A Fétida

O Bengone

M Romeo

F Scheurer

R Robles

N Lorente

L Limot
email: limot@ipcms.unistra.fr

Single-spin quantum sensing: A molecule-on-tip approach

Quantum sensing is a key component of quantum technology, enabling highly sensitive magnetometry. We combined a nickelocene molecule with scanning tunneling microscopy to perform versatile spin sensing of magnetic surfaces, namely of model Co islands on Cu(111) of different thickness. We demonstrate that atomic-scale sensitivity to spin polarization and orientation is possible due to direct exchange coupling between the Nc-tip and the Co surfaces. We find that magnetic exchange maps lead to unique signatures, which are well described by computed spin density maps. These advancements improve our ability to probe magnetic properties at the atomic level.

Quantum sensors can achieve sensitive magnetometry through changes in their spin states or quantum coherence when they interact with a magnetic target. Examples include SQUID magnetometers employing nitrogen vacancy centers in diamond [1] or carbon nanotubes [2]. The inclusion of magnetic field gradients into magnetometers through scanning probe techniques has allowed for the detection of single spins with a remarkable resolution of up to 10 nm [3][4][5]. Nevertheless, a paradigm shift is needed to achieve the ultimate aim of atomic-scale resolution in magnetometry.

A possible approach involves using a single magnetic molecule as a spin sensor. Molecules offer the advantage of a tunable electronic structure, making them wellsuited for fulfilling the design requirements of a quantum sensor [6]. Notably, recent advancements in scanning tunneling microscopy (STM) techniques have enabled the detection of a molecular spin on a surface through electron spin resonance (ESR) [7,8]. This breakthrough enables the detection of exchange and dipolar fields (> 10 mT) in the immediate vicinity of the molecular environment [9,10]. To freely access all surface locations, a more advantageous strategy involves attaching the molecule to the STM tip apex. To date, this has been successfully demonstrated solely with the spin-S = 1 nickelocene molecule [Ni(C 5 H 5 ) 2 , see Fig. 2] [11], albeit at the cost of limiting sensitivity to exchange fields > 1 T [12,13].

In contrast to ESR, the spin states of a nickelocene tip (referred to as the Nc-tip) are in fact monitored through spin excitations arising from the inelastic component of the tunneling current [14]. Sub-angstrom precision in sample spin-sensing is made possible by the exchange interaction occurring across vacuum between the Nc-tip and the surface, which modifies the Nc spin states [12? , 13]. In this study, we examined prototypical ferromagnetic Co islands that were grown on a pristine Cu(111) surface, including monolayer-thick islands that remained unexplored. Our findings, supported by ab initio calcula-tions, reveal that the thickness of the ferromagnetic layer exerts control over the orientation of sample magnetization and spin polarization. Furthermore, we demonstrate that the spatial dependence of the exchange energy across the surface is well captured by computed spin-density maps. Our study demonstrates the effectiveness of a Nctip in probing surface magnetism, even in the absence of an external magnetic field.

Cobalt deposition results in the formation of triangular-like nanoislands (for details, see supplementary material) [19], which are two atomic layers in height [16,20,21]. The islands, which are denoted by 2 in Fig. 1a), display an apparent height of approximately 325 ± 10 pm and exhibit typical sizes spanning from 10 to 30 nm. Approximately 5% of the surface area is occupied by islands that are three layers high (denoted by 3 in Fig. 1a), characterized by an apparent height of 540 ± 10 pm. To promote the growth of one-layer high islands (referred to as 1 in Fig. 1b), we introduce strain relaxations in the Cu substrate by either depositing Co at sub-room temperatures or by lowering the annealing temperature of the Cu substrate [22]. The monolayer islands exhibit variable sizes within the range of 7 -20 nm and have an apparent height of approximately 140 ± 10 pm. Their morphology is triangular with truncated corners, which signifies that the diffusion barriers involved in their growth process differ from those of bilayer islands [22].

The islands exhibit similar electronic properties. In Fig. 1c, we present differential conductance (dI/dV ) spectra acquired by placing a metal tip at the center of the islands. They were recorded using a lock-in amplifier operating at a frequency of 6.2 kHz and a modulation of 5 mV rms. Across all the islands, a prominent feature is observed in their dI/dV spectra, situated at energies of -0.39 eV (1), -0.33 eV (2), and -0.36 eV (3). Based on our ab initio calculations (Fig. S1) [19], it is assigned to minority d z 2 states, in agreement with previous findings [20,21]. Their detection in vacuum, however, is possible due to their hybridization with s -p states. Furthermore, similar to the bi-and trilayer islands [17,18,20], the monolayer islands host a freeelectron-like surface state. Their confinement within the The feedback loop was opened at 0.5 V and 1 nA. The 2 ML spectrum was acquired on an "unfaulted" island [16]. Inset: Sketch of the tunnel junction. Constant-height dI/dV maps of a Co monolayer acquired at (d) -100 mV and (e) +300 mV, respectively. The feedback loop was opened in the center of Co at a distance of approximately 500 pm from tip-surface contact. The dispersive states do not have sufficient weight compared to the d states to be directly detected by tunneling spectroscopy [17,18].

islands manifests as a standing wave pattern (Fig. 1d-e), exhibiting continuous variations in wavelength across a broad bias range (see Fig. S2 for more wave patterns).

The dispersion in the monolayer islands has parabolic behavior with an onset energy of -0.28 ± 0.01 eV [19] and an effective mass of m * = (0.44 ± 0.02) m (m: free electron mass), consistent with that of the bi-and trilayer islands.

The magnetic properties differ for the islands. Their magnetism is explored by attaching a Nc molecule to the tip apex (see Fig. S3) [19], and by recording the inelastic signal through the second derivative, d 2 I/dV 2 , of the current I versus bias V (with a lock-in modulation of 500 µV rms). Figure 2a displays a series of distancedependent d 2 I/dV 2 spectra obtained above a monolayer island with the tip positioned directly above a Co atom at the island's center. Figure 2b presents the corresponding 2D intensity plot. To calibrate the tip-surface distance, we utilized current versus displacement traces above a Co atom (see Fig. S3), setting z = 0 at the tip-Co contact point. At a distance of z = 100 pm, the spectrum exhibits a peak and a dip at biases of +3.9 and -3.9 mV, respectively. These peaks and dips indicate inelastic scattering events, wherein tunneling electrons transfer momentum and energy to the spin states of Nc [11]. As the tip approaches, the peak shifts upward, and the dip shifts downward in energy by nearly 5 mV. At z ≤ 60 pm, a second peak (dip) emerges, initially as a high-energy (low-energy) shoulder to the peak (dip), then becoming fully resolved at z = 40 pm due to its more pronounced energy shift. Intriguingly, in the bilayer and a majority of trilayer islands (> 80%), we observe instead a progressive splitting of the peak and dip in the d 2 I/dV 2 spectrum as the tip approached a Co atom vertically (Fig. 2c and2d; refer to Fig. S6 for the bilayer case). However, a minority of trilayer islands (< 20%) exhibit a behavior similar to monolayer islands.

To rationalize these findings, we assign the z-axis as the out-of-plane direction of the Co surface, for now assuming the molecular axis of Nc along the z-axis [11,12]. We consider the spin Hamiltonian described by Eq. 1:

Ĥ = DS 2 z + gµ B Bex Ŝ, (1)
where Bex = E ex /gµ B represents the exchange field produced by the Co atom on the spin Ŝ of Nc [19], µ B denotes the Bohr magneton, and g = 1.89 [12]. The monolayer spectra are reproduced using a dynamical scattering model with an in-plane exchange field (solid red lines in Fig. 2a), Bex = B ex x in Eq. 1 [23]. In this configuration, the ground state |Ψ 0 ⟩ and the two excited states |Ψ 1 ⟩ and |Ψ 2 ⟩ consist of a mixing of the |M |-spin states (Fig. S4), where M denotes the magnetic quantum number along the z-axis. We find a magnetic anisotropy of D = 3.9 ± 0.1 meV, which is usual for Nc-tips [24], and, consistent with expectations, the same exchange energy for both spin excitations (Fig. S5).

Conversely, the bilayer and trilayer spectra indicate instead that the exchange field has out-of-plane orientation ( Bex = B ex ẑ in Eq. 1) [13]. In this configuration, the two excited states, |Ψ 1 ⟩ = |M = +1⟩ and |Ψ 2 ⟩ = |M = -1⟩, become Zeeman split (see Fig. S4). The simulated line shapes (solid red lines in Fig. 2c), accurately match the experimental data, but unlike the monolayer islands, a finite lifetime of τ ≃ 0.1 ns is accounted for the excited states (Fig. S7) [19,25]. The lifetime is nearly 1000 times longer than that of a magnetic atom on a metal surface [26].

To validate these observations, we conducted Density Functional Theory (DFT) calculations to determine the Magnetic Anisotropy Energy per Co atom (MAE) within the islands (refer to [19] for calculation details and an extended presentation of the results). The calculated MAE values (Tab. I) are consistent with the experimental findings, with one exception being the trilayer case, where the MAE is close to zero. Other effects not accounted for in the simulations, such as the size and shape, therefore likely favor out-of-plane magnetization in the trilayer islands. Additionally, the presence of Cu at the corners of the trilayer islands, as evidenced by the Nc-tip measurements (see Fig. S8), could also influence the magnetic properties [27]. Based on these findings, some variability in the orientation of magnetization for the trilayer islands can be expected, depending on the specifics of the sample preparation.

The simulated line shapes provide quantitative insights into the exchange energy at specific Nc-Co distances. Above all island types, the exchange energy has an exponential variation exp (-z/λ) with a decay length of λ = 46 ± 3 pm (Fig. 3a). Notably, the exchange energy above the monolayer island is nearly three times stronger when compared to the bi-and trilayer islands, which exhibit identical exchange energies. The exchange coupling also changed across the surface. To visualize these variations, we acquire a three-dimensional dataset in the form of d 2 I/dV 2 (x, y, V ) at a fixed distance of z = 20 pm above a region of interest. We then determine the exchange energy at each lateral tip position by fitting the spectra. The resulting image (Fig. 3b-c) is comparable to the magnetic interaction map obtained for a single atom using STM-ESR [28]. However, in contrast to STM-ESR, our method allows us to span over multiple surface atoms, as illustrated for monolayer and bilayer islands in Fig. 3b and 3c, respectively. These images reveal a magnetic corrugation with a periodicity of 254 ± 6 pm, corresponding to the in-plane Co-Co distance determined with a metal tip. The corrugation is approximately two times weaker in the monolayer island (Fig. 3d) compared to the bi-and trilayer islands (Fig. 3e). This observation is in agreement with the computed spin density maps (Fig. 4a and4b). As in the experimental exchange energy maps, the corrugation of the spin density is stronger for the bi-and the trilayer islands compared to the monolayer island. The difference can also be observed in the height profiles (Fig. 4c), and in the sections of the spin density in a ( 11 (dashed line), the isolines show a stronger corrugation for bi-and trilayers (Fig. 4e-f) than for the monolayer (Fig. 4d). The origin of this behavior is tracked down to the d z 2 orbital. While for the monolayer the orbital has a spin magnetization of 0.28 µ B , for the bilayer it has a higher spin magnetization of 0.42 µ B (0.43 µ B for the trilayer). At the same time, the monolayer has a higher average absolute value for the spin density (Fig. 4c) in agreement with the stronger exchange energy observed experimentally.

The cobalt islands can also show spin polarization as their magnetization induces a spin imbalance in the tunneling current. This leads to discernible variations in the relative heights of the peaks and dips within the d 2 I/dV 2 spectrum [13]. For instance, in the case of a trilayer island, the low-energy excitation exhibits a higher dip amplitude at negative bias compared to the peak at positive bias (Fig. 2c; see also Fig. S7). To quantitatively assess this imbalance, we derive values from our line shape fits in the form of a spin polarization P = (h + -h -)/(h + +h -), where h + and h -represent the heights of the peak and dip, respectively. The trilayer islands exhibit a spin polarization of +0.17 at the Fermi energy (Fig. 5a), while the bilayer islands remain non-polarized within the investigated z-range.

The spin polarization of the monolayer island shows instead some z-dependency (Fig. 5a), changing from P = +0.2 at z = 100 pm to P = +0.05 at z = 20 pm. This variation is attributed to the rotation of the molecular axis relative to the z-axis, as predicted by DFT calculations. In vacuum, the tilt angle is close to 10 • , but approaches 0 • when Nc comes into contact with the surface [12,24]. To gain a deeper understanding of this behavior, we introduce the tip polarization (η t ) and the sample polarization (η s ), expressing the spin polarization of the tunnel junction as P = η t η s . While η s reflects the net polarization in the sample's DOS, η t quantifies the spin momentum transfer during the spin excitation [29]. To compute η t , we introduce an angle θ between B ex and the molecular axis and determine the eigenvectors |Ψ 0 ⟩, |Ψ 1 ⟩, and |Ψ 2 ⟩ through Eq. 1. The variation of η t with respect to θ is presented in Fig. 5b. For small θ, the molecular axis of the Nc-tip aligns nearly parallel to the out-of-plane surface magnetization, and the measured spin polarization closely mirrors the sample polarization (P ≈ η s ). This corresponds to the behavior encountered in the bi-and trilayer islands. Conversely, as θ approaches 90 • , η t exhibits a pronounced angular dependence. This behavior is characteristic of the monolayer island, where the molecular axis of the Nc-tip is almost perpendicular to the in-plane surface magnetization. For a perfect perpendicular alignment, the tip polarization becomes η t = 0, resulting in a non-polarized tunnel junction (P = 0). However, even a relatively small tilt angle of 10 • , corresponding to θ = 80 • , is sufficient to elevate the tip polarization to +0.6. Assuming this tilt angle at z = 100 pm, we can estimate a sample in-plane polarization of η s = +0.33 for the monolayer.

In summary, our investigation demonstrates the high sensitivity of an Nc-tip to surface magnetism and spin transport. A three-dimensional dataset of spin-excitation spectra allows for the construction of spatial maps of the exchange interaction, which resemble computed spin density maps. The maps offer atomic-level information on the spin orientation of the sample, making them valuable for magnetic imaging, particularly for intricate spin textures. Spatial maps of the surface spin polarization can be constructed by using the amplitude of the inelastic peaks-a facet we intend to explore in forthcoming work. To expand the possibilities of Nc-based magnetometry, future work will involve combining conductance and force measurements in a Nc-functionalized STM-AFM setup.

FIG. 1 .

 1 FIG. 1. (a) Constant-current image of Co islands grown on (a) Cu(111), (b) "strained" Cu(111) (image size: 40×40 nm 2 , sample bias: 80 mV, tunnel current: 60 pA). Numbers indicate ML thickness. (c) Typical dI/dV spectra acquired in the center of 1, 2, and 3 ML cobalt islands. The 2-and 1-ML spectra are displaced upward by 10 and 20 nS, respectively.The feedback loop was opened at 0.5 V and 1 nA. The 2 ML spectrum was acquired on an "unfaulted" island[16]. Inset: Sketch of the tunnel junction. Constant-height dI/dV maps of a Co monolayer acquired at (d) -100 mV and (e) +300 mV, respectively. The feedback loop was opened in the center of Co at a distance of approximately 500 pm from tip-surface contact. The dispersive states do not have sufficient weight compared to the d states to be directly detected by tunneling spectroscopy[17,18].

FIG. 2 .

 2 FIG. 2. d 2 I/dV 2 spectra above a Co atom of a (a) monolayer and (c) trilayer island acquired at different tip-Co distances. The spectra are displaced vertically by 0.5 ms/V from one another. The vertical lines in panel (a) indicate the position of excitation |Ψ0⟩ → |Ψ2⟩. Red lines: Simulations based on the dynamical scattering model. 2D intensity plot of a series of distance-dependent d 2 I/dV 2 spectra above a Co atom of a (b) monolayer and (d) trilayer island. Inset: Sketch of the tunnel junction. Nickelocene comprises a single Ni atom sandwiched between two cyclopentadienyl rings (C5H5).

FIG. 3 .

 3 FIG. 3. (a) Exchange energy as a function of distance z extracted from the d 2 I/dV 2 spectra. The dashed lines is the exponential fit described in the text. The exchange field is given on the right y-axis. (b,c) Spatial maps of the exchange energy acquired at a fixed distance of z = 20 pm above a monolayer and a bilayer island, respectively. Lateral (vertical) tip drift between each spectrum acquisition was maintained < 10 pm (< 5 pm) by dynamically tracking a local maximum. (d,e) Height profiles of the layers acquired along the dashed lines indicated in (b) and (c). The dashed line in (d) is the height profile of a trilayer.

FIG. 4 .

 4 FIG. 4. (a)-(b) Computed spin density maps at a fixed distance of 300 pm above a bilayer and a monolayer, respectively. (c) Height profiles of the layers along the lines indicated in (a) and (b). The dashed line is the height profile of a trilayer. (d)-(f) Section of the spin density in a (11 2) plane passing through the surface Co atom for monolayer, bilayer and trilayer, respectively. Blue (red) isolines indicate positive (negative) values. The black line indicates the position of the surface Co atoms. The height profiles in panel (c) are extracted 300 pm above the Co atoms (the position is indicated by a dashed black line).

FIG. 5 .

 5 FIG. 5. (a) Spin polarization as a function of distance z extracted from the d 2 I/dV 2 spectra. The dashed lines are a guide to the eye. (b) Computed value of ηt as a function of the angle θ. The insets sketch the two extremes where θ = 0 • (left) and θ = 90 • (right). The light-yellow areas highlight a 10 • tilt.

TABLE I .

 I DFT-computed magnetic moment of the topmost Co atom and MAE per Co atom for the three islands. More details are given in [19]. Positive MAE corresponds to an inplane orientation of the magnetization.

		Mag. moment (µB) MAE per Co atom (meV)
	1 ML	1.75	+0.628
	2 ML	1.76	-0.208
	3 ML	1.84	+0.005

ACKNOWLEDGMENTS L.L. gratefully thanks M. Ternes for providing his spin Hamiltonian solver. The Strasbourg authors acknowledge support from the EU's Horizon 2020 research and innovation programme under the Marie Sk lodowska-Curie grant 847471, from the International Center for Frontier Research in Chemistry (Strasbourg), and from the High Performance Computing Center of the University of Strasbourg. Part of the computing resources were funded by the Equipex Equip@Meso project (Programme Investissements d'Avenir) and the CPER Alsacalcul/Big Data. R.B. and N.L. thank financial support from projects RTI2018-097895-B-C44, PID2021-127917NB-I00 funded by MCIN/AEI/10.13039/501100011033, from project QUAN-000021-01 funded by the Gipuzkoa Provincial Council and from project IT-1527-22 funded by the Basque Government. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.


Mathieu Legras

Giulia Ghisleni

Rabouant Soilihi

Enzo Celmar

Guillaume Balavoine
email: guillaume.balavoine@ijm.fr

Fast Cycling Culture of the Marine Annelid Platynereis dumerilii

Platynereis dumerilii, a marine annelid, is a model animal that has gained popularity in various fields such as developmental biology, biological rhythms, nervous system organization and physiology, behaviour, reproductive biology, and epigenetic regulation. The transparency of P. dumerilii tissues at all developmental stages makes it easy to perform live microscopic imaging of all cell types. In addition, the slow-evolving genome of P. dumerilii and its phylogenetic position as a representative of the vast branch of Lophotrochozoans add to its evolutionary significance. Although P. dumerilii is amenable to transgenesis and CRISPR-Cas9 knockouts, its relatively long and indefinite life cycle, as well as its semelparous reproduction have been hindrances to its adoption as a reverse genetics model. To overcome this limitation, an adapted culturing method has been developed allowing much faster life cycling, with median reproductive age at 15 weeks instead of 6-8 months using the traditional protocol. A low worm density in boxes and a strictly controlled feeding regime are important factors for the rapid growth and health of the worms. Moreover, a genetic selection for fast-reproducing individuals has been applied to isolate a "Fast Forward" strain that can be used for egg microinjection. This culture method has several advantages, such as being much more compact, not requiring air bubbling or an artificial moonlight regime for synchronized sexual maturation, and necessitating only limited water change. A full protocol for worm care and handling is provided.

Introduction

During the process of experimental design, the choice of the most appropriate model organism is a crucial step that determines the validity of the study. Gregor Mendel, the pioneering author of some of the most famous genetics experiments on plants of the genus Pisum, stated that the selection of an unsuitable model can make the results questionable from the outset [START_REF] Abbott | Experiments on plant hybrids by Gregor Mendel[END_REF].

The pea plant was not only physiologically and morphologically suitable for his "Experiments on Plant Hybrids", but its representational power has made it a model organism by definition, [START_REF] Ankeny | Model Organisms[END_REF][START_REF] Irion | Developmental genetics with model organisms[END_REF]. Knowledge derived from a model organism can be projected and generalized to a broader range of systems, and this, together with the integration of multidisciplinary approaches, allows for a 360-degree comparative study between species across the tree of life. For example, in the case of bilaterian animals, studying mice, chickens, fish, and sea urchins (which belong to the deuterostomes) together with C. elegans and D. melanogaster (from the ecdysozoans group) has enabled a comparative approach to regulatory cladistics that has revealed the shared nature of signalling pathways and transcription factors among all modern Bilaterians [START_REF] Pires-Dasilva | The evolution of signalling pathways in animal development[END_REF]. The evolution of phylum-specific body plans and morphological structures is generally not due to the rise of new developmental genes, but we can expect and predict that novel regulation circuits have developed [START_REF] Peterson | Special feature: regulatory evolution and the origin of the bilaterians[END_REF]. Investigating how developmental genes are regulated in different lineages can therefore provide new evolutionary insights into the beginnings of the history of Bilaterians.

In this context, the marine annelid Platynereis dumerilii (Özpolat et al., 2021) compensates for the longstanding lack of representatives of the lophotrochozoans, which is the third branch of bilaterians, alongside deuterostomes and ecdysozoans, that has impeded a reliable approach to bilaterian comparative genetics. Carl Hauenschild was the first scientist to begin culturing P. dumerilii in 1953 and standardize its culture conditions [START_REF] Hauenschild | Platynereis dumerilii. Mikroskopische Anatomie, Fortpflanzung, Entwicklung[END_REF]. The potential of P. dumerilii as an emerging animal model lies in the study of its molecular and developmental processes. It is considered to have retained some ancestral characteristics and, as a polychaete annelid, to have only slightly deviated from Urbilateria, the last common ancestor of all Bilaterians [START_REF] Balavoine | Segment formation in annelids: Patterns, processes and evolution[END_REF][START_REF] Tessmar-Raible | Emerging systems: Between vertebrates and arthropods, the Lophotrochozoa[END_REF]. Earthworms and leeches have been used as lophotrochozoan animal models, but they show derived features within the annelid clade. They lack many of the head structures and trunk appendages, and their life cycle lacks a larval phase and metamorphosis, which are ancestral developmental traits of annelids [START_REF] Struck | Platyzoan paraphyly based on phylogenomic data supports a noncoelomate ancestry of spiralia[END_REF]. In contrast, all these characteristics are present in the polychaete family nereididae, to which P. dumerilii belongs, making it a suitable and strategic model for providing insight into the deep morphological and developmental past of all bilaterians. P. dumerilii is found in all European seas. When managing hundreds or thousands of individuals, the fact that they are undemanding, small-sized, and easily manipulated is crucial. Regarding its life cycle, a zygote can become a sexually mature adult in a few months under culture conditions, or about 1 year in the wild. The ability to culture P. dumerilii in the laboratory for the full life cycle, its rapid and highly synchronized external embryogenesis, and the transparency of embryos and juveniles (which allows for live imaging and the use of optogenetic tools) represent a significant advantage for studying reproductive biology, development, and regeneration. according to the pelagic/benthic behavior of the worm. From the fertilized egg to the 48 hpf stage, the organism has a diameter of about 160 µm. In the 72h stage, the swimming larva is about 250 µm long, while at 6 days it reaches about 400 µm (A. H. L. [START_REF] Fischer | The normal development of Platynereis dumerilii (Nereididae, Annelida)[END_REF].

The life cycle stages, and the development have already been well described (A. [START_REF] Fischer | The polychaete Platynereis dumerilii (Annelida): A laboratory animal with spiralian cleavage, lifelong segment proliferation and a mixed benthic/pelagic life cycle[END_REF][START_REF] Fischer | The normal development of Platynereis dumerilii (Nereididae, Annelida)[END_REF][START_REF] Hauenschild | Platynereis dumerilii. Mikroskopische Anatomie, Fortpflanzung, Entwicklung[END_REF][START_REF] Hempelmann | Zur Naturgeschichte von Nereis dumerilii Aud. et Edw[END_REF]. The following description is just a summary, with stages given at a culturing temperature of 18°C. A single mating of P. dumerilii can result in thousands of eggs (160 µm diameter). Once fertilized, the eggs develop into larval stages in a synchronized manner. The zygote contains lipid droplets, cortical granules, protein yolk granules, and secrete a protective jelly to avoid polyspermy (A. [START_REF] Fischer | The polychaete Platynereis dumerilii (Annelida): A laboratory animal with spiralian cleavage, lifelong segment proliferation and a mixed benthic/pelagic life cycle[END_REF] and protect the embryo from predation. Copious jelly secretion indicates successful fertilization. During the early stages of development, P. dumerilii embryos exhibit spiral cleavage. The first divisions are highly asymmetric, and cells can be identified based on their position and size. During this spiral phase, the lipid droplets fuse to form four large lipid droplets, which indicate healthy development. Larvae with more or less than four lipid droplets will not develop normally.

By 24 hours post-fertilization (hpf), the trochophore larvae are free from their jelly, start swimming with the help of a ciliary belt, and exhibit positive phototaxis. At this stage, larvae are planktonic and do not feed. The only food source is the yolk contained in their macromeres. By 4 days, the nectochaete larvae have produced a head, a pygidium (posterior end), tentacular cirri, anal cirri, and three chaetigerous segments with parapodia. The larvae then move from the pelagic zone to the benthic zone and create a mucus tube to spend most of their life in it. At 6 days, they begin feeding (mostly at night when they leave the tube) and add new segments to the posterior end from the segment addition zone (SAZ), located just anterior to the pygidium [START_REF] Balavoine | Segment formation in annelids: Patterns, processes and evolution[END_REF][START_REF] Fischer | The normal development of Platynereis dumerilii (Nereididae, Annelida)[END_REF]. From now on, the developmental pace of juveniles will vary between individuals. The size of the worm depends on its food regime and quantity of food intake. Before sexual maturation, gonial clusters begin to populate the body of the worms when they reach around 40 segments [START_REF] Kuehn | Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii[END_REF]. Gametes start to populate the coelom at approximately 50 segments in length but become mature at ~70-80 segments in size [START_REF] Fischer | Stages and stage distribution in early oogenesis in the Annelid, Platynereis dumerilii[END_REF][START_REF] Fischer | The structure of symplasmic early oocytes and their enveloping sheath cells in the polychaete, Platynereis dumerilii[END_REF]. During sexual metamorphosis, the worms stop feeding and their gut degenerates. Their eyes increase in size and extensive changes occur in their muscles, parapodia, and body color, as they transition from a sexually immature atoke form to a sexually mature epitoke form. Sexually mature worms need to swim very fast, so their muscles degenerate to make room for a new epitokous muscle type. Oocytes are yellow and give this color to mature females. Males are bicolor due to the white sperm that colors the anterior part of the worm, while the posterior part is red due to blood capillaries filled with hemoglobin [START_REF] Andreatta | Corazonin signaling integrates energy homeostasis and lunar phase to regulate aspects of growth and sexual maturation in Platynereis[END_REF][START_REF] Fischer | The normal development of Platynereis dumerilii (Nereididae, Annelida)[END_REF]. Once epitoky is complete, spawning of P. dumerilii peaks around 1 week after the full moon phase [START_REF] Zantke | Circadian and Circalunar Clock Interactions in a Marine Annelid[END_REF]. The worms leave their tubes and move into pelagic water to look for a partner. Aroused by pheromones [START_REF] Zeeck | Sex pheromones in a marine polychaete: Biologically active compounds from female Platynereis dumerilii[END_REF][START_REF] Zeeck | Uric Acid: The Sperm-Release Pheromone of the Marine Polychaete Platynereis dumerilii[END_REF], the mating couple starts a nuptial dance (circular fast swimming) and the female releases all of her oocytes into the water. The male fertilizes them externally and the worms die shortly after the release of gametes. All of these developmental stages can be influenced by changes in temperature (A. [START_REF] Fischer | The polychaete Platynereis dumerilii (Annelida): A laboratory animal with spiralian cleavage, lifelong segment proliferation and a mixed benthic/pelagic life cycle[END_REF].

Several studies have demonstrated the suitability of P. dumerilii for reverse genetics experiments, including transgenesis using DNA transposase (Backfisch et al., 2013(Backfisch et al., , 2014) ) and targeted gene knockouts with TALENs or Cas9 (Bannister et al., 2014;Bezares-Calderón et al., 2018). However, despite its potential as a genetic model organism, there are still challenges to making P. dumerilii a widely used "fruit fly of the sea." One of these challenges is the extensive genetic polymorphism present in laboratory strains, even after decades of maintenance in various European and American laboratories. Non-inbred strains still exhibit a high proportion of SNPs (Single Nucleotide Polymorphism). This is particularly relevant to genome editing by CRISPR-Cas9, as a PCR polymorphism study must be conducted for each targeted gene. Furthermore, reproduction in P. dumerilii occurs only once at the end of the life cycle, and adults die immediately after spawning. This makes it difficult to maintain homozygous strains, as adults of both sexes must be obtained on the same day, requiring large batches of transgenic animals to be produced and maintained until they reach sexual maturity.

The main challenge remains the relatively long reproductive cycle of P. dumerilii. In nature, the reproduction period is seasonal, implying that most worms live for about a year on average. In the laboratory, temperature (generally 18°C) and light regimes (8/16 hrs of night/day) are used to simulate the end of springtime. To replicate the role of moonlight in synchronizing the swarming of male and female adults, an artificial moon, usually in the form of a small bulb, is placed in the culture room for one week every four weeks. These conditions, combined with regular feeding, sometimes induce sexual maturation at a much younger age. However, the age of reproduction remains highly variable and is not correlated between worms from the same parental batch. A typical culture containing around 30 juvenile worms will usually produce adults whose ages vary between 4 and 12 months, making it difficult to create and maintain transgenic strains.

We hypothesize that three factors contribute to the variability in sexual maturation age. Firstly, the highly variable density of worms in culture boxes may be a factor. P. dumerilii juveniles are typically kept in flat plastic alimentary boxes with a bottom surface area of around 500 cm 2 . Young worms spin silk tubes at the bottom of the box in which they spend most of the day. Instead of being randomly distributed, the tubes are usually built in positions that maximize distances between worms, suggesting territorial behaviour. This behaviour is likely induced by antagonistic interactions between juvenile worms, which can be observed frequently as wandering worms are attacked and bitten by their neighbours. Secondly, the quantity of food delivered is not adapted to the number of worms in the boxes. Too little food results in slow growth, while too much food can lead to water fouling with the same effect or even worm death by asphyxia. In classical culture methods, water must be changed every two weeks to tackle fouling. Finally, the extensive genetic polymorphism observed in our historical culture population (which we call the polymorphic strain) and verified in numerous PCR studies on various genes has led us to hypothesize that genetic factors may also be involved in the variability of maturation age.

In this article, we describe how we have established a new protocol for culturing a selected strain of P. dumerilii called Fast Forward (FF) by controlling density, adapting food delivery, and selecting worms based on their maturation age. Sexual maturation for this strain begins at 12 weeks, and most worms mature before 18 weeks. This protocol places P. dumerilii among the small minority of animal models in which transgenic and genome editing techniques can be developed with ease in the future.

Most importantly, we have significantly simplified the culture conditions by eliminating the need for a moon cycle and reducing the frequency of water changes, thereby reducing staffing requirements and enabling the rearing of several transgenic strains in limited lab space.

Materials and Methods

Making new batches of larvae

Swimming adults are collected from low density boxes every morning. Male worms display a bicoloured white/red pattern, whereas female worms are yellow/orange. Only adults actively swimming are chosen daily for reproduction. To make all fertilizations and keep the embryos/larvae up to ten days after fertilization, small beakers (diameter 9.5 cm, height 5.5 cm) are used.

Approximately 150 ml of NFSW is added in each beaker, along with one pair of worms. All worms are manipulated using plastic pipettes (Samco scientific). Ideally, the release of gametes occurs in the next few minutes. Sometimes, this release does not happen spontaneously even though both individuals are active. In such cases, spawning can be achieved by gently pressing the male with a fine paintbrush to force the release of a small amount of sperm. This, in turn, triggers the release of the female oocytes. Once both adults have released their gametes, most of the NFSW used for fertilization must be poured out to prevent overnight fouling by the male sperm. This is done easily since the fertilized eggs sediment quickly at the bottom of the beaker. A new volume of 150 ml of clean NFSW is then added. The efficiency of the fertilization is checked by the formation of a transparent jelly coat around the eggs. Thirty minutes post fertilization, the eggs adopt a characteristic hexagonal spatial distribution while being pushed apart by the jelly. To prevent fouling of the water due to the decomposition of the jelly coat, 1.5 ml of 100x penicillin:streptomycin mix is added. Fertilized batches are incubated overnight at 20°C. The next day, at 24 hours post-fertilization (24 hpf), the larvae must be dejellified (getting rid of the jelly coat). The content of the beaker is poured into a large 80 m sieve (see lab-made equipment section), allowing the jelly to sieve through with the seawater. After two quick rinses with NFSW, the sieve is returned upside down to the original beaker and 150 ml NFSW is poured over the whole sieve to recover all swimming larvae. Small worms are incubated at 20°C until they reach 4 dpf. They are then fed with 3 ml of 1x frozen microalgae (Tetraselmis marina, Instant Algae™, Table 1). Three-segment small worms should start to feed and settle at the bottom of the beaker quickly.

Transplanting new boxes of juvenile worms

To achieve maximum survival of juvenile worms, it is important to use healthy batches of young worms. At 10 dpf, the beaker containing young worms should consist primarily of feeding individuals, which can be checked by a gut full of algae and the budding of a fourth segment in some. For further culturing, we will use these healthy and rapidly growing 4-segment young worms and avoid 3segment worms and those with an empty gut. The boxes used for the main phase of growth are polypropylene containers, that are 6.5 cm high, and can be easily stacked in incubators or on the bench. These containers have lids that are not completely airtight, ensuring vital gas exchange for the worms. Typically, four boxes are transplanted from each batch of young worms. Each box is filled with 500 ml of NFSW and supplemented with 3ml of 1x frozen algae and one cm 2 of old box algal mat (see commensals section). Twenty young worms are transplanted into each box, achieving the optimal density of 300 individuals.m -2 . Young worms can be selected using a P20 hand pipette with bevelled pipette tips. Worms reflexively grip the inside of the tip with their bristled appendages, so it is important to set the hand pipette at a very small volume (1 l) to pipette individual worms and expel them out of the tip efficiently. The boxes are incubated at 20°C, with a 16-hour day / 8-hour night light regime, for the entire remaining life cycle.

Food regime

The food regime is inspired from the classical culture system [START_REF] Hauenschild | Platynereis dumerilii. Mikroskopische Anatomie, Fortpflanzung, Entwicklung[END_REF]. Four different types of food are used successively to ensure rapid and healthy growth. The quantities mentioned must be strictly followed, as overfeeding can cause water fouling and be a major cause of death for juvenile worms. The microalgae typically used for feeding early juvenile stages are live Tetraselmis marina. To avoid time consuming lab culture of this green alga, commercial fish hatchery food (Instant Algae™, Reed Mariculture) is used. The algae are diluted 40 times in NFSW, aliquoted in 50 ml Falcon tubes and kept frozen at -20°C. Sera Micron® (Sera) is the food chosen for older worms, as its particle diameter is well suited for the worm's mouth at this stage and it is highly nutritious for rapid growth. It is resuspended (1% w/v) in NFSW and must be kept frozen if not used immediately.

Tetramin® flakes are fed to sub-adults. They are ground to a powder using a mortar and are suspended in NFSW (1% w/v). Tetramin® suspension can be kept frozen if not used on the same day.

Finally, frozen organic spinach is used to supplement both Sera® and Tetramin® regimes, in a 10% w/v suspension that is ground with a food blender. Food distribution is made according to table 1

Worm growth monitoring and water change

Young worms are not visible to the naked eye during the first three weeks of culturing in polypropylene boxes. After 3-4 weeks, they start spinning silk tubes that are large enough to be observed at the bottom of the containers. Therefore, it is recommended to check after one month that growth is normal, and no mortality of young worms has occurred. At two months (61 days), a water change is necessary. At this age, worms are firmly settled in their silk tubes during the day and the old water can be safely poured out into an intermediate container before being discarded.

Occasional runaway worms can be put back into their box with a plastic Pasteur Pipette. 500 ml of fresh NFSW is added to each box. Worms are counted at this 2-month stage to ensure that there are P. dumerilii cultures are anything but axenic. Boxes contain several other organisms, some of which are likely beneficial for the growth and maintenance of the annelid, while others may grow to the point of interfering with the well-being of the worms. These co-cultured organisms are typically passed down from generation to generation when transplanting worms to new boxes with pipettes and are not detected due to their microscopic size. Alternatively, they also come with the natural sea water (in our case, filtered natural sea water from the bay of Cancale in Brittany), which, despite filtering, is not sterile. It is important to mention that we have never observed cases of internal parasitism, such as by myxozoans [START_REF] Rangel | Morphology, Molecular Data, and Development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a Polychaete Alternate Host, Nereis diversicolor[END_REF], in our culture. This indicates that the filtration quality of the sea water we used has been satisfactory in this respect. In this paragraph, we will give a brief description of these commensals. We will describe how to transfer them from boxes to boxes if they may be a complementary source of food for the worms, help clean out excess distributed food, or help maintain the homeostasis of the box in any other respect. We will also describe how to get rid of unwanted commensals.

In the older boxes, the bottom gradually becomes covered with green filamentous algae and cyanobacteria mats (figure 3). Small worms feed on filamentous algae, as the algae gets cleared near the extremities of the worm tubes. Additionally, these photosynthetic organisms are likely important in maintaining a satisfactory level of dissolved oxygen in the water, as we never place air diffusers at any stage of this culture. We have observed that small worms settle in tubes earlier in boxes where we ensure that filamentous algae and other components of the mat grow rapidly. To obtain quick growth of the mat, we inoculate boxes with small pieces of the mat from a 4-month-old box. It is important to ensure that this old box does not contain any unwanted commensals (such as Dimorphilus worms, see below) or small P. dumerilii larvae or tiny juveniles from spontaneous reproduction, which can occur even when boxes are checked for adults daily. The mat is scraped from the box bottom using a plastic scraper. A small piece roughly 1 cm 2 is sufficient to inoculate a new box. This is done at the same time as new boxes are populated with little 10-day worms (figure 2).

Unicellular eukaryotes are also present in our culture boxes, consisting of two types: hypotrich ciliates wandering on the box bottom and swimming flagellated unicells (mostly dinoflagellates). In boxes, their proliferation is harmless to the worms and may even benefit the culture by cleaning out excess food. These unicellular eukaryotes are propagated from one generation of boxes to the next by pipetting adults or seeding filamentous algae, as practiced in the lab. These organisms do not require extensive monitoring, except for one exception. In the beakers containing larvae and small worms fed with Tetraselmis, dinoflagellates can proliferate excessively, rendering the water cloudy, depleting the medium of oxygen, and ultimately killing the small worms. If the recommended quantity of food and culture time in these small beakers with a high density of worms is followed (no more than 10 days), this problem is unlikely to become uncontrollable.

Lastly, small metazoans are sometimes co-cultured with P. dumerilii. Nematodes have been observed

on several occasions, but they never reach high densities and do not affect the wellbeing of P.

Data analyses and graphs.

All analyses were performed in 'R 4.1.3' and RStudio. Graphs were created using the R package 'ggplot2 v3.4.1'.

Results

Figure 4. Cumulative plots of mature worms in each generation of the FF strain selection. "Classical" refers to the application of the typical food regime [START_REF] Hauenschild | Platynereis dumerilii. Mikroskopische Anatomie, Fortpflanzung, Entwicklung[END_REF] to the original polymorphic strain. FF(1-8)G refers to the eight generations of selected worms . "Control" refers to the application of the final retained protocol to the polymorphic strain. The density, food regime and general statistics for each generation are described in 4. The food regimes are (A) classical regime, 1 st month with 3ml microalgae twice per week, 2 nd month with 3ml Sera micron twice per week, 3 rd month with mixed Tetramin twice per week; (B) , 1 st month with 3ml microalgae twice per week, 2 nd month with 1ml Sera micron twice per week, 3 rd month with mixed Tetramin/spinach as shown in Table 1; (C) Sera micron reduced to two weeks and spinach once per week from the 2 nd month, as described in Table 1.

Selection of fast reproducing individuals

An initial selection of four pairs of early-reproducing worms was made from the polymorphic stock.

The worms obtained from these four pairs were called FF1G (1st generation). All crosses were made between worms of the same generation, which were further referred to as FF2G, FF3G, and so on.

The dates of emergence of all mature worms were systematically recorded in each generation up to 130 days after fertilization. Only the earliest pairs of mature worms of each generation were retained for spawning the next generation. Importantly, we systematically avoided brother/sister crosses. The rationale for this restriction is to preserve as much of the initial polymorphism as possible to prevent the co-selection of deleterious mutations and the rapid selection of sub-optimal combinations of genes in terms of maturation age. Another crucial factor in this process was the selection of fastgrowing juvenile worms from 10 dpf batches to populate low-density boxes (see Materials and Methods). From FF5G onward, we exclusively selected the 10-day post-fertilization worms that had developed a fourth segment after six days of feeding with microalgae (figure 2). This is a second level of selection that may have influenced the final gene frequencies of the FF population. Eight generations were obtained in this way over a two-year period. Excluding the problem that affected FF4G, likely due to the food regime (see next section), the median age of maturing worms decreased from generation to generation (Figure 4) but did not improve after FF6G.

To determine whether genetic selection plays a role in fast cycling, we raised animals from the polymorphic stock using the same density and food conditions as those used in the last three generations of selected worms (FF6G-8G). The maturation curve of the polymorphic worms (Figure 2, control) does not appear significantly different from that of the selected worms FF6G-8G. The time of the first appearance of maturation for the polymorphic worms is 2 weeks later compared to FF6G-8G

(Figure 4, Supplementary Figure 1), but the median age of maturation for the polymorphic worms is the youngest obtained in the study (figure 4). The maturation frequency of animals over time for the polymorphic and first generations of selected worms (FF1G-2G) displays two peaks at 4-week intervals, even without the application of an artificial moonlight regime (Supplementary Figure 1).

Single waves of maturation are then observed in selected worms (FF3G-8G). The bimodal maturation in unselected worms could be linked to an intrinsic circalunar maturation rhythm [START_REF] Zantke | Circadian and Circalunar Clock Interactions in a Marine Annelid[END_REF], but this is merely a hypothesis. The overall sex ratio in selected worms was not significantly different from parity (Table 2) but there is a significant tendency for earlier male maturation (Supplementary Figure 1), resulting in a slight excess of males in the first 3-4 weeks of mature collection. This discrepancy was not displayed in the control experiment, suggesting that it might be the result of genetic selection. This is again a hypothesis as the determinism of sex in Platynereis is not currently known. The earliest maturing individuals were 60 days old but did not produce viable offspring. The earliest mature animals involved in successful reproduction were 68-70 days old.

Between 7 and 10 viable batches of eggs were selected for beginning the next generation of FF worms. The average age of the parents used for these maintenance reproductions decreased rapidly (Table 2 and Figure 4 in blue) and stabilized around 12 weeks (FF6G-8G).

We modified the density and food regime of the worm boxes over the two-year period, and unlike genetic selection, these two parameters appeared crucial in maintaining a fast-cycling culture, as explained below.

Feeding and water change

The classical food regime utilized for our polymorphic P. dumerilii culture is based on the methods developed by Hauenschild and Fischer. Initially, we provided Tetraselmis marina microalgae obtained from a lab culture at the Institut Jacques Monod (courtesy of P. Kerner) to little worms aged from 4 days to 1 month old. This was followed by a suspension of Sera micron® for the second month, and then a mixed regime of Tetramin® and organic spinach from the third month onwards. These various food items are adapted to the mouth size of the fast-growing worms. To simplify the procedure, we first tried to eliminate the organic spinach. Table 2 provides a timeline of the successive regimes and quantities of food delivered to the selected worms.

During the selection process, we encountered two issues that significantly impacted the viability and maturation age of the worms, leading us to gradually modify our approach. The first issue was that some of the worms in certain boxes were unable to spin silk tubes. These worms would wander for the first two months and eventually assume a curled position with reduced mobility and feeding activity. This problem culminated in the FF4G generation, resulting in high mortality and a decrease in maturation (Figure 2). Changing the water did not correct this syndrome, and since it was not due to water fouling, we hypothesized that it might be caused by nutritional deficiency. As a result, we added ground spinach for the last four generations, and the abnormal behavior decreased from the 5th generation onwards. We then decided to provide spinach from the beginning of the second month along with Sera micron, as we reasoned that spinach must contain vitamins or other essential nutrients that are not present in Sera micron. This new approach eliminated the syndrome.

The second problem was mortality due to water fouling, which mostly resulted from feeding the worms with Sera micron. Initially, we provided quantities of the suspension that were too large for the small number of worms (15-30) in each box. However, Sera micron, which is primarily made with Spirulina cyanobacteria, is a highly nutritious food and very efficient for fast growth of the worms.

Instead of eliminating it completely, we provided smaller quantities of the food suspension and limited the period of Sera micron to two weeks, as the worms quickly gained weight and could handle the larger particles of Tetramin. Initially, we attempted to raise the worms without performing timeconsuming water changes to simplify the culture process. We believed that with a small number of worms, a controlled feeding regime, and the continued presence of dense populations of ciliates and flagellates in the boxes to clear unconsumed food, water fouling should be minimized. However, we reintroduced water changes at 60 days post-fertilization (dpf) to prevent water fouling in a few boxes that could have resulted in mass worm death. Water fouling typically results in yellowish and cloudy water. In contrast, the accumulated faeces do not seem to cause any problems for the worms despite the revolting look of old boxes. The box bottoms are also typically covered with green filamentous algae inoculated from the previous generation of boxes, which the worms occasionally feed on without issue (see below).

The last change we conducted related to food was replacing lab grown fresh Tetraselmis marina microalgae with commercially frozen microalgae of the same species (see Materials and Methods).

We calculated a dilution factor for the microalgae that was equivalent to the dose of fresh algae we had been using. Half of the FF8G boxes were fed fresh algae, and the other half were fed frozen algae.

We took care to compare boxes from the same parents. After 6 weeks of growth, we did not observe any significant differences in growth between the two types of food (Supplementary Figure 2).

Therefore, the time-consuming task of culturing the microalgae can be replaced with relatively inexpensive commercial algae, given the small quantities used.

Worm density

Worm density appears to be the most important factor for fast maturation. The traditional method for raising worms involves alternating between high-density and low-density boxes [START_REF] Kuehn | A scalable culturing system for the marine annelid Platynereis dumerilii[END_REF]. Batches of small juvenile worms obtained from a single pair of worms in beakers are transplanted into one to three growth boxes, depending on the perceived density of worms. These worms will produce a dense layer of small tubes at the bottom of the box after a few weeks.

However, their growth is slow, and maturation will not occur if the density remains above one hundred worms per box. Increasing food doses does not help because small worms seem to inhibit each other's growth. Therefore, high-density boxes are transplanted into low-density boxes. The recommended density of worms for fast growth has typically been 30 worms per square box (around 500 individuals.m -2 ) (ref). Consequently, we decided to transplant low-density boxes directly from beakers containing 10 dpf feeding and growing little worms. Once food and water fouling problems were resolved, it became evident that the overall survival of these small, selected juveniles up to maturation age was excellent, approaching 100% in most cases, and their growth was very quick. To determine the optimal worm density for growth and maturation, we compared boxes of the FF6G generation that received different numbers of small worms (Table 2). Based on these data, we determined that the optimal density was 20 worms per box (300 individuals.m -2 ), significantly less than the previous conditions used.

Discussion

As described above, the life cycle of P. dumerilii is lengthy and complex. This has hindered the widespread adoption of this model on a global scale. In response to this challenge, we propose in this article a new and fast-reproducing strain called "Fast-Forward" (FF). The "Three Rs" principle outlined in Directive 2010/63/EU of the European Union legislation reflects the scientific community's current trend to Replace, Reduce, and Refine animal experimentation. The protocol presented in this article aims to reduce the resources required to culture P. dumerilii while accelerating and simplifying the management of its culture conditions. According to the Refinement principle of the "Three Rs", we anticipate that the proposed modifications to the care practices will alleviate the distress experienced by the animal, thus reducing the variability of scientific results. Improving data quality indirectly contributes to the Reduction principle, as fewer animals are required to obtain valuable results.

However, the use of live animals remains a widely used strategy in developmental genetics, and it is an area that must be improved in the future.

Suitability of the present culture method for different types of biological studies

The original laboratory culture of P. dumerilii was developed in 1953 in Germany by Carl Hauenshild and Albrecht Fischer (A. [START_REF] Fischer | The polychaete Platynereis dumerilii (Annelida): A laboratory animal with spiralian cleavage, lifelong segment proliferation and a mixed benthic/pelagic life cycle[END_REF][START_REF] Hauenschild | Platynereis dumerilii. Mikroskopische Anatomie, Fortpflanzung, Entwicklung[END_REF]. Since then, all laboratories that have developed cultures appear to have used offshoots of this original German culture. This strain initially included a mixture of Atlantic and Mediterranean worms, which may partly explain the high level of genetic polymorphism. A complete culture method was posted online for a long time on the Platynereis.de website, which was cited in many previous articles and helped many groups establish their own culture. Although this website is no longer available, a copy of the method can be found on the Platynereis.com website. Several groups have since attempted to simplify the method. A recent effort has resulted in a "scalable culturing system" [START_REF] Kuehn | A scalable culturing system for the marine annelid Platynereis dumerilii[END_REF] for starting a small-scale culture without a dedicated thermostatic room. This method simplifies the food regime, alternating between Spirulina powder for the youngest worms and Sera micron for juveniles up to sexual maturation.

Our technique offers several steps for simplification, enabling the compaction of culture and preservation of wild-type stock and several different genetically modified strains in a limited space.

We use flat boxes that can be stacked with at least two boxes on each shelf, without hindering gas exchange into the water. Piling up to four boxes has also been done without any issues. However, it is important to use plastic boxes with non-airtight lids (refer to Materials and Methods). We do not use air bubbling at any stage, due to the low-density of the culture and carefully managed food regime.

Compared to earlier methods, our technique significantly reduces the need for water changes. With only one water change required when the worms reach two months (instead of every two weeks), a low-density box of worms can complete its cycle using only one litre of NFSW for a maximum of four months. This minimizes the workload, expenses, and natural resources required, making it possible to maintain bigger cultures in the future.

The main advantage of this culture technique is the acceleration of the reproduction cycle, which is a crucial step in establishing P. dumerilii as a valuable model for transgenesis and genome editing techniques. Measuring the average life cycle of P. dumerilii has seldom been done in the past, but previous methods have clearly resulted in much longer life spans and, consequently, reproduction cycles. The culture technique used at the Institut Jacques Monod for previous works since 2009, following the Hauenschild/Fischer method, involved using high-density boxes for an extended period (3-4 months) before worms were transferred to low-density boxes for maturation. As a result, very few worms matured before 4 months, and even after dispatching to low-density boxes, few worms matured before 5-6 months. The recently proposed scaled-down, cost-effective in-lab culture [START_REF] Kuehn | A scalable culturing system for the marine annelid Platynereis dumerilii[END_REF] does not focus on speeding up the cycle and maintains high-density boxes (more than 300 worms/box) for two months after fertilization. A work on corazonin effects on growth and maturation [START_REF] Andreatta | Corazonin signaling integrates energy homeostasis and lunar phase to regulate aspects of growth and sexual maturation in Platynereis[END_REF] mentions a median maturation time for control polymorphic worms of around 8 months, compared with 3.5 months with our FF strain and rearing protocol. In contrast to these lengthy periods of culturing at high density, the crucial parameters manipulated in the current technique are the immediate establishment of low-density boxes and the careful selection of fast-growing small worms at ten days.

This culturing technique may not be suitable for all experimental purposes. For instance, some teams study biological rhythms, especially those that are controlled by the lunar cycle [START_REF] Poehn | A Cryptochrome adopts distinct moon-and sunlight states and functions as sun-versus moonlight interpreter in monthly oscillator entrainment[END_REF].

The complete suppression of the artificial moon cycle and the maturation of most worms within a single moon cycle (28 days) are clearly inadequate for these types of studies. Others investigate timerelated processes such as regeneration [START_REF] Vervoort | Studying Annelida Regeneration Using Platynereis dumerilii[END_REF] with recorded timelines under carefully controlled culture conditions. Using the FF strain and fast-cycling protocol, it will be necessary to test the capabilities of the selected worms in the specific process being studied and record new standard time courses accordingly. The elimination of high-density boxes means that the culture method does not allow for keeping large numbers of medium-sized juveniles, which may be impractical for teams working on processes occurring at these stages.

Comparison with other metazoan models

Table 2. Reproductive characteristics of animal laboratory models used for transgenesis/genome editing experiments. References: (a) [START_REF] Butt | The intensive production of lumbricus terrestris L. for soil amelioration[END_REF], (b) [START_REF] Akazawa | Development of a novel heterologous gene expression system using earthworms[END_REF], (c) [START_REF] Dominguez | Biology and Ecology of Earthworm Species Used for Vermicomposting[END_REF], (d) [START_REF] Shankland | Description of the Californian leech Helobdella robusta sp.nov., and comparison with Helobdella triserialis on the basis of morphology, embryology, and experimental breeding[END_REF], (e) [START_REF] Iyer | Reproductive differences among species, and between individuals and cohorts, in the leech genus Helobdella (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae), with implications for reproductive resource allocation in hermaphrodites[END_REF], (f) [START_REF] Williams | Towards a systems-level understanding of development in the marine annelid Platynereis dumerilii[END_REF], (g) (A. H. L. [START_REF] Fischer | The normal development of Platynereis dumerilii (Nereididae, Annelida)[END_REF], (h) [START_REF] Seaver | Annelid models I: Capitella teleta[END_REF], (i) [START_REF] Ebert | Ecology, Epidemiology and Evolution of Parasitism in Daphnia[END_REF], (j) [START_REF] Yamaguchi | Drosophila as a Model Organism[END_REF], (k) [START_REF] Schröder | From development to biodiversity -Tribolium castaneum, an insect model organism for short germband development[END_REF], (l) [START_REF] Howe | The effects of temperature and humidity on the oviposition rate of Tribolium castaneum (Hbst.) (Coleoptera, Tenebrionidae)[END_REF], (m) [START_REF] Lawrence | Generation time of zebrafish (Danio rerio) and medakas (Oryzias latipes) housed in the same aquaculture facility[END_REF], (n) [START_REF] Wittbrodt | Medaka -A model organism from the Far East[END_REF], (o) [START_REF] Jeffery | Astyanax surface and cave fish morphs[END_REF], (p) [START_REF] Blackburn | Modeling congenital kidney diseases in Xenopus laevis[END_REF], (q) [START_REF] Beck | An amphibian with ambition: a new role for Xenopus in the 21st century[END_REF], (r) [START_REF] Wolf | A molecular approach to fertilization[END_REF], (s) [START_REF] Tilley | The use of transgenics in the laboratory axolotl[END_REF], (t) (Phifer-Rixey & Nachman, 2015), (u) [START_REF] Weber | Maternal behaviour in Mus musculus sp.: An ethological review[END_REF], (v) [START_REF] Darling | Rising starlet: the starlet sea anemone, Nematostella vectensis[END_REF], (w) [START_REF] Kaliszewicz | Interference of asexual and sexual reproduction in the green hydra[END_REF], (x) [START_REF] Massaro | Development and population growth of Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa) in the laboratory[END_REF], (y) [START_REF] Frank | The hydroid Hydractinia: A versatile, informative cnidarian representative[END_REF], (z) [START_REF] Lechable | An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica[END_REF], (aa) [START_REF] Iglesias | The Completed Life Cycle of the Octopus (Octopus vulgaris, Cuvier) under Culture Conditions: Paralarval Rearing using Artemia and Zoeae, and First Data on Juvenile Growth up to 8 Months of Age[END_REF], (bb) [START_REF] Meneely | Working with Worms: Caenorhabditis elegans as a Model Organism[END_REF] Table 2 provides an overview of current metazoan models that have been acclimated in laboratory settings for experiments involving genetics and reverse genetics. This list includes a mix of models that have been developed for a long time, some for more than a century, with well-established protocols and numerous publications utilizing these techniques (such as the house mouse, fruit fly, and Caenorhabditis elegans), as well as several other models where efforts to develop genetic approaches are still in their early stages. Only a few models are available for studying the vast diversity of genetically dependent biological phenomena, as the duration of the life cycle is often a significant obstacle.

D. melanogaster and C. elegans are exceptions in this regard, as they depend on ephemeral food resources in the wild, specifically rotting fruits. As a result of selection pressure, they have evolved exceptionally fast development, growth, and sex maturation processes. However, these widely used models with fast life cycles are correlated with rapid evolution at the developmental, morphological, and molecular levels, making them "long evolutionary branch" models. Therefore, there is great interest in developing alternative model organisms with a short evolutionary branch for genetic investigations. These models can provide insights into the emergence of major anatomical features, cellular processes, and genetic machineries, simply because they are less derived in these respects compared to well established models.

The current list of short evolutionary branch metazoans includes notable species such as amphioxus [START_REF] Louis | How much does the amphioxus genome represent the ancestor of chordates?[END_REF], hemichordate worms [START_REF] Simakov | Hemichordate genomes and deuterostome origins[END_REF], P. dumerilii (Özpolat et al., 2021), the myriapod Strigamia maritima [START_REF] Chipman | The First Myriapod Genome Sequence Reveals Conservative Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima[END_REF], and the sea anemone Nematostella vectensis [START_REF] Putnam | Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization[END_REF], representing chordates, deuterostomes, annelids, arthropods, and cnidarians, respectively. Most of these species occupy key phylogenetic positions for reconstructing ancestral states in the metazoan tree. Phylogeny and evolution are not the only motivations for developing new genetic models, as some species exhibit unique derived characters that are worth exploring at the genomic/genetic level, such as the tardigrade Hypsibius exemplaris [START_REF] Yoshida | Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus[END_REF] and its ability to undergo anhydrobiosis.

Our new protocol places P. dumerilii in a better position for development as a high-performing genetic model in the future. The median life cycle obtained with this method (around 15 weeks) places P. dumerilii in a comparable position to the widely used fish model Danio rerio (zebrafish, table

2). Among annelids, the fast culturing of P. dumerilii places it on par in terms of culture conditions with other models, including both clitellates and "polychaetes" that have been developed in the past.

While clitellates have interesting developmental features such as large size and easily micro-injectable teloblasts, they are derived annelids, adapted to freshwater or terrestrial lifestyles. Among "polychaetes", Capitella teleta [START_REF] Seaver | Annelid models I: Capitella teleta[END_REF] has emerged with many publications in the last two decades. It belongs to the large clade Sedentaria. Lineage tracing by micro-injection [START_REF] Meyer | A comprehensive fate map by intracellular injection of identified blastomeres in the marine polychaete Capitella teleta[END_REF] and genome editing by CRISPR-Cas9 [START_REF] Neal | CRISPR/CAS9 mutagenesis of a single r-opsin gene blocks phototaxis in a marine larva[END_REF] have been used successfully in this species. More recently, the other sedentarian Owenia fusiformis [START_REF] Carrillo-Baltodano | Early embryogenesis and organogenesis in the annelid Owenia fusiformis[END_REF][START_REF] Helm | Owenia fusiformis -A basally branching annelid suitable for studying ancestral features of annelid neural development[END_REF] has also been shown to possess valuable characteristics for diverse biological studies. P. dumerilii however displays two main advantages over these models: being an errantia of the family Nereididae, it is widely considered to possess a more ancestral anatomy than the sedentarians and the reproduction by epitokous swarming allows for complete control of the reproduction event and easy manipulation of thousands of eggs and embryos, obtained daily using the fast-cycling culture method described here.

Culture size

The packing of boxes in incubators allows for easy scalability without the need to dedicate a thermostatic room of adequate size and shape for culture. The elimination of the lunar cycle and the rapid maturation of worms after 10 weeks are also important factors. There is no need for two rooms with alternating lunar cycles to obtain mature individuals over a monthly period. The maturation peaks will only depend on the age of the boxes. Mature worms will be used for three main purposes: colony maintenance, crosses with transgenic/CRISPR-edited worms, and micro-injection. We have generally been selecting mature worms aged less than 90 days to start the next generation of the FF population, and this selection is currently maintained. Worms older than 90 days are used for genetics and micro-injection. However, P. dumerilii still displays some unfavourable characteristics for genetic experiments, such as having 2n = 28 chromosomes, which limits the possibility of complex genetic combinations. Maintaining homozygous strains will be challenging as it requires two homozygous adults of opposite sexes on the same day, which in turn requires a large pool of worms for each strain. Therefore, the solution for keeping homozygous strains may lie in the development of sperm (or even embryo) freezing techniques (Olive and Wang, 1997). Transgenic lines expressing fluorescence as heterozygotes are maintained by crossing transgenic worms with wild-type FF individuals and selecting transgenic progeny under an epifluorescence microscope as early as 10 days post-fertilization. Our group in Paris has successfully maintained three different strains in this manner with only four boxes, each containing 20 worms, for each strain (Balavoine, unpublished).

In the current culture conditions, assuming an initial seeding of 20 worms per box and a conservative collection efficiency of 60%, we can calculate a productivity of approximately 1-1.2 worms/box/week over a 10-week period. There will be an unproductive period of 8-10 weeks following, as a new generation of boxes is growing. For teams that require wild-type worms throughout the year, which will be the case if multiple transgenic strains need to be maintained, the number of boxes will need to be doubled with a shift of 8-10 weeks between the seeding of low-density boxes. This shift can be easily achieved by maintaining batches of the same generation of small worms in high-density boxes (>300 worms) from 10 dpf on, fed exclusively with algae, for 8-10 weeks. The worms will remain very small until they are used to create new low-density boxes.

The current study may not represent the ultimate effort in establishing the most efficient culturing system for P. dumerilii, but it is a significant step towards reducing the variability in the age of sexual maturation. We believe that there is still much progress to be made in improving the food regime, particularly during the early settlement of the worms. One potential solution could be to draw inspiration from a recent small-scale culture published by [START_REF] Kuehn | A scalable culturing system for the marine annelid Platynereis dumerilii[END_REF], where they reduced the food regime to two items, as opposed to the four items utilized in our present method. Another area for improvement could be the replacement of natural filtered sea water, which can be costly for facilities not located near the sea, with artificial sea water. Hopefully, our findings, combined with advancements in developing efficient transgenesis, will encourage more groups in the future to adopt this remarkable model animal for their own studies and establish their own culture of P.

dumerilii.

Figure 1 .

 1 Figure 1. Platynereis dumerilii's life cycle. The different life stages are represented and categorized

Figure 2 .

 2 Figure 2. Platynereis dumerilii Fast Forward strain Protocol overview. The overall progression to make the selection of FF individuals and reach the proposed protocol is described in the Results section.

Figure 3 .

 3 Figure 3. Commensals commonly found in Platynereis dumerilii's culture boxes. Cyanobacteria and filamentous green algae are constitutive of the mat. Dimorphilus, nematodes and ciliates feed at the surface of the mat. Dinoflagellates are free swimming above the mat.

  dumerilii. However, more concerning are the proliferations of the tiny annelid Dimorphilus gyrociliatus[START_REF] Martín-Durán | Conservative route to genome compaction in a miniature annelid[END_REF]. These limbless, gliding annelids, which are less than 1 mm long, can be detected by examining the box bottoms with a stereomicroscope. In some of our lowdensity culture boxes, they have reached enormous densities, hindering the growth of P. dumerilii and potentially causing their death. To eliminate them, we transferred all P. dumerilii individuals to a new box. Each worm had to be rinsed with seawater several times to ensure no D. gyrociliatus remained. It is also important to follow simple rules to eliminate these unwanted commensals from the culture: the beakers containing the small 10-day worms used to populate new boxes should be checked for any contaminating D. gyrociliatus, and plastic pipettes used to collect adults should be discarded and replaced when collecting worms from boxes of a new generation.

Table 1 . Weekly schedule for the distribution of food in individual boxes

 1 .

	Worm ages	monday	wednesday	friday
	4-30 dpf	3 ml algae		3ml algae
	31-45 dpf 1ml Sera micron® 3 ml spinach 1ml Sera micron®
	> 46 dpf	3ml Tetramin®	3ml spinach	3ml Tetramin®

. All volumes are suspensions in NFSW at the concentration indicated in the text. Worm ages are in days post fertilization (dpf).

Table 2 .

 2 The dashed lines and numbers in red indicate when 50 % of the initially transplanted worms have matured (median maturation age). Numbers in blue indicate the average age of worms selected to spawn the entire next generation.

Table 2 . Main statistics of the successive generations of the FF strain selection

 2

. Columns are named as in figure

Acknowledgements

We thank the CNRS, the University Paris Cité and the Institut Jacques Monod for funding this research. In addition, the Balavoine group was financially supported by the Agence Nationale de la Recherche (grant PRCI TELOBLAST ANR-16-CE91-0007) and the Fondation ARC pour la recherche sur le cancer (grant LSP 190375). GG was supported by a Master student fellowship of the EUR G.E.N.E. graduate school (#ANR-17-EURE-0013) that is part of the Université Paris Cité IdEx #ANR-18-IDEX-0001 funded by the French Government through its "Investments for the Future" program. We want to thank P. Kerner for his help in microalgal supplies and tips. We acknowledge the staff of the animal facility of the institute J. Monod for help in worm husbandry.

Contributions

ML, GG, and GB shared most of the worm husbandry, data collection, curation and analysis. RS and EC helped with worm husbandry. ML, GG and GB wrote the manuscript. GB designed the study. All figure hand drawings are made by GG.

Supplementary information

Supplementary figure 1. Sex ratio in mature animals across the selected FF generation. FF(2-8)G are the selected generation for which significant numbers of matures have been obtained. "poly" is the control culture with polymorphic worms raised with the same density and food regimen as the last three selected FF generations.

Supplementary figure 2. Growth in function of early algae food.

Two alternative food regimes were applied on FF9G worms, one with one month of freshly grown microalgae (AF) and the other one with one month of frozen algae (IA, Instant Algae™) at two months post fertilization. As a proxy for growth, the length of silk tubes (closely representing worm length) covering the bottom of the boxes were measured using imageJ.