Yescas Tonatiuh

Anthony González

Victor Leonard

Alexandra Gaude

Annick Delplanque

Pierre Barre

Lorna Rougé

Francoise Garnier

Jacques Bienvenu

Marc Bienvenu

Zelsmann

Tonatiuh Yescas González
email: tonatiuh.yescasgonzalez@cea.fr

Anthony Leonard

Victor Gaude

Alexandra Delplanque

Annick Barre

Pierre Rougé

Lorna Garnier

Françoise Bienvenu

Marc Zelsmann

Emmanuel Picard

David Peyrade

IgE detection in allergic patient's serum by absorption analysis of biofunctionalised microparticles

Keywords: particle-based immunoassay, optical absorption, magnetophoresis, colloids, visible light spectrum

Introduction

Allergic diseases are a concern worldwide as they have become more prevalent in the last five decades [START_REF]White Book on Allergy: Update 2013, Executive Summary[END_REF][START_REF] Sicherer | Food allergy: Epidemiology, pathogenesis, diagnosis, and treatment[END_REF], even at a higher rate in developing countries [START_REF] Asher | Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys[END_REF], which has guided research to improve the performance of detection techniques and treatment procedures. Certain prospects include personalised medicine approaches to diminish the cases of misdiagnosis and increase the effectiveness of immunotherapy [START_REF] Ferrando | Personalized medicine in allergy[END_REF][START_REF] Ortega | Pharmacogenetics: Implications of race and ethnicity on defining genetic profiles for personalized medicine[END_REF], and component-resolved diagnosis to identify specific clinical phenotypes [START_REF] Tuano | Utility of Component-Resolved Diagnostics in Food Allergy[END_REF][START_REF] Treudler | Overview of component resolved diagnostics[END_REF]. Regarding protein-functionalised micro and nanoparticles, considerable therapeutic results have been obtained from their implementation as a preventive measure to protect against sensitization and reducing anaphylactic reactivity [START_REF] Chien | Microparticles encapsulated with antigen protect against sensitization and reduce anaphylactic reactivity in a food allergy model[END_REF][START_REF] San Román | Co-delivery of ovalbumin and CpG motifs into microparticles protected sensitized mice from anaphylaxis[END_REF], as well as carriers for drug delivery [START_REF] Ferrer | New pharmaceutical dosage forms for allergy treatment[END_REF][START_REF] Muzzarelli | Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers[END_REF]. In the diagnostic field, polystyrene (PS) particles have been used to increase the detection limit of enzyme-linked fluorescence immunoassays for antibody concentration measurements in serum samples [START_REF] Ohashi | A micro-ELISA system for the rapid and sensitive measurement of total and specific immunoglobulin E and clinical application to allergy diagnosis[END_REF][START_REF] Yasukawa | Flow sandwich-type immunoassay in microfluidic devices based on negative dielectrophoresis[END_REF]. Similarly, superparamagnetic (SM) particles are of particular interest as immuno-supports to increase the protein capture rate [START_REF] Teste | Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis[END_REF][START_REF] Speroni | Magnetic particles functionalized with PAMAM-dendrimers and antibodies: A new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods[END_REF] and as capturing support in agglutination tests by agglomerating fluorophores already reacting with antibodies (secondary interaction) or by directly emitting light (primary interaction) [START_REF] Agrawal | Single-bead immunoassays using magnetic microparticles and spectral-shifting quantum dots[END_REF][START_REF] Li | Molecularly imprinted magnetic microparticles for the simultaneous detection and extraction of Rhodamine B, Sensors Actuators[END_REF]. Therefore, micro and nano particles are relevant in allergy diagnostics research due to plurifunctionality and increased system resolution.

Most particle-based immunoassays rely on fluorescence, as this type of labelling presents a linear signal behaviour, enabling the distinction between faint differences in protein concentration down to single protein resolution. For instance, digital ELISA applies fluorescence detection on an isolated microbead acting as support and labelled by reporter fluorophores achieving 14fg/mL resolution [START_REF] Rissin | Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations[END_REF], while flow cytometry assays are capable of multiplexing such techniques [START_REF] Mejia-Ariza | DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles[END_REF][START_REF] Vignali | Multiplexed particle-based flow cytometric assays[END_REF]. Whereas the sensibility is considerable and it is possible to precisely characterize these particle complexes to gather quantitative results, it relies on a large set of sensors and a very controlled environment to detect extremely faint light signals, heavily limiting device miniaturization and affordability for multiplexed immunoassays. Thus, an approach involving broad optical detection of pigmented microparticles in suspension could offer a compact inexpensive benchtop device for detection of different antibodies in serum, providing quantitative test results with a working range similar to those of clinical laboratories.

In this work, we present a protein detection mechanism based on visible light absorption of PS-SM particle complexes in free suspension that have reacted with the antibodies in serum, analogous to indirect ELISA tests. Firstly, SEM imaging is used to visualize the surface topography of our particles and how they interact with antibodies in serum for different allergens. Then, we evaluate the minimum detectable protein concentration of our absorption analysis by comparing a single allergen with a control protein. Finally, we estimate the specificity of our setup by comparing the response of two major alimentary allergens that also exhibit cross-reactivity in clinical tests.

Materials and methods

The setup performs a volumetric immunoassay using dyed PS microparticles, with immobilised allergen extracts on the surface, which serve as immunosupports for isolation of human antibodies in serum (IgE) and as optical signal. After 1 hour, the particles are centrifuged and transferred to a suspension containing SM microparticles with secondary antibodies specific to IgE (aIgE), which results in a stable PS-IgE-SM complex after 30 minutes if the antibodies recognised the allergen on the PS particle, as depicted in Fig. 1a. The suspension is analysed by a spectrometer, gathering continuously its absorption signal in the visible light spectrum, as depicted in Fig. 1b. The formed complexes are then gathered by magnetophoresis, when we apply a planar magnetic field on the suspension. As particles migrate towards the magnet, their concentration within the illumination region decreases, showing a decrease in signal as in Fig. 1c. The stabilised absorption level after ~50s is related to the concentration of IgE in serum. For the experiments, serum samples from 1 patient strongly allergic to peanut and 1 strongly allergic to hazelnut, which had been previously diagnosed by skin-prick test, serve as sources of IgE. The IgE concentration was measured by a commercial immunoassay test (ImmunoCAP, Thermo Fisher Scientific).

Allergen extract isolation, purification and quantification

Allergen extracts of peanut (Arachis hypogea -Ara) and hazelnut (Corylus avellana -Cor) were obtained by initial cryogenic milling of the nut seeds to obtain a fine powder. Then, using a high-speed benchtop homogenizer (FastPrep, MP Biomedicals) along with a lysis buffer (FastPROTEIN, MP Biomedicals) to isolate the proteins present in food. The purity of the preparation was checked by SDS-PAGE in 12.5% acrylamide gels followed by Coomassie blue staining [START_REF] Rougé | Characterization of IgE-binding epitopes of peanut (Arachis hypogaea) PNA lectin allergen cross-reacting with other structurally related legume lectins[END_REF], with qualitative validation of the presence of allergens by Western blot testing with allergic patient's sera. The protein concentration was estimated by BCA analysis (Pierce BCA, Thermo Fisher Scientific) of the isolated solution.

Microparticle preparation

Commercially available microbeads were used, following commonly used binding protocols [START_REF]Covalent Coupling[END_REF]. As immunosupport, we used 6µm carboxylate blue-dyed PS spheres (Polybead, Polysciences, Inc.). Apart from the allergen extracts previously described, bovine serum albumin (BSA) was also immobilised on some particles to act as control. For all proteins, to guarantee a significant surface area coating we added 500 µg per 1x10 5 beads. The carboxyl groups on surface require an initial activation, which was done in acidic conditions (MES buffer, 100 mM, pH 5.0) by addition of EDC and sulfo-NHS during 30 minutes. After activation, the protein solution was added to the suspension in neutral medium (PBS buffer, pH 7.4) during 3 hours. To prevent unspecific interaction, the uncoupled active sites on the bead were blocked with a primary amine source (Tris buffer, 200 mM, pH 9.0) during 24 hours. Finally, the particles were stored at 3-5 °C (PBS-TBN 0.1% (w/v) BSA buffer, pH 7.4).

Concerning the magnetic sorting of PS particles, we used 1µm tosyl-group SM beads (Dynabeads MyOne, Thermo Fisher Scientific), consisting of magnetic nanocrystals encased in a PS matrix. The recognition of IgE was done by commercial goat anti-human IgE antibodies (Sigma-Aldrich Merck), adding 200 µg per 1x10 7 beads. The tosyl groups offer a straightforward coupling to the proteins, requiring only the reaction with the protein for 24 hours (sodium phosphate buffer, 100 mM, pH 7.4). The blocking and storage steps are identical to those of PS particles.

Serum reaction

The particles were suspended in a reaction buffer (PBS-TBN 2% (w/v) BSA buffer, pH 7.4) at a concentration of 1x10 5 beads/mL, to which several volumes of serum were added. After 1h for IgE isolation, the colloidal sample was centrifuged and resuspended in PBS-TBN 0.1%. Finally, SM particles were put in the suspension at a concentration of 1x10 7 beads/mL.

SEM imaging

In view of the high accumulation of electrostatic charge in non-conductive specimens, while avoiding metallic coating that would disrupt the features on our samples, we took images from a SEM (ULTRA plus, Zeiss) at a low acceleration voltage (1kV, 30 piA current, 4-7 mm distance) using the type I and type II secondary electrons signals [START_REF] Hortolà | Secondary-electron SEM bioimaging of human erythrocytes in bloodstains on high-carbon steel substrate without specimen preparation[END_REF].

Spectrometer characteristics

The absorption detection in the visible light spectrum was performed by a commercial optical-fibre-based spectrometer (QE65000, Ocean Optics), using a Peltier cooler for thermoregulation. The fibre's core diameter was 400 µm in order to collect the signal from several particles and the data analysis was performed at a wavelength of 523 nm (±15nm), as the illumination source was a colour LED. As optical reference, we used diluted serum in the same proportion as the tests.

Results and discussion

Immobilisation of active allergens on surface

For the protein bioconjugation, the molecular Ara allergens present on suspension after purification are Ara h1, Ara h2, Ara h3, Ara h8 and Ara h9, whereas the Cor extraction resulted in the presence of Cor a1, Cor a8, Cor a9 and Cor a14. Further validation of the coating protocol was necessary for our specimens in order to optimise the procedure, which can be seen in the micrographs of Fig. 2. Globally, all PS particles with Ara allergen extracts (PS-Ara) exhibit fusiform elongated irregular regions on their surface of ~150±50 nm in length along the largest axis, which during antibody isolation result in large protuberances of ~200±20 nm in length and 80±10 nm in height, as shown in Fig. 2a and Fig. 2b, respectively. In contrast, PS beads with Cor allergen extract (PS-Cor) present almost undiscernible topographical features of 30±15 nm in size as evidenced in Fig. 2c. However, after IgE isolation as shown in Fig. 2d, uniformly-distributed filiform groupings become apparent. Comparatively, the commercially available PS beads display an homogeneously corrugated surface, with some larger granular bumps of 90±20 nm in diameter, as showcased in Fig. 2e.

Regarding the particle complex, depicted in Fig. 2f, the elevated number of SM beads make possible the magnetophoresis of a high proportion of reactive PS species (~85% throughout all tests). Nonetheless, the hydrostatic nature of our experiment favours agglomeration of the magnetic particles, which in turn increases the probability of dragging non-recognised PS beads. Yet, the magnetic sorting is highly selective and does not affect strongly the unstable interactions in the absence of IgE, particularly clear when comparing to the control suspension (PS-BSA).

Optical detection of protein interaction

Initially, the sensibility of the immunoassay was determined, as illustrated in Fig. 3a, by comparing the results of PS-BSA and PS-Ara at λ = 523nm, reaching different final absorption levels 40 seconds after applying the magnetic field, related to the migration of particle complexes. Evaluating the reaction with several concentrations of Ara-specific IgE [START_REF] Tuano | Utility of Component-Resolved Diagnostics in Food Allergy[END_REF][START_REF] Ohashi | A micro-ELISA system for the rapid and sensitive measurement of total and specific immunoglobulin E and clinical application to allergy diagnosis[END_REF][START_REF] Rissin | Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations[END_REF][START_REF] Van Hage | ImmunoCAP assays: Pros and cons in allergology[END_REF][START_REF] Barre | Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes[END_REF]36, 42 ng/mL), from the original serum sample of ~250 ng/mL, we obtained the graph seen in Fig. 3b. The lowest detectable concentration is 24 ng/mL, lying outside the exclusion region with the absorption being linearly dependent to protein concentration. The performance is in the range of commonly used techniques in clinical labs, but 100 times less sensible than reported ImmunoCAP specifications. Notwithstanding, inter-assay variability and working range is similar, while affordability of the detector mechanism are superior in our design [START_REF] Van Hage | ImmunoCAP assays: Pros and cons in allergology[END_REF][START_REF] Wang | Correlation of serum allergy (IgE) tests performed by different assay systems[END_REF]. Also, these results put in evidence the minimum working conditions, so the sensibility can be addressed by adding more particles to the procedure to increase the SNR.

As cross-reactions are common in immunological tests, determining the effect it has on our procedure is necessary. This was accomplished by contrasting the reaction between PS-BSA, PS-Ara, and PS-Cor to antibodies specific to Ara and Cor, separately at 3 critical concentrations around our detection limit: 12, 24, 36 ng/mL. Some degree of false detection is expected as several studies show a high incidence of cross-reactivity [START_REF] Masthoff | Peanut allergy is common among hazelnut-sensitized subjects but is not primarily the result of IgE cross-reactivity[END_REF][START_REF] Rougé | Mapping and conformational analysis of IgE-binding epitopic regions on the molecular surface of the major Ara h 3 legumin allergen of peanut (Arachis hypogaea)[END_REF][START_REF] Barre | Homology modelling and conformational analysis of IgE-binding epitopes of Ara h 3 and other legumin allergens with a cupin fold from tree nuts[END_REF]. Yet, selective interaction should be preserved as the beads offer other major molecular allergens for Ara and Cor that have shown no interaction with other IgE species [START_REF] Barre | Molecular modelling of the major peanut allergen Ara h 1 and other homotrimeric allergens of the cupin superfamily: A structural basis for their IgE-binding cross-reactivity[END_REF][START_REF] Barre | Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes[END_REF][START_REF] Beyer | Predictive values of component-specific IgE for the outcome of peanut and hazelnut food challenges in children[END_REF].

For Ara-sepecific IgE, Fig. 4a shows that PS-Cor presents some degree of antibody recognition, while PS-Ara displays an almost identical result as in the previous test. Their absorption signals are undiscernible at 24 ng/mL and begin to diverge much more significantly from 36 ng/mL onward. Even when the previous purification measurements during extraction and subsequent SEM images showed only presence of Cor allergens on PS-Cor, this result is consistent with the findings of Barre and coworkers [START_REF] Barre | Homology modelling and conformational analysis of IgE-binding epitopes of Ara h 3 and other legumin allergens with a cupin fold from tree nuts[END_REF] regarding structural similarities. It has been reported that Ara h3 and Cor a9 share 67% of amino acids, which could explain the false positive. Concerning the analysis of Cor-sspecific IgE, the response from PS-Ara was slightly different than that of control PS-BSA, but within the exclusion values, as observed in Fig. 4b. Meaning, PS-Cor was the only detectable signal and its absorption behaviour mimics the result for PS-Ara in our initial test.

Conclusion

Correct allergy diagnosis has become more relevant as the disease becomes more prevalent worldwide. We have developed and offered a proof-of-concept for a real time system by which the IgE concentration in a person's serum can be estimated through visible light absorption analysis of colloids. The proposed protocol offers a detection limit of 24 ng/mL for physiological samples, with a current linear detection range comparable to commercially used techniques. With respect to the specificity, it is susceptible to false detection due to crossreactivity of similarly structured allergens, but increasing the types of molecular allergens immobilised on the particle's surface yields an improvement in particle interaction accuracy. Although its sensibility is not comparable to current research whose focus is single-protein resolution, its characteristic miniaturisation and affordability would provide a benchtop device for several developing regions. Development of a prototype is being presently developed, along with further clinical tests to validate these results along a larger population.

Fig. 1 .

 1 Fig. 1. Schematic description of the microbead-based IgE detection by visible light absorption. (a) Preparation of the PS/SM particle suspension with human IgE, contemplating a reaction time of ~1.5h. (b) Absorption analysis of colloids for portable detection of antibodies within 1min. (c) Discernible signals created by different proteins immobilised on the PS surface, determined by the IgE concentration in solution.

Fig. 2 .

 2 Fig. 2. Characteristic particle-protein interaction seen on SEM imaging. The superficial topography of PS-Ara (a) before and (b) after interaction with IgE in serum is distinct to that of PS-Cor (c) before and (d) after serum reaction. Both specimens are also distinguishable from (e) a PS bead without bonded proteins. (f) View of a dragged particle complex by magnetophoresis: SM particles recognised IgE isolated by PS-Ara. Scale bars: 1 µm.

Fig. 3 .

 3 Fig. 3. Absorption of light by our particle-based immunoassay. (a) The absorption decay and stabilised level of the PS particles of interest is distinguishable from non-recognised particles; (b) showing an absorption difference proportional toIgE concentration. Results (for n = 3 repetitions) in (a) are normalised to their maximum; results in (b), to the measured absorption of 1x10 5 PS particles/mL.

Fig. 4 .

 4 Fig. 4. Effect of cross-reaction (molecules Ara h3 and Cor a9) on our immunoassay: (a) test of patient's serum allergic to Ara with several IgE concentrations, (b) test with Cor-allergic serum at differing antibody concentration. Results (for n = 3 repetitions) are normalised to the measured absorption of 1x10 5 PS particles/mL.

Acknowledgements

This work has been partially supported by the LabEx Minos ANR-10-LABX-55-01.


Flore Samaran
email: flore.samaran@ensta-bretagne.fr

Adrien Berne

Emmanuelle C Leroy

Sérgio Moreira

Kathleen M Stafford

Marcia Maia

Jean-Yves Royer

Antarctic blue whales (Balaenoptera musculus intermedia) recorded at the Equator in the Atlantic Ocean

. In general, these whales

spend the austral summer around the Antarctic and migrate northwards in winter, although there is evidence from both whaling-era data and modern passive acoustic monitoring that some animals remain at high latitudes in winter [START_REF] Hinton | Major Barrett-Hamilton relating to the whales of South Georgia[END_REF][START_REF] Širović | Seasonality of blue and fin whale calls and the influence of sea ice in the Western Antarctic Peninsula[END_REF][START_REF] Thomisch | Spatiotemporal patterns in acoustic presence and distribution of Antarctic blue whales Balaenoptera musculus intermedia in the Weddell Sea[END_REF]. The northernmost-documented wintering destinations of Antarctic blue whales include the west coast of Africa up to Angola (Best 1998, Figueiredo and[START_REF] Figueiredo | Blue whales Balaenoptera musculus off Angola: recent sightings and evaluation of whaling data[END_REF], the eastern tropical Pacific [START_REF] Stafford | Antarctic-type blue whale calls recorded at low latitudes in the Indian and eastern Pacific Oceans[END_REF], off northern New Zealand [START_REF] Mcdonald | An acoustic survey of baleen whales off Great Barrier Island, New Zealand[END_REF], and the Indian Ocean near the Diego Garcia atoll [START_REF] Stafford | Antarctic-type blue whale calls recorded at low latitudes in the Indian and eastern Pacific Oceans[END_REF].

Most current knowledge about the occurrence of Antarctic blue whales comes from passive acoustic data sets from hydrophones deployed from high to low latitudes (Antarctic deployments reviewed in [START_REF] Van Opzeeland | Towards Collective circum-Antarctic Passive Acoustic Monitoring: The Southern Ocean Hydrophone Network (SOHN)[END_REF]; for lower latitudes see [START_REF] Stafford | Antarctic-type blue whale calls recorded at low latitudes in the Indian and eastern Pacific Oceans[END_REF][START_REF] Mcdonald | An acoustic survey of baleen whales off Great Barrier Island, New Zealand[END_REF], Samaran et al. 2013[START_REF] Tsang-Hin-Sun | Low-frequency sound level in the Southern Indian Ocean[END_REF][START_REF] Leroy | Seasonal and diel vocalization patterns of Antarctic blue whale (Balaenoptera musculus intermedia) in the southern Indian Ocean: A multi-year and multi-site Study[END_REF]. This approach has proved very effective for monitoring Antarctic blue whales as they produce a distinct low frequency signal, known as the Z-call, easily recognizable from its Z-shape in the time-frequency domain (Fig. 1A; [START_REF] Ljungblad | A comparison of sounds attributed to pygmy blue whales (Balaenoptera musculus brevicauda) recorded south of the Madagascar Plateau and those attributed to "true" blue whales (Balaenoptera musculus) recorded off Antarctica[END_REF][START_REF] Rankin | Vocalisations of Antarctic blue whales, Balaenoptera musculus intermedia, recorded during the 2001/2002 and 2002/2003 IWC/SOWER circumpolar cruises, Area V, Antarctica[END_REF][START_REF] Širović | Blue and fin whale acoustic presence around Antarctica during 2003 and 2004[END_REF].

Densely produced Z-calls from distant Antarctic blue whales can create a 'chorus' of energy between 28 and 26 Hz (Fig. 1B). Both calls and choruses have been used to identify regions and seasons of occurrence for this species (e.g., [START_REF] Širović | Blue and fin whale acoustic presence around Antarctica during 2003 and 2004[END_REF][START_REF] Thomisch | Spatiotemporal patterns in acoustic presence and distribution of Antarctic blue whales Balaenoptera musculus intermedia in the Weddell Sea[END_REF][START_REF] Leroy | Seasonal and diel vocalization patterns of Antarctic blue whale (Balaenoptera musculus intermedia) in the southern Indian Ocean: A multi-year and multi-site Study[END_REF]. Here, we present the first evidence of the surprising seasonal presence of Antarctic blue whales at the Equator in the Atlantic Ocean.

In 2013, three hydrophones were deployed on moorings in the equatorial Atlantic Ocean during the COLMEIA-HYDRO acoustic experiment [START_REF] Maia | COLMEIA cruise[END_REF]Fig. 2). The instruments, developed by the Laboratoire Geosciences Océan (Brest, France), consisted of hydrophones connected to autonomous acquisition and storage systems [START_REF] D'eu | Long-term autonomous hydrophones for large-scale hydroacoustic monitoring of the oceans[END_REF]. Each instrument was moored in the axis of the sound fixing and ranging (SOFAR) channel. The acoustic data were digitized at a sample rate of 240 Hz using a 24-bit analog-to-digital conversion. The hydrophone sensitivity was -163.5 dB re: 1V/Pa, including preamplifier gain. Upon recovery, the data were downloaded and examined via long-term spectrograms. The records at the three sites clearly show a band of energy between 26 and 26.5 Hz throughout the year (Fig. 3). This band indicates the presence of the Antarctic blue whale chorus resulting from the first unit of Z-calls. The energetic bandwidth near 17-22 Hz represents a combination of Antarctic blue whales Z-calls, fin whale 20 Hz-pulses, and possibly Atlantic blue whale calls (16-18 Hz, Mellinger and Clark 2003).

To more closely analyze the presence of Antarctic blue whale calls, two metrics were used: the number of distinct Z-calls or call counts, and the power of the Antarctic blue whale chorus.

Indeed, a lack of individual call detections does not necessarily mean an absence of calling whales; a "loud" Antarctic blue whale chorus suggests that whales are present in the area, but not close enough to the hydrophone for individual calls to be distinct. Z-calls were automatically detected using the "Z-detector", a subspace-detection algorithm specifically developed to detect Antarctic blue whale Z-calls [START_REF] Socheleau | Automated detection of Antarctic blue whale calls[END_REF]. Because potentially masking signals (airguns, ship noise and Antarctic blue whale chorus) occur in the data set and can therefore result in false detections of Antarctic blue whale Z-calls, all detections were visually checked. All false detections were deleted and not considered further in this analysis. The number of remaining calls detected certainly underestimated the total number of recorded calls, since faint or noise-masked calls were either deleted or not detected. The total number of detected calls were summed and plotted per week (Fig. 4). To complement the observation, the chorus level (in dB re 1 μ Pa 2 /Hz) was calculated as the energy in the frequency band of 25.5-26.5 Hz minus the noise level in the 30-33 Hz frequency range, just above the chorus (see [START_REF] Leroy | Seasonal and diel vocalization patterns of Antarctic blue whale (Balaenoptera musculus intermedia) in the southern Indian Ocean: A multi-year and multi-site Study[END_REF]. This Chorus to Noise-without-chorus Ratio (CNR) was then averaged per week (Fig. 4).

The CNR at all sites showed a clear seasonal variation with a peak from May to July 2013 (Fig. 4). The CNR levels were very similar among the 3 sites except for a few weeks between mid-May and mid-June where the level was somewhat higher at the eastern site (C4).

At C2 and C4, very few and only faint individual Z-calls were detected, while at C5, over 1,144 individual clear Z-calls were detected. The calls recorded at C5 were not detected at the same time, or within the time frame required to travel from one hydrophone to another, as the faint calls recorded at C2 and C4. Calls in the vicinity of C5 were regularly detected during the austral winter (June-September; Fig. 4). The greatest number of detections, however, occurred over a 2-day period in July (15-16 July) with a total of 637 calls (56% of the total number of detections).

During 9 consecutive hours 382 Antarctic blue whale Z-calls were detected with high signal-tonoise ratios suggesting the singing blue whale(s) was (were) relatively close to the hydrophone.

During this short period, Z-calls were repeated at very regular inter-call intervals (mean 73.5 s from the start of a call to the start of the next call). Songs were sometimes interrupted by longer intervals of ~189 s (116 -311 s, n=32). The CNR peak occurred a month before the distinct series of calls (mid-June 2013).

Detection ranges of Antarctic blue whale individual Z-calls and chorus are difficult to estimate.

The range will depend on the environmental conditions (ambient noise levels, sound propagation) that can change seasonally, and on the depth and source levels of calling whales. Estimated detection distances for individual blue whale calls from other hydrophones deployed in the SOFAR channel have ranged from 10-200 km [START_REF] Stafford | Seasonal variability and detection range modeling of baleen whale calls in the Gulf of Alaska, 1999-2002[END_REF][START_REF] Samaran | Source level estimation of two blue whale subspecies in southwestern Indian Ocean[END_REF].

Based on simple Monte Carlo estimations for the COLMEIA location, individual Z-calls with good SNR were likely produced by whales within a few tens kilometers of the hydrophones.

Beyond this distance, clear Z-calls can occasionally be distinguished but propagation effects likely alter the shape of the signal and/or merge many Z-calls into a chorus making individual Z-calls indistinguishable. Beyond a few hundred kilometers, Z-calls contribute to the ocean soundscape only as a chorus (R. Emmetière, pers. comm. 2017 1 ). As the loudest series of individual calls and songs were only recorded on one of the three hydrophones, and the two closest hydrophones were 360 km apart, the whale(s) producing the loudest songs could not have been more than half this distance away (i.e., 180 km) or they would have been recorded on more than a single hydrophone. Even though we cannot tell how far, within this 180-km radius, the calling animals were from the hydrophones, the detection of distinct, loud individual Z-calls and songs suggests that Antarctic blue whales were present near our hydrophones at the Equator and were contributing to the underwater soundscape.

The COLMEIA-HYDRO data are of particular interest since they were collected in a region with a high seasonal productivity, but where only few catches, sightings, or strandings of blue whales are reported [START_REF] Branch | Past and present distribution, densities and movements of blue whales Balaenoptera musculus in the Southern Hemisphere and northern Indian Ocean[END_REF]. Only a single blue whale was recorded from the northeastern South Atlantic in whaling records off modern-day Gabon in 1926 [START_REF] Mackintosh | The southern stocks of whalebone whales[END_REF]. Three blue whales were sighted off the coast of Paraíba (7˚S), during commercial hunting seasons, between 1966seasons, between and 1981seasons, between (da Rocha 1983) ) and two blue whales were captured commercially in Paraíba, in 1948 and1965, and one in Rio de Janeiro (23˚S), in 1962 [START_REF] Zerbini | A review of the occurrence and distribution of whales of the Genus Balaenoptera along the Brazilian coast[END_REF]. In 1992, a 23 m long female stranded in Rio Grande do Sul (30˚S; Dalla Rosa and Secchi 1997).

Most recently, an adult and a young blue whale were sighted twice on 27 and 31 July, 2011, in Rio Grande do Norte (6˚S; de Oliveira 2015). It is not known whether these were Antarctic blue whales.

The COLMEIA-HYDRO records provide the northern-most evidence for a presence of Antarctic blue whales and the longest seasonality of this species at low latitudes. Based on their calling patterns, the bulk of the detected loud calls were likely produced by relatively few animals. One could assume that the songs recorded on 15 and 16 July 2013 were from a single individual in the vicinity of the hydrophone. In the South Atlantic, Antarctic blue whales are recorded yearround near Ascension Island (7°56'S 14°25'W; [START_REF] Haver | The not-so-silent world: Measuring Arctic, Equatorial, and Antarctic soundscapes in the Atlantic Ocean[END_REF]). In the Indian and eastern tropical Pacific Oceans, Antarctic blue whale calls were recorded at 8°S only in the austral winter and in each of these locations, in limited numbers and over relatively few days [START_REF] Stafford | Antarctic-type blue whale calls recorded at low latitudes in the Indian and eastern Pacific Oceans[END_REF]). However, the records presented here come one to two decades after these studies, and suggest that in the interim the global population of Antarctic blue whales has continued to grow and spread. The circum-Antarctic blue whale population was estimated at 2,280 individuals in 1998 [START_REF] Branch | Abundance of Antarctic blue whales south of 60 S from three complete circumpolar sets of surveys[END_REF], and its best growth rate at 7.3% per year [START_REF] Branch | Evidence for increases in Antarctic blue whales based on Bayesian modelling[END_REF]). In the intervening 2 decades since then, the population may have increased to well over 8,000 Antarctic blue whales. It is therefore entirely possible that this species has expanded its habitat both spatially and temporally around Antarctica and as far north as the Equator in the Atlantic, Pacific and Indian Oceans. However, the absence of individual Z-calls at 4°S in the Indian Ocean, in 2012-2013 [START_REF] Leroy | Seasonal and diel vocalization patterns of Antarctic blue whale (Balaenoptera musculus intermedia) in the southern Indian Ocean: A multi-year and multi-site Study[END_REF], suggest that Antarctic blue whales have not yet migrated that far north.

Figure 1 :

 1 FIGURESA)B)

Figure 2 :Figure 3 :

 23 Figure 2: Locations of the hydrophones during the COLMEIA-HYDRO acoustic experiment in the Equatorial Atlantic Ocean. Hydrophone deployments: C2: 01°20'N 031°21'W from 02 Feb to 22 Aug 2013; C5: 00°10'N 027°47'W from 25 Feb to 26 Dec; C4: 00°25'N 024°39'W from 21 Feb to 13 Sep 2013.

Figure 4 :

 4 Figure 4: Chorus to Noise-without-chorus Ratio (CNR) level for all sites and weekly number of Z-calls detected at the C5 station over the available weeks of data.

ACKNOWLEDGMENTS

The authors wish to thank the captains and crews of RV Atalante and NPAOC Araguari for the deployment and recovery, resp., of the COLMEIA-HYDRO hydrophones. These expeditions were funded by the French Ministry of Research and the Brazilian Navy, with additional support for the hydroacoustic experiment from CNRS-INSU, the Labex Mer and the Regional Council of Brittany. We are grateful to SECIRM, Brazilian Navy, for their help and support to this project.

LITERATURE CITED