# The Core Story: Intertwined Maternal and Progeny Signatures Enclosed in the Otolith

Clara Anne Lord, Sophie Berland, Vincent Hay, Philippe Keith

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Clara Anne Lord, Sophie Berland, Vincent Hay, Philippe Keith. The Core Story: Intertwined Maternal and Progeny Signatures Enclosed in the Otolith. Minerals, 2024, 14 (11), pp.1176. 10.3390/min14111176. [https://doi.org/10.3390/min14111176](https://doi.org/10.3390/min14111176)

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Article

# The Core Story: Intertwined Maternal and Progeny Signatures Enclosed in the Otolith

Clara Anne Lord *, Sophie Berland, Vincent Hay and Philippe Keith

UMR 8067, Biologie Des Organismes Et Écosystèmes Aquatiques (BOREA), Sorbonne Université, Muséum National d'Histoire Naturelle, Université de Caen Normandie, Université Des Antilles, CNRS, IRD, CP26, 43 Rue Cuvier, 75005 Paris, France; sophie.berland@mnhn.fr (S.B.); vincent.hay@sorbonne-universite.fr (V.H.); philippe.keith@mnhn.fr (P.K.)

* Correspondence: clara.lord-daunay@sorbonne-universite.fr

**Abstract:** Analytical imaging via synchrotron analysis was used to study sagittal otoliths of four freshwater pipefish species exhibiting amphidromous or freshwater resident lifestyles. We undertook a hyperfine elemental scanning of a 25 µm diameter length area centred on the core, namely the pre-hatch zone, corresponding to the *sagitta* formed during embryogenesis. We analysed calcium (Ca), strontium (Sr), manganese (Mn), chromium (Cr), cobalt (Co), nickel (Ni) and sulphur (S), with the latter serving to count growth increments. Whatever the species, a Ca pit consistently marked the core; the amphidromous species exhibited Mn enrichment at the core and the *sagitta* formed a week before hatching. These results illustrate common otolith formation mechanistic processes. Sr and heavy metals' signals in the pre-hatch zone were compared to their signal in the edge of the otolith, an area encompassing the adult freshwater (adFW) environmental signature. We found that Co and Ni otolith signals had no relation to environmental conditions, but it is worth looking into Cr as a marker of early-life environment. While we caution against the use of a Sr:Ca ratio for life history interpretations at the core because of the Ca pit, the Sr signal analysis uncovered possible variations in the parental behaviour that potentially trigger newly-hatched juvenile migration to sea or residency. Finally, our results demonstrate that the microchemistry of otolith formation during early development is a melting of maternal signal during egg yolk elaboration, of the environmental signal during egg incubation, and of individual physiology during early growth.

**Keywords:** elemental environmental signature; freshwater pipefish; otoliths; core; trace element incorporation; synchrotron XRF 2D scanning imaging

## 1. Introduction

Biomineralization is a widespread process among living organisms. It leads to the elaboration of a variety of solid structures such as, for example, shells, teeth, coral, spicules, bone, pearls, or otoliths. These biominerals are of both organic and inorganic composition and their formation is under physical, physiological, and biological control and it is complex to untangle the magnitude and role of each controlling process from one another in the formation of biomineralized structures [1]. The small amount of organic material exerts a crucial control on the mineralization process, contributing to the determination of the size, crystal morphology, specific crystallographic orientation, and properties of the particles formed [2]. Although the organisation of biomineralized structures is globally known, the precise mechanisms controlling crystal nucleation remain to be studied and understood. Even with this lack of knowledge, biominerals are recurrently used to study the biological object itself, i.e., the organism responsible for the formation of such a structure [3]. Otoliths are remarkable paired calcified structures in the inner ears of bony fishes [4]. They are used in a wide number of studies ranging from species determination, stock assessment, population connectivity and dynamics [5,6], habitat use and fish migration, age estimation, pollution assessment to life cycle determination [7]. Recent studies on pipefish otoliths,

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**Citation:** Lord, C.A.; Berland, S.; Hay, V.; Keith, P. The Core Story: Intertwined Maternal and Progeny Signatures Enclosed in the Otolith. *Minerals* **2024**, *14*, 1176. https://doi.org/10.3390/min14111176

Academic Editor: Athanasios Godelitsas

Received: 11 October 2024

Revised: 12 November 2024

Accepted: 18 November 2024

Published: 20 November 2024

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**Copyright:** © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
using synchrotron-based analysis methods, have shown that the otolith is under the control
of complex processes, which may sometimes lead to biological misinterpretation [1,8].

Otoliths start forming during embryogenesis [9]. The primordium is the element
around which the otolith will grow continuously during the life of the fish; it is therefore
the growth centre of the otolith and is also called core. Otolith cores are composed of
material deposited prior to active feeding and are considered to represent the portion of the
otolith formed at the spawning site while the egg sac, elaborated by the mother, is being
absorbed [10,11]. Chemical analysis of the otolith core may then provide the link between
a mother and its progeny. The core is only a few microns in diameter [9], making its
study highly challenging. However, a few researchers have undertaken it with the hope of
retrieving maternal information and elements on very early life [11–13]. While the growth
of the otolith is well documented, the formation of the core and the growth of the otolith
during embryogenesis is still quite unknown, and partitioning the maternal influence from
the environmental influence during egg incubation remains difficult to assess.

Fish have three pairs of otoliths: the *sagittae*, the *lapillii*, and the *asterici*. The *astericus*
is very rarely used to retrieve biological information. It is often the smallest and it also is
formed later than the other two during embryogenesis [4,14]. The *lapillus* is sometimes
used and, as some authors state, better suited for the study of early-life events [15–17].
However, the most used otolith is the *sagitta*. It is generally the biggest otolith, enabling
easier analysis [14]. Recent research shows that both *sagitta* of a given individual render
the same general information in terms of element incorporation and localization, and in
terms of age determination [8]; between the *sagitta* and the *lapillus* of an individual, the
global information on life history traits is the same, although there are differences in age
determination, with a lower age determined with the *lapillus*. In the present paper, we
chose to focus on the study of core formation and embryonic growth of sagittal otoliths as,
based on previous research, they seem to be slightly more reliable than *lapilli* [8].

The study of the core, because it is so challenging, necessitates highly precise methods
to retrieve information. Recent research has shown that 2D element mapping methods are
most suited for precise data acquisition [1, 8, 17, 19]. Synchrotron-based XRF mapping, for
instance, has allowed for the understanding of otolith biomineralization processes and the
incorporation of elements in the otolith lattice, such as strontium (Sr), heavy metals like
iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), or zinc (Zn), the latter having an important
role in the growth of the otolith as it is a cofactor of biomineralization enzymes [8, 20, 21].
Additionally, a new synchrotron-based XRF method for counting growth increments was
recently developed based on the counting of sulphur peaks along a growth transect: each
sulphur peak corresponds to one growth increment [18]. Because these recent papers
have helped unravel new elements of otolith growth mechanisms and biomineralization
processes, we found it appropriate to tackle the core of the otoliths using this method, as it
is precise enough to map elements at a hyperfine scale and allows for the analysis of areas
with micrometric dimensions.

We analysed the core of freshwater pipefish *sagitta*. We focused on four species in-
habiting tropical island streams: *Microphis brachyurus* (Bleeker, 1854) (Sri Lanka to French
Polynesia), *Microphis nicoleae* Haÿ et al. 2023 (Papua New Guinea and Solomon Islands),
*Microphis torrentius* Jordan and Seale, 1906 (Samoa, Vanuatu and New Caledonia), and
*Microphis cruentus* Dawson and Fourmanoir, 1981 (New Caledonian endemic). *M. brachyu-*
*rus, M. nicoleae* and *M. torrentius* are amphidromous [1,18]: the adults live in freshwater
and the eggs, carried by the adult male, hatch in freshwater. The newly hatched juveniles
migrate to the sea where they can stay up to 40 days [18]; they then return to freshwater
to settle and reproduce. *M. cruentus* has been categorized as a facultative amphidromous
species. Indeed, some individuals complete their life cycle in freshwater without ever
going to the sea [1]. In many species considered to be obligate migratory species, there
can be variations in the migratory modalities [22–24]. Our sampling is thus composed of
both amphidromous and resident specimens, for which the analysis of the core could help
unravel a potential parental behaviour leading to these differences in migratory behaviour.
The different geographical provenance of individuals will help assess the feasibility of using the otolith core for tracing the natal origin of individuals, especially in the case of migrating diadromous species. Indeed, amphidromous individuals do not necessarily live where they were born, except for resident species like *M. cruentus*. The amphidromous life cycle is an adaptation to the colonisation of tropical island rivers; the dispersal phase at sea enables individuals to recruit to rivers potentially far from their natal stream. Juveniles can then, after the marine phase, either recruit back to their natal stream or find a new environment to colonise. The study of the otolith core of these amphidromous pipefish may help characterise migration capacities of the different species and to assess whether the elements measured at the core can really be used to interpret biological facts on the early-life history of fishes.

The aim of the present work is to use synchrotron XRF scanning imaging to tackle the question of exploiting the otolith core region to uncover potential early-life environmental signatures. We studied a 25 µm diameter length area centred on the core of different pipefish species, including individuals with different geographical provenance and experiencing diadromous or resident lifestyles, using synchrotron-based XRF hyperfine scale mapping. This method allows characterising the core and the embryonic otolith in terms of calcium carbonate mineral and in terms of its elemental composition from which common processes relating to otolith formation will be proposed. We also compared elemental composition of the core with the information contained in the edge of the otolith, known to encompass the signature of the adult life environment. Variations in elemental composition between the samples studied will allow assessment of the use of precise otolith core analysis for the understanding of the migratory behaviour of amphidromous species, and retracing of environmental conditions at hatching and maternal influence on progeny.

## 2. Materials and Methods

### 2.1. *Sample Preparation*

Otoliths were obtained from the Muséum national d'Histoire naturelle (MNHN, Paris, France) collections of freshwater Syngnathidae, sampled from different locations in the Indo-Pacific realm and preserved in 95% ethanol, a preservation medium that has been shown not to hamper microchemical analysis [25]. Sample preparation was thoroughly described in our previous works [1,8,18] since the specimens used in the present study were selected in a set of pipefish analysed using the synchrotron XRF method to characterize their life cycle, i.e., whether they were amphidromous or freshwater residents. We used a total of 13 specimens: seven *Microphis brachyurus* (three from Japan, one from Tahiti in French Polynesia, and three from New Caledonia); two *Microphis nicoleae* (one from Papua New Guinea—PNG—and one from the Solomon Islands); two *Microphis torrentius* and two *Microphis cruentus*, all from New Caledonia (Table 1). *M. brachyurus*, *M. nicoleae*, and *M. torrentius* specimens are all amphidromous, while *M. cruentus* specimens are both freshwater residents, in the context of facultative amphidromous species.

The otoliths (*sagittae*) were removed from the saccules of the inner ear of each sample, thoroughly rinsed with MilliQ water and kept dry until they were individually embedded in epoxy resin (Araldite 2020, Escil, Chassieu, France). The embedded otolith was cut on either side of its core along the longest transverse axis using a low-speed diamond-bladed saw (Buehler, Leinfelden-Echterdingen, Germany) and ground-on carbide silicon abrasive discs of decreasing grain size (Escil, Chassieu, France). A slice less than 200 µm thick was obtained, exposing a frontal section down to the core. The slice was checked by light microscopy. The slices were used for emitted X-ray fluorescence spectrometry analysis at the NANOSCOPIUM beamline of Synchrotron SOLEIL (St Aubin, France), and for Raman microspectrometry at the CRC Raman facility (MNHN, Paris, France). As both methods are non-destructive, they could be carried out consecutively on the same samples. The otolith analysis was performed, starting with XRF analysis, to retrieve the elemental distribution data from otoliths that had not been submitted to high-energy radiations beforehand, to prevent from any interference risk, even though mineralogy changes are unlikely. The
slices were mounted on X-ray transparent rings and displayed in an in-house mounting device to expose the otolith cross section perpendicular to the synchrotron beam.

<table><caption>**Table 1.** Sampling. Collection number: National Museum of Natural History of Paris collection identification; Scan ID: Synchrotron scan identification corresponding to each *sagitta* (country, river, scan number, species code); SL: Standard length of the fish in mm; Sex: F: female, M: male; sampling location (country, island and river).</caption><thead><tr><th>Species</th><th>Collection Number</th><th>Scan ID</th><th>SL (mm)</th><th>Sex</th><th>Country</th><th>Island</th><th>River</th></tr></thead><tbody><tr><td>M. brachyurus</td><td>MNHN-IC-2023-0276</td><td>JAP.Genka.8067.bra</td><td>175.75</td><td>F</td><td>Japan</td><td>Okinawa</td><td>Genka</td></tr><tr><td>M. brachyurus</td><td>MNHN-IC-2021-0320</td><td>JAP.Genka.8079.bra</td><td>187.81</td><td>F</td><td>Japan</td><td>Okinawa</td><td>Genka</td></tr><tr><td>M. brachyurus</td><td>MNHN-IC-2023-0275</td><td>JAP.Teima.8091.bra</td><td>165.12</td><td>F</td><td>Japan</td><td>Okinawa</td><td>Teima</td></tr><tr><td>M. nicoleae</td><td>MNHN-IC-2023-0446</td><td>PNG.Gavu.3939.nic</td><td>69.41</td><td>F</td><td>Papua New Guinea</td><td>New Britain</td><td>Gavuvu</td></tr><tr><td>M. nicoleae</td><td>MNHN-IC-2021-0336</td><td>SOL.Ov.3918.nic</td><td>64.37</td><td>F</td><td>Solomon Islands</td><td>Ranongga</td><td>Ovana</td></tr><tr><td>M. brachyurus</td><td>MNHN-IC-2023-0051</td><td>POL.Pap.3913.bra</td><td>105.02</td><td>F</td><td>French Polynesia</td><td>Tahiti</td><td>Papenoo</td></tr><tr><td>M. brachyurus</td><td>MNHN-IC-2023-0273</td><td>NC.Creek.8072.bra</td><td>124.32</td><td>F</td><td>New Caledonia</td><td>Grande Terre</td><td>Creek Baie</td></tr><tr><td>M. brachyurus</td><td>MNHN-IC-2023-0272</td><td>NC.PwH.8075.bra</td><td>168.60</td><td>M</td><td>New Caledonia</td><td>Grande Terre</td><td>Pwé Hiit</td></tr><tr><td>M. torrentius</td><td>MNHN-IC-2021-0334</td><td>NC.PwH.8084.tor</td><td>86.15</td><td>M</td><td>New Caledonia</td><td>Grande Terre</td><td>Pwé Hiit</td></tr><tr><td>M. torrentius</td><td>MNHN-IC-2021-0335</td><td>NC.Ga.8077.tor</td><td>89.69</td><td>M</td><td>New Caledonia</td><td>Grande Terre</td><td>Garana</td></tr><tr><td>M. brachyurus</td><td>MNHN-IC-2023-0277</td><td>NC.Ga.8081.bra</td><td>120.41</td><td>F</td><td>New Caledonia</td><td>Grande Terre</td><td>Garana</td></tr><tr><td>M. cruentus</td><td>MNHN-IC-2021-0349</td><td>NC.Ouen.8069.cru</td><td>110.23</td><td>F</td><td>New Caledonia</td><td>Grande Terre</td><td>Ouenghi</td></tr><tr><td>M. cruentus</td><td>MNHN-IC-2021-0349</td><td>NC.Ouen.8071.cru</td><td>168.60</td><td>F</td><td>New Caledonia</td><td>Grande Terre</td><td>Ouenghi</td></tr><tr><td>M. brachyurus</td><td>larva *sagitta*</td><td>NC.PwH.8092.bra</td><td>-</td><td>-</td><td>New Caledonia</td><td>Grande Terre</td><td>Pwé Hiit</td></tr></tbody></table>

Beside the otoliths sections from the specimen in the adulthood, the *sagitta* of a *M. brachyurus* larva which had been caught in freshwater at hatching and had therefore never undertaken its migration to the sea (Table 1) was analysed. There was no preparation for the larva *sagitta*, except it had been preserved in 95% ethanol. The *sagitta* was analysed in toto. An X-ray transparent loop (Dual Thickness MicroMounts, MiTeGen, LLC, Ithaca, NY, USA, 30 µm) was used to handle the larva *sagitta* (hereafter named ‘larva’) up to its placement in the mounting setup. The set up is equipped with X and Y motorized plates allowing sample displacement with 100 nm accuracy perpendicular to the beam.

## 2.2. Synchrotron-Induced X-Ray Fluorescence Data Acquisition

The X-ray fluorescence spectrometry (XRF) was performed by scanning the full-size areas of the otolith cross sections at the Nanoscopium Nanoprobe CX3-scanning spectro-microscopy beamline of the Synchrotron Soleil (Saint-Aubin, France). The beamline allows the focusing of a high-energy and coherent X-ray beam for diffraction analysis and imaging with 0.5 µm spatial resolution. The method has been described in detail in former works [1,8,18]. Briefly, data, consisting in XRF emitted energy in the 2–16 keV range covered by the beamline, were simultaneously collected under continuous scanning in FLYSCAN mode [26]. All the samples were subjected to the same conditions: spot size of 0.5 µm and a dwell time of 40 ms, providing the same calibration. The output data consists of
hyperspectral datasets stored in Hierarchical Data Format (HDF) for each sample that can
be loaded as images (scanning image) to be processed using imaging methods.

## 2.3. Specific Element Distribution Characterization

The sample XRF scanning data from the hyperspectral dataset were processed using the open source PyMCA software (PyMca 5.9.2 http://pymca.sourceforge.net/, accessed on 15 September 2024) [27], as its libraries allow XRF data processing and spectra fitting. The samples' z-values (signal intensity) were displayed along the emission energy range course in the x-axis. After appropriate spectrum fit with the Ca and Sr strong peaks, target element abundance (count) was computed from under the Kα peak of each target element separately. For the elements used in the present study, the Kα relevant lines were at (in eV units): 231 (S); 541 (Cr); 590 (Mn); 693 (Co); 748 (Ni). The broad peaks at 369 and 1416 (ev) sign for the Ca and Sr signal, respectively. Each element signal imaging and counting was performed on subsets of the hyperspectral dataset corresponding to restricted intervals centred at the target Kα band, namely 217:233, 533:545, 577:597, 686:706, and 725:754 for S, Cr, Mn, Co, and Ni, respectively. This method of counting Kα peak restricted area has the benefit of limiting interference, in case neighbour element lines overlap, and ensuring the target trace elements signal addressing.

As a corollary, all the counts produced in the present study are in arbitrary units. However, confidence in the comparison of results is underpinned by the fact that the same parameters were applied to all the samples, from data acquisition to extraction, and the synchrotron XRF method proved capable of elemental quantification [1]; the counts can be compared among the samples with confidence.

## 2.4. Region of Interest Retrieval

Scanning image of the sample can be restored from the compiled hyperspectral dataset using the Fiji software (Fiji Is Just ImageJ) (http://fiji.sc/cgi-bin/gitweb.cgi, accessed on 20 September 2024) and shareware for image processing package with self-updating properties [28] using 'importHDF5' tool. The data are produced as a stack of the entire samples' z-values series. Navigation through the images stack allowed making the subsets' element-specific scanning export as z-profile data by summing across the ad hoc energy range (see above). Each element course over the sample for imaging and computation was made using the Fiji software tools.

Ca distribution images were used to pinpoint the core position and draw a circle 25 µm in diameter around the very spot of the ore to specify the pre-hatch zone as a region of interest (roi), which is displayed in each element course's subsets. The adult freshwater (adFW) roi was designed at the outer rim of the otolith section, as described in [18].

## 2.5. Computing

The large amount of data resulting from Synchrotron-XRF acquisition is released as compiled outputs thanks to in-house developed data reduction pipeline and can be processed on standard working stations by using an image analysis program, such as Fiji software, e.g., for hyperstack subsets acquisition, and the collaborative open source R project available on the Comprehensive R Archive Network (https://cran.r-project.org/, accessed on 24 September 2024) [R Development Core Team, 2010, version 4.2.3] for data manipulation, e.g., for summing the z-values over the two areas of interest to allow their comparison on graphs or for topographic display. For addressing samples and roi comparison, sum signal intensity was reported to the pixel size and referred to as "normalized results" in the figure legends.

## 2.6. Raman Microspectrometry

Raman analyses were done at the CRC MNHN laboratory using an inVia Raman confocal microspectrometer (Renishaw, Wotton-under-Edge, UK) equipped with a 532 nm laser source set to 2.1 mW. The spectra were acquired with a 5% power filter, which was
used to avoid causing heating damage to the sample. Raman spectrum was acquired at the core spot of the otolith cross section on the samples. The motorized microscope stage of the device monitored the positioning. To encompass the Raman aragonitic signature range (i.e., vibrational band at 705 and 1084 cm⁻¹), data were collected centred at 1100 cm⁻¹ (170 at 1930 cm⁻¹) with a spectral resolution of 2 cm⁻¹. The analysis was the co-addition of three spectra accumulated at up to 1 s exposure.

**3. Results**

Figure 1 is a summary of each step of the XRF otolith microchemistry analysis undertaken in the present work. On each image obtained for each otolith, a 25 µm diameter area, centred on the core, was drawn (Figure 1A). This 25 µm diameter dimension was chosen as the *sagitta* of the larva studied, caught just at hatching (Table 1), was circular and was approximately 29 µm in diameter (Figure 1B). We chose a slightly lower dimension to make sure we focused on a zone which grew during embryogenesis. This very region of interest was named the ‘pre-hatch’ zone; it includes the core and the biomeralized material, which was accreted around the core during embryogenesis. We first focused on calcium (Ca), as it is the main constitutive element of otoliths [4] (Figure 1A,C). We then studied Sr distribution, as it is widely used to study fish environmental conditions; more specifically, Sr is a marker of salinity [29–31]. Sr distribution was analysed on its own and relative to Ca, as the Sr:Ca ratio is most used in research on diadromous fish (Figure 1C). The cumulated synchrotron-based X-ray fluorescence emissions in the otolith samples over the 25 µm disc was plotted (Figure 1D). Energy intensities (x-axis) are displayed with their count (y-axis). The broad peaks at 366 eV and 1399 eV are characteristic X-ray emissions from Ca and Sr, respectively. The XRF peak intensities allow the identification of other elements which are worth analysing. Sulphur marks the presence of proteins and is used to count otolith growth increments [18]. This method is then used to assess the presence and number of growth increments in the embryonic otolith. One of the aims of the present work is to assess whether the pre-hatch otolith is a reliable marker of early-life environment, so we chose heavy metals which are environmental marker candidates: chromium (Cr), manganese (Mn), cobalt (Co), and nickel (Ni). The latter two were concomitantly chosen as they exhibit close emission peaks. Cr and Mn peaks are distinct from one another and from any other element peak (Figure 1D). Each of the chosen elements was individually mapped for each otolith. We also tackled the question of the use of studying the otolith pre-hatch region to uncover potential early-life environmental signatures by comparing elemental composition of the core with the information contained in the outermost area of the otolith, known to encompass the adult environmental signature, and was named hereafter the ‘adult freshwater environment’ area (adFW) (Figure 1E).

**3.1. Raman Deduced CaCO₃ Mineral in Aragonite Form at the Otolith Core**

The Raman analysis was spotted at the core position of the otoliths and resulted in the assessment that it is composed of CaCO₃ crystallised under the aragonite form (Figure 2). In this respect, the spectra show the characteristic aragonite bands i.e., vibrational bands at 705 and 1084 cm⁻¹.
<figure><img src="image_6.png" /></figure>

**Figure 1.** Summary of the steps of the XRF otolith microchemistry analysis. (A) Ca XRF scanning image of the cross section of the otolith. A circle 25 µm in diameter around the core (dark spot) specifies the pre-hatch region of interest (blue line delimitation). (B) Sr XRF scanning image of the sagitta, the dimensions of which were used for sizing the included pre-hatch zone. (C) 3D reconstruction of z-values of Ca (left), Sr (mid) XRF signal (arbitrary units), and Sr:Ca z-values ratio (right) of the pre-hatch zone. The surface plots show uneven Ca z-values at the centre of the pre-hatch zone, i.e., the core position when Sr z-values stay still, resulting in a Sr:Ca ratio 'pit' at the core place. (D) Sample z-values (signal intensity, log units) displayed along the emission energy range course. After appropriate spectrum fit (Ni fit shown in red line), the target element signature peaks position (coloured dotted lines with corresponding element symbol above) was used to design the restricted interval subsets of the hyper-spectral dataset. (E) Schematic drawing of the regions of interest positioning, pre-hatch area, and adult freshwater area (adFW), on the otolith section.

3.2. *Sr Driven Pre-Hatch Area Zonation*

For the 13 otoliths analysed, the Ca surface plots of the 25 µm pre-hatch zone show a Ca depression, or pit, right at the core (Figures 1C and S1).
<figure><img src="image_7.png" /><figcaption>**Figure 2.** Raman signature at the core. The picture shows the location of the spectrum acquisition: the crossing centre of the guides targeting the core. The spectrum acquired (below) is dominated by the broad band observed at 1084 cm⁻¹ with contribution of a vibrational response at 705 cm⁻¹, the characteristic bands of CaCO₃ crystalized in the aragonite form.</figcaption></figure>

Each otolith analysed showed a peak of Sr:Ca at the core (see maps in [1]). Sr surface intensity plots were drawn to see whether there was an actual Sr peak at the core without the influence of Ca, but for all the otoliths analysed, the Sr signal is smooth and uniform on the entire surface of the pre-hatch area, as shown in the Sr surface plot (Figure 1C). Because of the Ca pit, as shown on the Sr:Ca surface plot, the Sr:Ca ratio is higher at the core (Figure 1C), although the quantity of Sr is not actually higher at the core. The Sr:Ca signal is an artefact caused by the lower quantity of Ca at the core.

### 3.3. *Mn Patch at the Core*

The otoliths of *M. brachyurus* (including the larva), *M. nicoleae*, and *M. torrentius*, which have an amphidromous life cycle, show a peak of Mn at the core, right at the location of the Ca pit or in its immediate vicinity (Figure 3A,B). It is slightly to the side for *M. nicoleae* caught in PNG (Figure 3A) and quite scattered for *M. nicoleae* caught in the Solomon Islands. For most specimens, the Mn signal at the core is consistently higher than in any other region of the otolith where there are no other high concentration patches of Mn. To illustrate this affirmation, we have compared the Mn signal intensity of the pre-hatch area to the external rim band in the adult region (adFW). The Mn of the pre-hatch area is always higher than the adFW (Figure 4). The two *M. cruentus* specimens (freshwater residents) analysed, however, seem to lack this Mn peak at the core (Figure 3B), although the Mn signal is still higher in the pre-hatch zone than in the adFW zone (Figure 4).
<figure><img src="image_8.png" /><figcaption>Figure 3. *Cont.*</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 3.** Element distribution maps (Mn: manganese, Cr: chromium, Co: cobalt, Ni: nickel) at the pre-hatch zone. (A) Samples from Japan (Jap), Papua New Guinea (PNG), Solomon Islands (SOL), and French Polynesia (POL). First line shows projection of the element distribution from the in toto larva *sagitta*. Other lines show the XRF imaging of the selected element at the pre-hatch zone from full-grown individuals. (B) Samples from New Caledonia (NC). Lines show the XRF imaging of the selected element at the pre-hatch zone from full-grown individuals. Note that *M. cruentus* (last and penultimate lanes) are freshwater resident individuals. Sample labels include country, river, XRF scan number, and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*) (see Table 1). The white line is the core delimitation in the pre-hatch zone.</figcaption></figure>
<figure><img src="image_10.png" /></figure>

**Figure 4.** Comparison of the manganese (Mn) signal intensity in the pre-hatch area (blue) versus the adult freshwater area adFW (grey) (summed z-values, arbitrary units, normalized results). Sample labels include country, river, XRF scan number, and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*) (see Table 1).

### 3.4. Chromium, Cobalt and Nickel in the Pre-Hatch Zone

Cr, Co, and Ni individual maps show that these trace metal elements exhibit a similar behaviour in all the otoliths analysed (Figure 3A,B). Cr is present, but the signal is scattered over the entire surface analysed and, wherever present, the signal's intensity is low compared to the other metals analysed. The Cr signal is quite present in the larval otolith (Figure 3B). Co and Ni have approximately the same distribution and signal in all otoliths analysed, whatever the species, and there does not seem to be any difference between species. The similar Co and Ni signals could be due to peak partial superimposition (Figure 1D), leading to mixed signals of the two elements. However, the repeatability of the results for Co and Ni validates the methodology used.

### 3.5. Growth Increments Within the Pre-Hatch Zone

Sulphur marks the presence of proteins and is therefore used to estimate the number of growth increments [18]. The c. 2 µm wide transect was drawn from the centre of the core to the edge of the pre-hatch zone analysed (Figure 5). The Mn plot shows a clear peak at the core (Figure 5), as explained in the result Section 3.3 (Figure 3); it was drawn to compare with S intensity at the core. Indeed, S at the core is low (Figure 5). The S-peak profiles obtained enabled counting of the number of increments; there are, for example, seven S-peaks for the *M. brachyurus* specimen from French Polynesia (3913) (Figure 5). Interestingly, all the counts are very close and there are seven or eight increments in the pre-hatch zone for all the otoliths analysed (Figures 5 and S2).

### 3.6. Pre-Hatch vs. adFW Elemental Signatures

To test the use of the core region to deduce maternal origin of amphidromous pipefish, we compared the elemental signals inherent to the pre-hatch zone and to the adFW zone of the otolith, as defined by Lord et al. [8] (Figure 1E).
<figure><img src="image_11.png" /></figure>

**Figure 5.** Growth increments within the pre-hatch zone (specimen POL.Pap.3913.bra). **Left:** Study transect from the core (red arrow) to the edge of the pre-hatch area; **Middle:** Manganese (Mn) intensity plot showing a clear peak at the core (red arrow); **Right:** Sulphur (S) intensity plot showing the S peaks along the study transect. No peak is present at the core (red arrow). There are seven S-peaks positioned with strokes and counting along the transect. Increment counts are given for each sample in the table on the left of the figure. Sample labels include country, river, XRF scan number, and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*) (see Table 1).

### 3.6.1. Strontium

The Sr signal intensity is greater for the pre-hatch zone than for the adFW zone for all the individuals analysed (Figure 6). The signal always appears shifted to the highest intensities. We can group individuals according to their pre-hatch/adFW Sr profile similarities: (i) the two *M. nicoleae* specimens (SOL.Ov.3918.nic; PNG.Gavu.3939.nic), *M. brachyrurus* (NC.Ga.8081.bra) and *M. torrentius* (NC.Ga.8077.tor) specimens caught in the Garana River (NC) and the three *M. brachyurus* specimens from Japan (JAP.Genka.8067.bra; JAP.Genka.8079.bra; JAP.Teima.8091.bra). These specimens show a wide gap between in their Sr profiles between the pre-hatch zone and the adFW; (ii) *M. brachyurus* from French Polynesia (POL.Pap.3913.bra), *M. brachyrurus* and *M. torrentius* caught in the Pwé Hitt River (NC.PwH.8075.bra; NC.PwH.8084.tor), and the *M. brachyurus* caught in the Creek Baie River (NC.Creek.8072.bra). These specimens show quite a spread-out adFW Sr profile. (iii) The two *M. cruentus* specimens (NC.Ouen.8069.cru; NC.Ouen.8071.cru) show very little difference between the pre-hatch and the adFW Sr profiles. Their pre-hatch and adFW signals only show a slight shift, both exhibiting Sr signals of low intensity. These results show that two individuals caught in the same river have the same profile and that individuals may have similar Sr profiles although sampled in different locations.

### 3.6.2. Heavy Metals

Apart from the individuals sampled in the Pwé Hitt River and in the Garana River in New Caledonia, the Cr signal (Figure 7) is similar for all individuals analysed, both in the pre-hatch zone and in the adFW zone, with very little difference between both zones of the otolith. For the four New Caledonian specimens mentioned above, the Cr signal is considerably higher in the adult zone compared to all other individuals, and it is even higher in the pre-hatch zone of the *M. torrentius* specimen compared to its adFW zone and compared to the pre-hatch zone region of all other samples.
<figure><img src="image_12.png" /><figcaption>**Figure 6.** Comparison of the strontium (Sr) signal intensity in the pre-hatch area (blue) vs. the adult freshwater (adFW) zone (grey) (summed z-values, arbitrary units, normalized results). Sample labels include country, river, XRF scan number, and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*) (see Table 1).</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 7.** Comparison of the heavy metal signals between the pre-hatch region (blue) and the adult freshwater (adFW) zone (grey) (normalized results) for each of the individuals. Cr: chromium; Co: cobalt; Ni: nickel. Sample labels include country, river, XRF scan number, and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*) (see Table 1).</figcaption></figure>
Co and Ni show similar profiles (Figure 7). This validates the method used considering Co and Ni have very close emission energies and because their signal peaks partially overlap (see Figure 1D). The difference between the pre-hatch zone region and the adFW zone is very variable and seems to be individual-dependent. Indeed, Co and Ni can have very different signals in the adult zone even for fish sampled in the same river, i.e., the two specimens from Garana River in NC or the two *M. cruentus* specimens have very different adFW profiles.

# 4. Discussion

## 4.1. Mineralisation at the Core

It is widely known that otoliths are calcium carbonate biomineralized structures. Raman analysis confirmed that, at the earliest time of the otolith formation, the crystal was arranged under the aragonite form, the most common polymorph in fish otoliths [4]. All the otoliths analysed showed a pit of Ca at the core. Otolith formation requires bringing together organic and inorganic components in time and space [32]. The pit of Ca can be explained by the fact that otolith nucleation is probably initiated by organic molecules. Indeed, reports from previous studies, for example on zebrafish embryos, show that the core is probably nucleated by spherules made of rearranged glycogen particles [9]. Glycogen molecules seem to play a key role in initiating the formation of hard-calcified tissues [33–35]. Polysaccharides have been reported to be involved in the initiation of biomineralization processes for a long time. For instance, carbonate microbialites, which consist of sedimentary aggregates associated with microbial mats and can be considered among the oldest pieces of evidence of life on Earth, exhibit aragonite crystal arrangements laid down in an organic matrix rich in polysaccharides, which play an important role in crystal formation [36]. Chitin, one of the most abundant polysaccharides in living beings, offers a wide range of biomineralization scaffolding among protozoans, animals, and even plants [2].

As for many processes in life, basic tools are used in highly different contexts for similar results, and biomineralisation processes, in particular, support parallel evolution based on basic toolkits [37]. It is not surprising to find polysaccharides implicated in the formation of otoliths. In addition, proteins have also been identified to be involved in the control of biomineralization [38–40]. These proteins are usually highly negatively charged and contain carboxylate, sulfate, or phosphate as functional groups, which may bind Ca²⁺ ions and control crystal nucleation and growth [2]. It would then seem that a combination of polysaccharides and proteins are at the origin of otolith nucleation. These would be proteoglycans, which consist of a protein core to which highly negatively charged linear polysaccharide side chains are covalently attached. Proteoglycans, which have not yet been characterized, have also been found to be involved in the calcification process of fish otoliths [41,42].

## 4.2. Mn Signature

All the otoliths analysed, including the larva and apart from the two *M. cruentus* specimens, exhibited a peak of Mn at the core. This peak is superimposed to the Ca pit, although its shape is not circular. Mn incorporation could be a substitution for Ca, given the similar ionic radius between both elements [43] and the fact that both Mn and Ca can form 2+ ions. However, given the circular growth of the embryonic *sagitta*, the shape of the Mn aggregation near the core seen on the maps is surprising, and it might correspond to the sequestration of Mn in the newly-formed otolith. Indeed, during the biomineralisation process of otoliths, elements may be unevenly incorporated in the crystal at a hyperfine scale, leading to mosaic elemental patterns, as seen for Sr or heavy metals in pipefish otoliths [1,8], and not unlike what is observed for Mn at the core and its vicinity.

The elevated Mn signal at the core has been observed for freshwater and marine fish species [10,44–47]. Herrings (*Clupea harengus* L.) and sprats (*Sprattus sprattus*), marine fish of the Clupeidae family, both show elevated Mn concentrations in the otolith core,
concentrations which did not reflect the environmental conditions in which the embryos developed [45]. Indeed, as teleost fish eggs have some permeability [48], they may take up trace elements from their environment, such as Mn²⁺ ions. However, Brophy et al. [45] show that, even in Mn poor environments, the core is Mn rich. The same result was found for six other species, sampled in very different taxonomic groups [10]: these species inhabit marine temperate or tropical regions and exhibit various spawning modes (pelagic spawner, demersal nest brooder, ovoviviparous brooder), including an amphidromous Galaxiidae from New Zealand. Their results [10] indicate a peak of Mn at the core in 77% of otoliths analysed for the marine species, and in 95% of otoliths analysed of the amphidromous species. Ruttenberg et al. [10] used LA-ICPMS to analyse the otolith core and its surroundings and authors suggest that, for otoliths for which there was no Mn peak observed, the core must have unintentionally been altered during preparation procedures. Ref. [45] came to the same conclusion concerning otoliths which did not produce a distinct peak; they also blamed variations in the individual ablation events. In our present case, we demonstrate the constraint location of the Mn patch covering a few microns in the vicinity of the core, which has led to a stringent sample selection process and can support the ambivalent Mn core signature reported by these authors. There is a distinct peak at the core of each otolith analysed for *M. brachyurus*, *M. nicoleae*, and *M. torrentius*, confirming the results found in previous studies. The two *M. cruentus* specimens show no Mn peak. We could also suggest a manipulation artefact, but it is unlikely the two specimens exhibit the Ca pit, i.e., the precise location of the core. The *M. cruentus* individuals analysed are residents; their life cycle entirely takes place in freshwater, compared to the three other species studied, which are amphidromous and spend their juvenile life at sea [1]. The absence of Mn is, however, surprising considering that Mn enrichment of the core has been observed for many freshwater and marine species [10,44,45,47]. In the present case, the absence of the Mn peak could be a marker of a facultative amphidromous individual; or it could be species-specific. Mn enrichment at the core may be replaced by other elements, such as Ba or Mg [10]. Unfortunately, these elements are outside the scope of detection offered by the beam line we have used; we therefore could not test this hypothesis. As this remains highly speculative, it would be interesting to measure these other elements, maybe by size-exclusion chromatography-inductively coupled plasma spectrometry [10] to understand the lack of Mn in these individuals. As we only had two *M. cruentus* individuals, it is compulsory to increase the number of specimens in a future study and to also include other facultative amphidromous species [49–52]. Finally, Mn concentration at the core could simply depend on the Mn concentration of the yolk.

The role of Mn, and its high concentration, during the early life of fish remains un-
known, but a few hypotheses have been put forward. Mn is the 12th most abundant element
in the environment (Earth's crust, water, and organisms) [53] and it plays fundamental
roles in the metabolism and cell function of living organisms [54,55]. But Mn, similar to
many heavy metals, becomes toxic after a certain concentration threshold [56]. Mn has a
high affinity for lipids and toxic concentrations act on cell membrane rigidity by causing
tighter lipid packing [57]. Mn exposure causes endocrine disruptions in *Astyanax altiparanae*
(Teleostei: Characidae) females during the final ovarian maturation [58], and in a study on
pike (*Esox lucius*) and bream (*Abramis brama*), the highest concentrations of Mn were found
in the skin, gills, and gonads and in both fish species, with female gonads accumulating
higher amounts of Mn compared to male reproductive organs [59]. Oocyte growth in
oviparous species, such as fish, is characterized by maternal deposition of molecules, which
will enable the embryo to grow especially nutritive elements composing the yolk sac, such
as lipids [60]. The chemical composition and the concentration of trace elements inside
the egg seems to influence the chemical composition of the otolith core, taking over the
influence of the environment in which the eggs develop [61]. We might add that Mn²⁺ ions,
which seem to be in high concentration in the eggs compared to the surrounding water [62],
may be adsorbed by the highly negatively charged proteoglycans of the otolith core, thus
acting as an acellular magnet. Indeed, because of Mn's affinity for lipids and its capacity
to be stored in gonads, we hypothesise that the yolk is probably rich in Mn. Because of its potential harmful effect on cells, the Mn the embryo intakes from the yolk sac could be directed towards an acellular environment, i.e., trapped in the otoliths as soon as it starts forming, and that not only in *sagittae*, but also in *lapillus*, as Mn peaks have been reported in *lapillus* cores as well [63]. Newly formed otoliths would act as detoxification structures, thus protecting the embryo. Another possible explanation for the Mn peak at the core, which does not exclude the previous hypothesis of a detoxification process during yolk consumption, is that this metal may facilitate the assembly of the otolith core [64].

Brophy et al. [45] suggested that calcium carbonate would be initially deposited as calcite rather than aragonite, the main form of crystallization for teleost *sagittae* [4], since Mn has a higher affinity for calcite than for aragonite [65,66]. However, we have demonstrated, using the Raman method, that pipefish otoliths were composed of aragonite from the start.

### 4.3. *The Sr Core Story*

The Sr:Ca ratio exhibits a peak at the core, at the same location as the Ca pit. The Sr distribution over the pre-hatch zone is smooth, even over the Ca pit, and is representative of a freshwater environment [1,8,18]. The Sr:Ca peak observed is an artefact due to the Ca pit enhancing the Sr signal, but it does in fact not mark an environmental change. Indeed, the Sr:Ca ratio is widely used to trace marine environments. In the case of amphidromous species, the high Sr:Ca ratio at the core has always been interpreted as a higher concentration of Sr in the maternal environment. For instance, for freshwater diadromous species, this higher Sr:Ca ratio was interpreted as a migration of the adult reproducing individual in an estuarine location for the eggs to hatch closer to the sea [7,18]. With the new results obtained here, we can say that authors misinterpreted the Sr:Ca maps. Our results show that caution must be taken when using the Sr:Ca ratio at the core to interpret life events.

We analysed the Sr signal on its own, as it showed a smooth distribution at the core, and we compared its intensity between the pre-hatch region and the adFW region of the otolith. Only the signal is quantified, and it is comparable between samples because all samples were analysed under the same experimental conditions (time and beam). The results show that the Sr signal at the pre-hatch region is always greater than in the adFW zone, although the environmental signature for both zones should be identical since the growth of the otolith at the core and in the adult zone both occur when the fish is in freshwater. This first statement concerning the Sr profile and its repeatability validate the method. For migrating species, especially species that disperse just after hatching, like amphidromous species, the core is the only region of the otolith that can provide information on the provenance of the individual. Environment and otolith Sr usually have a 1:1 relationship [67]. The yolk is elaborated by maternal products, which are derived from the mother's environment and physiology. In the core, the Sr signature comes from both the yolk, which has a maternal heritage, and the ambient water of the incubation environment [13,61,68,69]. Unfortunately, the relative contribution of Sr from the yolk and from the surrounding water in the core is unknown [11]; it often leads to the exclusion of the core in the analysis of natal water [67,70,71], especially when studying species in which the maturation and spawning environments are distinct. Adults of amphidromous species live in freshwater [72], female pipefish lay their eggs in freshwater and the males incubate them in freshwater, so the maturation and spawning environments are the same. This explains why the Sr signal is smooth throughout the entire pre-hatch region of all our samples, as the mother and the eggs share the same environment; in addition, the Sr signal is characteristic of freshwater [1]. For the resident species, *M. cruentus*, endemic to New Caledonia, the pre-hatch region Sr intensity is only slightly higher than the adFW zone intensity. As these specimens remain their entire lives in the same river, we would expect the signals to overlap completely. However, the formation of the core and of the pre-hatch otolith is under the influence of physiological factors and depends on the bioavailability of elements present in the yolk sac, the egg medium, and the incubation environment. Furthermore, we identified a Ca pit, and Sr, as the second major component in the otolith
composition, is a good candidate to substitute to Ca. It is then possible that the Sr signature at the core may be artificially enhanced as the otolith starts to form. This could explain the slight Sr signal shift observed for *M. cruentus*. We can then consider that the Sr signal at the pre-hatch and in the adFW zones for the resident species are quite similar and that the maternal environmental signature is that of freshwater.

For *M. brachyurus*, *M. torrentius*, and *M. nicoleae*, the Sr intensity in the pre-hatch region is quite higher than in the adFW zone. In addition to the incorporation of Sr due to the specific physiology of the core formation, it seems that, unlike the resident species, eggs may have matured and/or were incubated in environments that were under the influence of higher salinity. These three species are amphidromous and spend their juvenile phase at sea [1]. Our results also show that different species, caught in very different locations, can show the same Sr profile, like, for example, *M. brachyurus* from French Polynesia and *M. brachyurus* from the Japanese Teima River. Our results on Sr core region composition do not allow linking individuals to a specific geographical location, but we uncovered similarities in life histories, or similarities in Sr integration dynamics. Among amphidromous species, there are a certain number, which exhibit a facultative amphidromous life cycle, even in open systems. There are indeed land-locked *Rhinogobius* spp., which are forced to complete their life cycle in freshwater [73]. But for species living in free-flowing streams, researchers have not proposed a plausible explanation for the existence of resident individuals. The Sr signal analysis of the pre-hatch region shows that the two *M. cruentus* residents were only under the influence of a freshwater signature at otolith formation; this means that the mothers matured their eggs in freshwater and that the eggs were incubated in freshwater, with no influence of salinity during embryogenesis. *M. brachyurus*, *M. torrentius*, and *M. nicoleae*, all migrating amphidromous species, show a higher pre-hatch Sr signal, showing that the mother matured its eggs and/or that the eggs were incubated in a freshwater environment, but under the influence of saline water, like estuaries. Although the juveniles of amphidromous species hatch in freshwater, the saline influence during egg formation and embryogenesis may be the physiological signal for downstream migration to the sea. In the absence of this signal, juveniles remain in freshwater and are therefore residents. Indeed, many physiological changes are triggered by changes in water salinity [74,75]. Also, the freshwater current flowing into the sea triggers amphidromous fish recruitment into rivers, and their upstream migration [76]. It has been noted before that maternal conditions early in life can have large effects on the adoption of particular strategies by individual fish [77,78]. Our results tend to confirm that parental behaviour seems to play a role in determining whether the juvenile migrates to sea or not. There are, however, also abiotic factors, such as stream and ocean hydrodynamics, that may mediate partial migration strategies of amphidromous species [52]. Furthermore, life-history characteristics, such as propensity to migrate, are heritable [79–82] and we can reflect upon the possible increasing proportion of freshwater residents among amphidromous species and its consequences on species maintenance in the distinctive tropical river ecosystems, knowing amphidromy is an adaptation these fragmented habitats [83].

### 4.4. *The Limits of Heavy Metals as Accurate Environmental Signatures*

We chose to analyse Cr, Co, and Ni (and Mn), as these metals are potential environmental signatures, being important constitutive elements of the Earth's crust [84]. They were also chosen as they are available in the detection spectrum of the Synchrotron beamline. Cr signals were quite similar for all individuals analysed, with generally little difference between the pre-hatch region and the adFW region. There were, however, four individuals which exhibited much higher Cr signals, both in the pre-hatch zone and especially in the adFW zone; they were sampled in the two New Caledonian rivers located in the North Province, the Pwé Hiit River, and the Garana River. There are many mining activities in New Caledonia, both past and present, which exploit nickel but also iron, gold, silver, or chromium. Mines are located throughout the country and Cr, which was exploited as chromite, was particularly extracted in sediments in the north, showing that northern
sediments are Cr rich. Toxic Cr is also released from nickel mining sediments in surface waters and Cr is released from sediments solely as toxic Cr(VI) [85]. Studies on heavy metals show that Cr is incorporated in the otolith proportionally to its concentration in the environment, unlike other heavy metals, such as Ni, Co, or vanadium [86]. Our results suggest that fish sampled in the Pwé Hiit and Garana rivers may have been exposed to high Cr concentrations, which were then incorporated into the otolith. The analysis of the *sagitta* of the *M. brachyurus* larva caught in the Pwé Hiit River also exhibits a strong Cr signal (Figure 3A), which is consistent with the analysis of the other individuals caught in the same river. Other rivers sampled in New Caledonia, i.e., Ouenghi River and Creek Baie du Nord, do not seem, at the time fish were caught, to have toxic Cr concentrations as the signals are the same as for French Polynesia, Japan, Solomon Islands, or Papua New Guinea.

Ni and Co concentration in the otolith is primarily due to their physiological roles as biomolecule co-factors, rather than because of the ambient environment [64,86]. This assumption is supported by our results. Indeed, Ni and Co signals are quite similar in each given otolith. This is not surprising, as Ni and Co are found together in natural environments. For instance, Co is recovered from sediments during Ni refining [87]. We assume that Co and Ni integration in the otolith is linked to physiology rather than to ambient concentration in the environment. Indeed, were it the case, fish caught in the same rivers would at least have similar signals in the adFW zone, which is not what we observed; the two *M. cruentus*, both residents and fished in the Ouenghi River, have very different adult zone Co and Ni signals. The same observation can be made for both specimens fished in the Garana River. Co and Ni incorporation is then due to individual growth dynamics. Furthermore, Co and Ni may not be as bioavailable as other heavy metals, such as Cr. Indeed, Co and Ni can be situated in the centre of metalloproteins and other complex biologically active molecules in organic material [88,89], preventing them from reaching the otolith. The accumulation of such metals into the otolith may also be prevented by the competition for these metals by biologically active metalloproteins in the endolymph [64]. We therefore caution against the employment of Co and Ni as environmental markers, although they were good candidates, especially in New Caledonia, which is particularly rich in Ni. However, Cr seems to be more adequate, especially to trace water pollution events, as toxic Cr is incorporated in otoliths. Further studies should be led to validate this heavy metal as an environmental marker.

### 4.5. Pre-Hatch Otolith Growth Dynamics

The larval otolith used in the present study to standardize the pre-hatch zone extent in the samples (Figure 1B) was deduced to exhibit almost a spherical shape (29 to 32 µm in diameter) by means of reflectometry and X-ray optical properties. In the adult specimens studied, growth increments were deduced from the S pattern in the pre-hatch zone. Early growth of the otolith during embryogenesis is centred on the core and quite regularly arranged centrifugally. Changes in growth directions occur later during development, leading to the usually elongated shape of the *sagitta* [90]. The number of increments found by counting the suite of S signal intensity peaks is consistent with the quite few related previous studies. Vestibular structures and sensory epithelia appear in the late embryonic stage [91,92]. The *sagitta* of the rainbow trout *Oncorhynchus mykiss* appears 12 days after fertilization [93], although the saccular otolith can appear earlier for other smaller fish species, such as the killifish (3 days after fertilization) [94], or the ayu (4 days after fertilization) [93]. Salem & Omura [93] reported the same number of incremental layers as us in the present study, counting seven increments in the rainbow trout *sagitta* that form during embryogenesis before hatching. We do not know for sure when vestibular structures appear in freshwater pipefish species, but our results indicate that *sagittae* would appear roughly one week before hatching. There are no studies describing the embryonic development of freshwater pipefish, but there are a few on marine Syngnathidae, reporting, e.g., *Syngnathus abaster*, larvae start hatching about two weeks after fertilization [95]. For
all Syngnathidae, the hatched larva stays in the male brood pouch until larval development is finished and the yolk sac completely consumed [96]. It is reasonable to assume that the embryonic development of pipefish before hatching lasts between 10 and 20 days and that the *sagitta* appears 7 to 8 days before hatching. To relate back to the Mn peak, and considering the time needed to integrate elements in the otolith [4], the period of a few days before the formation of the *sagitta* primordium would be sufficient to concentrate Mn in the vestibular zone ready to be integrated in the otolith.

**5. Conclusions**

The present work attempted to untangle the information contained in otoliths formed
during embryogenesis of four freshwater pipefish species. For a long time, researchers
have tried to retrieve information from the analysis of otolith cores, hoping it was an open
book about the individual's early-life history, a transgenerational story passed down from
mother to progeny. Indeed, the core region of otoliths is under the influence of maternally
inherited elements, but early-life processes may alter the maternal signal. In our study,
hyperfine elemental analysis led to uncovering several otolith formation processes common
to the four species studied, for instance: (i) the presence of a Ca pit characterizing the core;
(ii) Mn incorporation at the core region appearing as an unevenly distributed patch, which
probably is linked to Mn concentration in the yolk sack rather than to an environmental
signature; (iii) our results also bring new insights into the timing of otolith formation as, in
all species studied, they form one week before hatching, a process which may be common
to other fish species whatever the duration of embryogenesis. Mapping of heavy metals,
Cr, Co, and Ni, showed that Cr would be an appropriate environmental marker, revealing
conditions in which the mother elaborated eggs and/or in which they were incubated. Co
and Ni, however, seem to be markers of individual physiology and otolith growth dynamics
and should not be used to retrieve early-life information. Finally, a precise comparison
of the Sr signal found in the pre-hatch region of the otolith and in the edge of the otolith,
a reflection the adult freshwater habitat, showed that parent behaviour may have great
consequences on their newly hatched progeny's trigger to migrate to the sea or not. The
core story can be seen as a tangled bundle of threads, and cautious reading is the key to
untie the knots and unravel the origin of each piece of information.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/min14111176/s1, Figure S1: 3D reconstruction of z-values of calcium (Ca), of the pre-hatch zone. The surface plots show uneven Ca z-values at centre of the pre-hatch zone, i.e., the core position. Sample labels include country, river, XRF scan number and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*). Figure S2: Sulphur (S) intensity plot showing the S peaks along the study transect (see Figure 5). The core position is at the intersect of y- and z- axis. Sample labels include island, river, XRF scan number and species identifier (bra: *M. brachyurus*, nic: *M. nicoleae*, tor: *M. torrentius*, cru: *M. cruentus*) (see Table 1).

**Author Contributions:** Conceptualization, C.A.L., S.B. and P.K.; Methodology, S.B. and C.A.L.; Investigation, V.H. and S.B.; writing—original draft preparation, C.A.L. and S.B.; writing—review and editing, C.A.L., S.B. and P.K.; supervision, P.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data available at https://doi.org/10.48579/PRO/IXCHAF (accessed on 30 September 2024).

**Acknowledgments:** The authors gratefully acknowledge O. Belhadj, head of vibrational spectroscopy equipment set (Raman, FTIR) for her scientific advice and her technical assistance, as well as CRC MNHN laboratory for Raman beamline time. The Synchrotron SOLEIL, and especially the "NANOSCOPIUM" beamline crew K. Medjoubi and A. Somogyi are also greatly acknowledged for the provision of synchrotron radiation facilities, for assistance in using beamline and monitoring the Proposals (20191306 and 20210141). Special thanks for their continuous help, outstanding tech-
nical support and constructive suggestions. For the samples from French Polynesia, we would like to thank V. Mazel (Ichtyo-Pacific). For those from PNG, thanks to G. Kaipu (PNG NRI) for the research permit (N°018196), N. Gowep (CEPA) for the export permit and our friends, P. Amick, B. Ruli (Live & Learn), J. Anamiato (National Museum and art Gallery of PNG) and D. Vaghelo (WNB Provincial Govt, Environment section). We want to thank all the responsible Chiefs of the areas investigated for their kind permission, and the village communities who have always heartily helped in research field prospecting of rivers. For the Solomon Islands, we would like to thank D. Boseto (ESSI) for his invaluable help on the field, the landowners and tribes of the Solomon Islands for allowing the expedition team to enter their customary lands, and the Government for the support and facilitation of the legal process. For New Caledonia, we would like to thank the New Caledonian Government and the New Caledonian North and South Provinces, the DAFE (C. Fort), J.J. Cassan, and N. Charpin from the NGO "Vies d'Ô Douce" for his help on the field. We also would like to thank K. Maeda and H. Kobayashi for specimens from Japan.

**Conflicts of Interest:** The authors declare no conflicts of interest.

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