccharides. The uninfected control group in each plate allowed measuring the background

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signal in the ELISA assay. When cells reached confluence, the medium was replaced by fresh

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culture medium containing or not sulphated polysaccharides and incubated at 37°C for 2 h in 5%

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CO2. The medium was then removed and cells were infected with 2 x 105 spores/well of

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Encephalitozoon cuniculi (pre-incubated or not with sulphated polysaccharides during 2 h at 37°C)

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and incubated in a 5% CO2 atmosphere at 37°C for 1 h. After a quick wash of the cells with PBS,

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fresh culture medium, containing or not polysaccharides, was finally added and plates were

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incubated at 37°C in 5% CO2. After 6-7 days, the culture medium was removed and cells were fixed

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with methanol at -80°C for 30 min. Methanol was removed and cells were saturated with 10 mM

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Tris-2% BSA (Sigma-Aldrich) overnight. Then cells were incubated at 37°C for 2h with an anti-E.

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cuniculi rabbit serum (from naturally infected rabbits) diluted at 1/1,000 in an antibody dilution

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buffer (10 mM Tris pH=7.4, 150 mM NaCl, 0,05% Tween 20, 0,2% BSA; Sigma-Aldrich). Cells

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were washed five times with washing buffer (10 mM Tris pH=7.4, 0.05% Tween 20) and incubated

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at 37°C for 1 h with an anti-rabbit antibody (Anti-Rabbit IgG AP Conjugate, Promega) diluted at

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1/10,000 in antibody dilution buffer. The cells were washed five times with washing buffer and the

220

substrate solution (0.1 mM 4-méthylumbelliferyl phosphate (MUP), 1 mM MgCl2, 50 mM Na2CO3

221

pH=9,8; Sigma-Aldrich) was added. After incubation at 37°C for 30 min in gentle shaking, the

222

reaction was stopped by the addition of 3M NaOH and the fluorescence was measured with a

223

Fluoroskan Ascent FL (Thermolabsystems®), using excitation and detection wavelengths of 355 nm

224

and 460 nm respectively.

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225 226

2.5

Honeybee artificial rearing and experimental procedures

227

All experiments were performed on the european honeybee Apis mellifera. Two frames of sealed

228

brood were taken in one colony and placed in an incubator in the dark at 33°C with 60% relative

229

humidity. Emerging honeybees were collected, confined to laboratory cages (Pain type) in groups

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of 45, and maintained in the incubator for five days. During this time, the caged honeybees were fed

231

with 50% (w/v) sucrose syrup supplemented with 1% (w/v) proteins (Provita'bee, Biové

232

Laboratory) ad libitum. To mimic the hive environment as much as possible, little pieces of wax (5

233

x 8 cm) and Beeboost® (Pherotech, Delta, BC, Canada) releasing a queen's mandibular pheromone,

234

were placed in each cage. After five days of feeding, four experimental groups were created: (i)

235

uninfected controls, (ii) infected with N. ceranae, (iii), infected with N. ceranae fed with syrup

236

supplemented with fumagillin (1 µg.mL-1), and (iv) infected with N. ceranae fed with syrup

237

supplemented with sulphated polysaccharides diluted at 200 µg.mL-1, except PM which were

238

diluted at 100 µg.mL-1. Three replicates were done for each experimental group. Treatments

239

(fumagillin and polysaccharides) were administrated 2 days before the infection that occurred 5

240

days after the emergence of the bees from the brood.

241

Honeybees were individually infected (see below honeybee infection) and fed ad libitum during 19

242

days with 50% (w/v) sucrose syrup with 1% (w/v) proteins (Provita'bee, Biové laboratory)

243

supplemented or not with fumagillin (1 µg.mL-1) or sulphated polysaccharides according to their

244

experimental group. Each two days, feeders were replaced. Throughout the experiment, each cage

245

was checked every day and any dead honeybees removed and counted. At the end of the

246

experiment, honeybees were sacrificed and the abdomen was dissected and homogenized in

247

distilled water using a manual tissue grinder. The resulting suspension was cleaned by

248

centrifugation at 300 x g for 10 min and pellet re-suspended in distilled water. The parasite load was

249

determined by counting with a hematocytometer chamber.

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230

250

251

2.6

Honeybee infection

252

Spores of N. ceranae were obtained from experimentally-infected honeybees. After sacrifice, the

253

abdomen of infected honeybees was dissected and homogenized in distilled water using a

254

stomacher Bag Mixer 400P (InterScience, Saint-Nom, France). The suspension was filtered through

255

a 100 µm cell strainer (BD Biosciences, New Jersey, USA) and the resulting suspension was

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centrifuged at 300 x g for 5 min to collect spores contaminated by pollen. The pollen was removed

257

by at least 3 centrifugation steps of 30 s at 100 x g. Spores in the supernatants were then pooled

258

after a centrifugation step at 300 x g for 5 min and re-suspended in distilled water. The spore

259

concentration was determined by counting with a hematocytometer chamber. At 5 days post-

260

emergence, caged bees were starved for 1 h, CO2 anaesthetized and individually-infected with

261

125,000 spores diluted in 3 µL of 50% (w/v) sucrose syrup. Non-infected bees were similarly

262

treated without N. ceranae spores in the sucrose solution.

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263 2.7

Statistical analysis

265

The effect of N. ceranae infection on honeybees was analyzed with a Kaplan-Meier survival

266

analysis taking into consideration all groups followed by a Cox-Mantel test to determine the

267

significant difference between the infected group and others by using Statistica 7.0 software

268

(StatSoft inc., Tulsa, USA). N. ceranae development success (i.e. number of spores produced) was

269

analyzed for each treatment by comparing to the infected group by using a Kolmogorov-Smirnov

270

test, a non-parametric test. The significance thresholds were deemed as significant for p<0,05.

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271

3.

Results

273

3.1

Production and extraction of sulphated polysaccharides from macro- and microalgae

274

Macro- and microalgae were chosen as sources of polysaccharides with the objective to test

275

sulphated polysaccharides of vegetable origin. After their extraction and partial purification they

276

were characterized for their composition (Table 1) and besides their sulfate content two families of

277

macromolecules were defined. The first one is composed of highly sulphated macromolecules

278

(between 15.1 and 33.8 % of sulfate content) including sulphated galactans (C1, C3 and H) and

279

ulvan (U) from Ulva lactuca mainly containing rhamnose and glucose residues. These

280

polysaccharides extracted from macroalgae have been abundantly described in the literature and

281

their monosaccharidic composition, sulfate content and purity were in accordance with those

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ip t Sulfate (%)

Proteins (%)

283 Monosaccharide composition 284 (molar ratios %) 285

28.2 ± 4

-

Gal 95, Fuc 03, Glc 01, Xyl 01 286

17.9 ± 0.3

-

287 Gal 56, Glc 41, Fuc 02, Xyl 01

17.6 ± 0.3

15.1 ± 0.3

7.1 ± 0.7

0.6 ± 0.3

33.8 ± 0.1

-

14.9 ± 0.8

1.7 ± 0.4

-

70.8 ± 1.2

17.9 ± 0.8

9.1 ± 0.3

-

86.1 ± 3.6

67.9 ±0.3

19.6 ± 1.0

8.4 ±1.5

-

69.3 ± 4.1

55.0 ± 1.0

12.9 ± 0.5

4.3 ± 1.1

6.0 ± 0.3

Neutral sugar (%)

Acidic sugar (%)

Iota carrageenan - C1

73.5 ± 2.1

70.4 ± 0.5

1.9 ±0.9

Iota carrageenan - C3

83.1 ± 1.8

72.9 ± 0.6

1.1 ±0.9

Ulva lactuca - U

67.3 ± 3.2

50.6 ± 0.5

Halymenia durvillei - H

75.9 ± 2.1

75.6 ± 0.1

Arthrospira platensis - S

37.6 ± 1.2

22.6 ± 0.9

Porphyridium purpureum- PP

88.0 ± 4.1

Porphyridium marinum - PM

Rhodella violacea - RC Rhodella maculata -RM

M

d

ep te

Ac c

Rhodella violacea - RL

an

Purity (%)

Origin

cr

Table 1: Structural characterization of polysaccharides.

us

282

52.8 ± 3.5

42.4 ± 2.0

8.2 ± 1.4

2.7 ±0.1

5.5 ± 0.5

58.7 ± 1.6

49.8 ± 0.3

9.7 ± 2.2

9.1 ±0.1

6.4 ± 0.6

Rha 52, Glc 30, Glu 09, Xyl 05 288 Gal 03 289 Gal 89, Ara 05, Glc 03, Fuc 02 Xyl 01 290 Glc 24, Rha 22, Xyl 15, Fuc 14 Glu 13, Gal 12 291 Gal 42, Xyl 30, Glc 25, Glu 02 292 Fuc 01 Gal 47, Xyl 30, Glc 20, Glu 02 293 Fuc 01 294 Xyl 45, Gal 39, Rha 08, Glu 03 Ara 03, Glc 01

295 Gal 52, Xyl 34, Glc 07, Glu 03 Rha 02, Ara 01 296 Gal 45, Xyl 42, Glu 05, Rha 05 297 Ara 02, Glc 01

298 299

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previously described (Delattre et al., 2011; Fenoradosoa et al., 2009; Lahaye et al., 1999). The

301

second family of polysaccharidic macromolecules gather together moderately sulphated

302

polysaccharides (between 1.7 and 9.1 % of sulfate content), all polysaccharides belonging to this

303

group are from microalgae. Their structure appeared as complex and composed of several kinds of

304

monosaccharides. Among the red marine microalgae, Porphyridium strains produced sulphated

305

glucoxylogalactans whereas Rhodella strains excreted sulphated galactoxylans or xylogalactans

306

partially associated with proteins. These results are in accordance with structures of

307

exopolysaccharides of red marine microalgae partially elucidated in previous studies (Arad & Levy-

308

Ontman, 2010; Pignolet, Jubeau, Vaca-Garcia, & Michaud, 2013; Villay et al., 2013). The strain of

309

cyanobacteria (A. platensis) tested as polysaccharide producer led to the obtaining of a weakly

310

sulphated polyanionic heteropolymer composed of 6 main monosaccharides as described by

311

Trabelsi et al. (2009).

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300

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312

3.2 Cytotoxic effects of sulphated polysaccharides on human foreskin fibroblast cells

314

Cytotoxicity assays were performed in order to determine the cytotoxicity of the 10 sulphated

315

polysaccharides at several concentrations from 50 to 200 µg.mL-1 (Figure S1/Table 2). This assay

316

allowed measuring the cell survival in presence of the polysaccharidic extracts and the

317

determination of the highest non cytotoxic concentrations. The sulphated polysaccharides showed

318

no cytotoxic effect on HFF cells at 200µg.mL-1, except RL and PM that seem to induce a low

319

toxicity (around 30%).

320 321

Table 2. Highest concentrations of sulphated polysaccharides without any cytotoxic effect on the HFF cell line.

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Compound

Cytotoxic effect

C1

>200 µg.mL-1

C3

>200 µg.mL-1

U

>200 µg.mL-1

H

>200 µg.mL-1

Page 17 of 30

13

S

>200 µg.mL-1

PP

>200 µg.mL-1

PM

>100 µg.mL-1

RL

>100 µg.mL-1

RC

>200 µg.mL-1

RM

>200 µg.mL-1

C1: Iota carrageenan from Rhodia, C3: Iota carrageenan from Ikeda, U: U. lactuca, H: H. durvillei, S: A. platensis, PP: P. purpureum, PM: P. marinum, RL: R. violacea LMGEIP001, RC: R. violacea CCAP 1388/5, RM: R. maculata.

326

3.3

327

The growth inhibition of microsporidia by polysaccharides was measured by an ELISA assay

328

performed on the human microsporidia E. cuniculi as no cell culture is available for N. ceranae. All

329

the polysaccharide solutions were used at a concentration of 200 µg.mL-1, except RL and PM which

330

were used at a concentration of 100 µg.mL-1. Fumagillin was tested as a positive control in order to

331

compare the effectiveness of the polysaccharides with a well-established antimicrosporidian

332

molecule. A control, composed of E. cuniculi-infected cells, without any antimicrosporidian

333

compound, was included to evaluate the standard growth of E. cuniculi. All the polysaccharides

334

tested were shown to reduce the growth of E. cuniculi with inhibition percentages ranging from

335

29.92% for RM to 99.39% for PP (Figure 1). Sulphated polysaccharides from P. purpureum (PP)

336

and P. marinum (PM) exhibited the greatest activities, respectively 99.39% and 90.33% of growth

337

inhibition in vitro; these values are 20% higher than those obtained with fumagillin (74.71%).

cr

ip t

322 323 324 325

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In vitro antimicrosporidian activities of sulphated polysaccharides

Page 18 of 30

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NI

I

F

C1

C3

U

H

S

PP

339

RL

RC

RM

cr

338

PM

ip t

Parasite growth (%)

100 90 80 70 60 50 40 30 20 10 0

Fig.1

us

340 3.4

Effect of sulphated polysaccharides on Nosema ceranae-infected honeybees

342

Two iota carrageeans (C1 and C3), two sulphated polysaccharides from Porphyridium species (PM

343

and PP) and one from Rhodella (RC) were selected to investigate their effectiveness in vivo on

344

encaged bees infected by N. ceranae. Survival analysis indicates a significant improvement in the

345

survival of honeybees fed with fumagillin (p=0.000), PM (p=0.001), C3 (p=0.005) and RC

346

(p=0.037), compared to the infected control during the infection (Figure 2 and Table S1).

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Page 19 of 30

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Complete

Censored

1,0

0,8

ip t

0,7

cr

0,6

0,5

us

Cumulative Proportion Surviving (%)

0,9

0,3 0

2

4

6

8

10

12

Days

14

16

18

20

22

M

347

an

0,4

Infected PM PP RC C1 C3 F

Fig.2

349

N. ceranae development was monitored as the number of spores present in the abdomen of

350

surviving honeybees at the end of experiment (day 19) (Figure 3).

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ip t cr us an

351 Fig.3

353

The statistical analysis revealed that the spore content was higher in infected control group than in

354

infected bees fed with fumagillin (p<0.001), PM (p<0.005), PP and C1 (p<0.05). The best inhibition

355

of parasite growth was obtained for fumagillin (a reduction of ~60% the parasite load) followed the

356

PM treatment, which reduces by ~30% the parasite load, (40.8x106 in the PM group versus

357

61.6x106 spores/bee in the infected control group). The honeybees supplemented with PP also

358

presented a significant decrease in their parasite load (-20.4%). Surprisingly, the polysaccharide C3

359

that improved the survival of the honeybees (Figure 2) induced an increase in parasite load

360

(+29.9%, p=0.005).

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361 362

4.

Discussion

363

Our results indicated that sulphated polysaccharides from both micro- and macroalgae could have

364

an antimicrosporidian action in vitro and could significantly reduce the parasite load and/or improve

365

the survival of N. ceranae-infected bees. These results confirmed those obtained by Hayman et al.

Page 21 of 30

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(2005) with exogenous glycosaminoglycans (GAGs) during the interaction of E. intestinalis with its

367

host cells and reinforced the hypothesis that microsporidia can adhere to targeted cells by the way

368

of host surface glycans. All polysaccharides tested here are natural sulphated biopolymers eco-

369

friendly produced by wild strains of macro- and microalgae, some of them such as carrageenans

370

being largely employed as food additives. Our data indicate that these polysaccharides could be

371

used as antiparasitic agents to protect honeybees against Nosema species. No direct correlation was

372

established between the sulfate content of polysaccharides and their antiparasitic activity. Indeed, in

373

vitro experiments with E. cuniculi revealed antiparasitic activities for all the 10 polysaccharides

374

with greatest ones for lowly sulphated exopolysaccharides of Porphyridium species. These results

375

were confirmed by in vivo experiments on honeybees infected by N. ceranae. The two

376

polysaccharides from Porphyridium species are glucoxylogalactans with low sulfate and uronic acid

377

contents compared to the majority of GAGs with the exception of hyaluronic acid which is not

378

sulphated. No hexosamine has been detected as constitutive monosaccharide whereas this kind of

379

monosaccharide is abundant in GAGs. However, exopolysaccharides from Porphyridium genus are

380

well known to possess sulfate groups linked to glucose and galactose in the 6 or 3 positions (Arad

381

& Levy-Ontman, 2010) as observed for sulfations of N-acetyl-hexosamines of GAGs. Other

382

polysaccharides with defined structures tested here such as carrageenans (C1 and C3) and the highly

383

sulphated galactan (H) have higher sulfate content but sulfation occurred on 2-O and/or 6-O

384

positions of galactopyranose residues (Delattre et al., 2011; Fenoradosoa et al., 2009). For ulvan,

385

sulfate groups were mostly found on xylose and rhamnose residues at 2-O and 3-O positions

386

(Lahaye et al., 1999). No data are available at this time for polysaccharides of Rhodella species. So

387

the position of sulfate groups rather than the sulfate content of polysaccharides could be the most

388

important parameter for an antiparasitic activity.

389

Sulphated polysaccharides have been described for their antiviral activity. For instance, fucoidans

390

extracted from the brown seaweed Adenocystis utricularis are able to block early events of viral

391

replication of HIV-1 in vitro (Trinchero et al., 2009). In addition, anti-malarial activity was also

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366

Page 22 of 30

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shown with sulphated polysaccharides extracted from seaweed (carageenans) or regioselectively

393

modified sulphated cellulose. These studies clearly indicated that sulphated polysaccharides can

394

interfere with a number of host receptors during the adhesion prior to the host cell invasion by

395

Plasmodium (Schwartz-Albiez et al., 2007). In Microsporidia, adhesion to the host cell is also a key

396

step in the invasion process. Indeed, the adherence of Encephalitozoon spores to host cells was

397

shown to precede infection and is mediated by host cell surface glycosaminoglycans (GAGs). When

398

adherence is inhibited by exogenous sulphated glycans (heparin, chondroitin sulfate A, chondroitin

399

sulfate B, mucin and dextran sulfate) the host cell infection significantly decreased (Hayman et al.,

400

2005). Two spore ligands that could be involved in host cell adherence were identified, EnP1 a

401

spore wall protein at the surface of E. cuniculi (a mammal’s parasite) and SWP-26 which was

402

identified in the spore wall of the silk worm’s parasite Nosema bombycis). Both surface proteins

403

contain in their sequence one or two Heparin Binding Motif (HBM) which could be involved in the

404

adhesion process (Li et al., 2009; Southern, Jolly, Lester, & Hayman, 2007). As the parasite load

405

decreases less with PM, PP and C1 compared with fumagillin, these three algal sulphated

406

polysaccharides could act by blocking the adhesion of the parasite instead of inhibiting the parasite

407

growth. In this case, the autoinfection process that occurs secondarily between neighbouring cells in

408

the midgut is not inhibited (Higes, García-Palencia, Martín-Hernández, & Meana, 2007). However

409

as the sulfated polysaccharides could act on the adhesion process, it could therefore reduce the

410

transmission of the parasite.

411

The protective effect of sulphated polysaccharides could be also related to the decrease of the

412

parasite load as it was shown for PM and C1, but also by a significant lower lethality without any

413

decrease of the parasite load as it was the case for C3. For this latter case, one explanation could be

414

related to the antioxidant properties of seaweed sulphated polysaccharides. Antioxidant properties

415

of carrageenans and ulvans appeared related to their sulfate content. For instance kappa, iota and

416

lambda carrageenans showed different antioxidant activities by inhibiting superoxide radical

417

formation (Rocha de Souza et al., 2007). Recent studies also have demonstrated that the total

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antioxidant capacity was significantly increased in N. ceranae-infected queen (Alaux et al., 2011).

419

The measure of activity of one of the antioxidative enzyme (GST) was increased 1.6-fold and 1.7-

420

fold, respectively, in midgut and fat body of infected honeybees compared to uninfected ones

421

suggesting that N. ceranae infection may lead to reactive oxygen species (ROS) production (Vidau

422

et al., 2011). Although the ROS might be required for gut homeostasis and immunity against

423

pathogens (Kim & Lee, 2014), an elevated quantity of ROS could be deleterious to the bees, so the

424

addition of antioxidant compounds, like sulphated iota carrageenans (C3), in food during N.

425

ceranae infection might improve the honeybee physiology and allow a decrease of lethality by

426

reducing ROS-induced damages.

427

Another hypothesis to explain that algal sulphated polysaccharides may improve survival in N.

428

ceranae-infected honeybees and/or decrease the parasite load is that these compounds possess

429

immunomodulatory activities by stimulating the immune response or in controlling immune cell

430

activity to mitigate associated negative effects such as inflammation (Chen, Wu, & Wen, 2008).

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431

ip t

418

To conclude, our experiments showed that algal sulphated polysaccharides could be used to improve the survival of N. ceranae-infected honeybees and reduce the parasite load. This could

433

represent an alternative strategy to control nosemosis as fumagillin is no longer licensed in several

434

countries.

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435

Acknowledgements

437

This work is supported by a public grant overseen by the French National Research Agency (ANR)

438

(Reference: ANR-12-EMMA-0030). M.R. acknowledges the support of a Fellowship from the

439

French National Research Agency (ANR-12-EMMA-0030). A.V. was supported by a grant from

440

the ‘‘Ministère de l’Education Nationale de l’Enseignement Supérieur et de la Recherche’’.

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Figure 1: Effect of sulphated polysaccharides on E. cuniculi proliferation. Parasite proliferation

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was measured at 7 days post-infection. Results originate from three independent experiments and

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are expressed as mean±SD. 100% activity on the y axis represents the maximum parasite

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proliferation.

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NI: Non-Infected cells, I: Infected cells, F: fumagillin, C1: Iota carrageenan Rhodia, C3: Iota

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carrageenan Ikeda, U: U. lactuca, H: H. durvillei, S: A. platensis, PP: P. purpureum,

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PM: P. marinum, RL: R. violacea LMGEIP001, RC: R. violacea CCAP 1388/5, RM: R. maculata.

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Figure 2: Effect of sulphated polysaccharides on N. ceranae-infected honeybee survival. Data

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indicate the cumulative proportion of surviving honeybees exposed to N. ceranae in the presence or

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not of polysaccharides. Bees were exposed to polysaccharides 2 days before infection with 125,000

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spores of N. ceranae per bee. Data from three replicates of 45 honeybees were analyzed with the

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Kaplan-Meier method. I: infected control group, PM: P. marinum, PP: P. purpureum, RC: R.

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violacea CCAP 1388/5, C1: Iota carrageenan Rhodia, C3: Iota carrageenan Ikeda, F: fumagillin.

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Figure 3: Mean number of N. ceranae spores per honeybee abdomen. The number of spores

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was determined from surviving honeybees on day 19, in response to fumagillin (F) and

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polysaccharide (PM, PP, RC, C1, C3) treatments (n=30/treatment). Significant differences (p<0.05)

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were obtained for PM, PP and C1 when comparing to the infected group by using the Kolmogorov-

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Smirnov test, a: p<0.005, b: p<0.05, c: p<0.001. PM: P. marinum, PP: P. purpureum, RC: R.

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violacea CCAP 1388/5, C1: Iota carrageenans Rhodia, C3: Iota carrageenans Ikeda, F: fumagillin.

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Supplementary data

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Figure S1: Dose-response analysis of polysaccharide toxicity against the HFF cell line using the sulforhodamine B (SRB) assay. SRB incorporation was measured 96h after addition of sulphated polysaccharides. Results are expressed as mean±SD. 100% activity on the y axis represents the maximum cell viability (growth of cells without products). A 20% DMSO-treated control has been introduced to have a positive control of cytotoxicity. F: Fumagillin, C1, C2, C3: Iota carrageenans, U: U. lactuca, H: H. durvillei, S: A. platensis, PP: P. purpureum, PM: P. marinum, RL: R. violacea LMGEIP001, RC: R. violacea CCAP 1388/5, RM: R. maculata.

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Supplementary table 1

Table S1: Evolution of mean survival of the different honeybees treated groups ± SD. Recording were made 4, 7, 10, 13, 16 and 19 days post infection.

day 7 96.3 ± 4.6 96.3 ± 4.6 98.5 ± 2.6 77.8 ± 5.9 97.8 ± 2.2 98.5 ± 2.6 91.9 ± 5.1 95.6 ± 2.2

day 16 81.5 ± 5.6 68.1 ± 14.3 85.2 ± 11.2 57.8 ± 8.0 80.0 ± 8.0 74.1 ± 4.6 83.0 ± 7.1 75.6 ± 11.1

day 19 76.3 ± 4.6 46.7 ± 13.5 74.1 ± 16.7 40.0 ± 8.0 63.0 ± 7.8 56.3 ± 6.8 65.9 ± 8.4 58.5 ± 18.9

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uninfected infected F C1 C3 PP PM RC

day 4 99.3 ± 1.3 97 ± 3.4 99.3 ± 1.3 94.1 ± 4.6 97.8 ± 2.2 99.3 ± 1.3 97.8 ± 0.0 96.3 ± 2.6

Mean survival ±SD day 10 day 13 94.1 ± 6.4 88.1 ± 7.1 91.1 ± 8.9 81.5 ± 9.0 97.8 ± 2.2 90.4 ± 11.0 77.0 ± 7.1 67.4 ± 9.3 95.6 ± 4.4 90.4 ± 5.1 96.3 ± 2.6 83.0 ± 6.8 89.6 ± 7.1 87.4 ± 7.1 93.3 ± 3.8 85.2 ± 7.1

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Treatment

F: fumagillin, C1: Iota carrageenans Rhodia, C3: Iota carrageenans Ikeda, PP: P. purpureum,

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PM: P. marinum, RC: R. violacea CCAP 1388/5.

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