Weijing Han

Rania El Botty

Elodie Montaudon

Laurent Malaquin

Frederic Deschaseaux

Nicolas Espagnolle

Elisabetta Marangoni

Paul Cottu

Gérard Zalcman

Maria Carla Parrini

Franck Assayag

Luc Sensebe

Pascal Silberzan

Anne Vincent-Salomon

Dutertre Guillaume

Sergio Roman-Roman

Stephanie Descroix
email: stephanie.descroix@curie.fr

Jacques Camonis

Stéphanie Descroix

In vitro bone metastasis dwelling in a 3D bioengineered niche

Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium deposition. We thus demonstrated that this device offers an adequate soil to seed breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patientderived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

Introduction

Cancer metastasis is a major evolutionary step in the natural history of cancer disease because it increases the burden of disease (through pain, invalidity, and other functional handicaps) and is also the cause of death for cancer patients, rather than the primary tumor [START_REF] Gupta | Cancer Metastasis: Building a Framework[END_REF][START_REF] Steeg | Metastasis: a therapeutic target for cancer[END_REF]. A metastatic cancer is considered as a completely distinct disease with its own biology and metabolisms, as opposed to being interpreted as an ectopic colony of the primary tumor with identical biological properties [START_REF] Chiang | Molecular Basis of Metastasis[END_REF].

The thorough study of the biology of metastasis faces many limitations, many of which are practical [START_REF] Chaffer | A Perspective on Cancer Cell Metastasis[END_REF][START_REF] Talmadge | AACR Centennial Series: The Biology of Cancer Metastasis: Historical Perspective[END_REF].

Experimental systems that tackle metastasis biology confront the same challenge as the biology of cancer in general, which is that establish an in vitro system respectful of the identity of initial cells. For instance, it is well known that in so-called "flat biology" cell lines that are derived from primary tumors and established in tissue culture plates vary in many aspects, including gene transcription, which has a huge impact on drug sensitivity [START_REF] Hidalgo | Patient-Derived Xenograft Models: An Emerging Platform for Translational Cancer Research[END_REF][START_REF] Tentler | Patient-derived tumour xenografts as models for oncology drug development[END_REF][START_REF] Cheung | Collective Invasion in Breast Cancer Requires a Conserved Basal Epithelial Program[END_REF]. Thus, new models or methods for cancer metastasis studies need to be developed. Several solutions have been proposed to address this issue, such as spheroids/organoids, tumor-on-chip, and mouse models, all of which have emerged as relevant experimental alternatives to study cancer-cell biology [START_REF] Baker | Deconstructing the third dimension-how 3D culture microenvironments alter cellular cues[END_REF][START_REF] Pampaloni | The third dimension bridges the gap between cell culture and live tissue[END_REF]. Nevertheless, these innovative methods also have unique limitations.

While spheroids/organoids are better biomimetic 3D models than monolayer cultures, fibroblasts or immune cells are still deprived of the native tumor microenvironments that play essential roles during metastasis. Tumor-on-chips [START_REF] Bhise | Organ-on-a-chip platforms for studying drug delivery systems[END_REF][START_REF] Nguyen | Dissecting Effects of Anti-cancer Drugs and Cancer-Associated Fibroblasts by On-Chip Reconstitution of Immunocompetent Tumor Microenvironments[END_REF] and the use of some porous scaffolds [START_REF] Reagan | Investigating osteogenic differentiation in multiple myeloma using a novel 3D bone marrow niche model[END_REF][START_REF] Qiao | Engineering 3D approaches to model the dynamic microenvironments of cancer bone metastasis[END_REF] offer interesting 3D models that could provide interactions between cancer cells and stromal cells. However, most of them lack the physiological and mechanical architecture of the native environment, which are proven critical factors in bone metastasis [START_REF] Villasante | Bioengineered human tumor within a bone niche[END_REF][START_REF] Bissell | Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression[END_REF][START_REF] Olechnowicz | Contributions of the Host Microenvironment to Cancer-Induced Bone Disease[END_REF]. The Achilles heel of mouse models is that they raise ethical problems regarding animal well-being, which is an issue that most countries refuse to ignore and are hardly representative of the autologous immune anti-cancer response in immune-compromised animals [START_REF] Hidalgo | Patient-Derived Xenograft Models: An Emerging Platform for Translational Cancer Research[END_REF][START_REF] Tentler | Patient-derived tumour xenografts as models for oncology drug development[END_REF].

Bone metastases are among the most debilitating metastases (as they include pain, fracture, and medullary compression) and the most common metastatic site of invasive breast carcinomas [START_REF] Weilbaecher | Cancer to bone: a fatal attraction[END_REF][START_REF] Weigelt | Breast cancer metastasis: markers and models[END_REF][START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF]. In this context, we focus on the development of a new 3D breast cancer bone metastasis model, in which a biomimetic bone microenvironment can be maintained in vitro and metastatic breast tumor cells can be well settled and colonize the scaffold while maintaining their biological properties. We first reconstituted an in vitro bone niche with structural and mechanical features of trabecular bone using 3D printing; it was further refined by in-situ differentiating human bone marrow-derived mesenchymal stem cells (MSCs) into osteogenic lineage with collagen matrix and mineralized calcium depositions, which are critical in bone homeostasis and regeneration [START_REF] Bobbert | Effects of bone substitute architecture and surface properties on cell response, angiogenesis, and structure of new bone[END_REF][START_REF] Kim | Stereolithographic bone scaffold design parameters: Osteogenic differentiation and signal expression[END_REF][START_REF] Sitarski | 3D Tissue Engineered in Vitro Models of Cancer in Bone[END_REF]. To assess if this bone niche represents a bona fide soil to seed bone-metastasis cells, breast metastatic patient-derived xenograft (PDX) cells which are considered as better approach than cell lines to achieve clinically relevant results, were inoculated inside the bone. These co-cultures revealed that breast metastatic cells colonized the scaffold and replicated the cells' cycling proficiency, in a similar manner to in vivo. Finally, our study demonstrates that, in these conditions, metastatic breast cancer cells exhibit chemo-resistance, which could imply that this engineered breast cancer bone-metastasis model is a strong alternative for studying bone-metastasis pathophysiology.

Materials and methods

MSC culture and differentiation

Bone marrow mononuclear cells from healthy donors were obtained from femoral heads, according to the guidelines of the ethical committee of Toulouse University (N° AC-2014-2384). After Ficoll-gradient density separation, bone marrow MSCs were isolated from mononuclear cells that had been cultured in a basal culture medium: alpha-minimum essential medium (α-MEM) supplemented with 1% (100 U/mL/100 mg/mL) penicillin/streptomycin, 10% fetal bovine serum (FBS), and 1 ng/ml FGF2 (basic culture medium) [START_REF] Brennan | Inferior In Vivo Osteogenesis and Superior Angiogeneis of Human Adipose-Derived Stem Cells Compared with Bone Marrow-Derived Stem Cells Cultured in Xeno-Free Conditions[END_REF]. After Passage 1, flow cytometry was performed to characterize the membrane phenotype of MSCs, as has been described elsewhere [START_REF] Naji | Endocytosis of indium-tin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells[END_REF].

In order to evaluate their osteogenic potential, bone marrow MSCs were induced in an osteogenic differentiation medium in two steps, after one week of maintenance in a basal culture medium. Expanded MSCs were cultured in a Dex-diff medium (basal αMEM supplemented by 1% penicillin/ streptomycin, 10% FBS, 50 μM ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM dexamethasone) for one week, and then in bone morphogenetic protein (BMP)-diff medium (basal α-MEM supplemented by 1% penicillin/streptomycin, 2% FBS, 50 μM ascorbic acid, 10 mM β-glycerol phosphate and 50 ng/mL BMP-4) for another week.

Reconstruction of a 3D-printed bone scaffold

The architecture of the bone scaffold was reconstructed from the trabecular part of the femoral epiphysis bone. First, a micro-tomography scanner was used to obtain the microstructure of the femoral epiphysis bone (Fig. 1a,b), with 10 μm Z resolution and 50 μm X,Y resolution [START_REF] Mézière | Experimental observation of ultrasound fast and slow waves through threedimensional printed trabecular bone phantoms[END_REF]. Second, the threshold of the micro-tomography images was adjusted to remove the air or marrow from the interstitial spaces that filled the femoral epiphysis, but the bone microstructures were not removed (Fig. 1c). Finally, the image stacks of the bone microstructure were transformed into a 3D STL file using ImageJ. After reconstruction, a cylinder with 10 mm diameter and 2 mm height was selected (Fig. 1d) and further assembled with fiducials, in order to clarify the XYZ position for further characterization and to identify the pillars platform under the cylinder to avoid the first overexposure during 3D printing (Fig. 1e).

The assembled 3D bone scaffold file was loaded into a 3D stereolithography system (DWS 028J+, Italy).

Bone scaffolds were then printed with 40 μm printing resolution in the Z-axis using DS-3000 biocompatible resin. Printed bone scaffolds were developed using isopropanol, dried under nitrogen stream, and cured for 12 minutes in a UV oven (DWS) (Fig. 1f). After UV exposure, the supporting pillar platform was manually removed and the microstructures composing the bone scaffolds were characterized using SEM (S3700, Hitachi) (Fig. 1g). This demonstrated that the trabecular microstructures were replicated accurately, as has been described elsewhere [START_REF] Mézière | Experimental observation of ultrasound fast and slow waves through threedimensional printed trabecular bone phantoms[END_REF]. Furthermore, the optical properties of the 3D-printed bone scaffolds were also checked by a confocal laser-scanning microscope (Leica SP8, Germany). DS-3000based bone scaffolds have strong auto-fluorescence and absorption in the violet band (405 nm) but not in the blue, green, or far-red ranges (data not shown).

Prior to use, bone scaffolds were sterilized in 70% ethanol for 30 minutes. Then, the bone scaffolds were washed with PBS and further pre-treated with a basic culture medium every 24 hours for 48 hours. Finally, the pre-treated scaffolds were coated in 10 µg/mL human fibronectin overnight, before use.

MSC cultivation and differentiation in 3D-printed bone scaffolds

After pre-treatment and surface coating, bone scaffolds were inserted into 15 mL tubes for further MSC seeding and cultivation. Primary MSCs, which were those with less than five passages, were trypsinized and harvested in a basal culture medium at a density of 0.5 million/mL. Then, 2 mL MSC suspensions were added into each 15 mL tube that contained a bone scaffold, which were then gently agitated every ten minutes for the first 30 minutes and then every 30 minutes for a further 1.5 hours. Thereafter, 8 mL of the basic culture medium was added. The basic culture medium was renewed twice per week over the following three weeks (control conditions).

In parallel, MSCs were also induced into osteogenic lineage. After the seeding, which is described above, MSCs were cultured in a basic culture medium for one week and then differentiated by adding a Dex-diff medium after the first week and a BMP-diff medium after the second week. All mediums were renewed twice per week for three weeks (differentiated conditions). Meanwhile, MSCs were also osteodifferentiated in a Petri dish following the same procedure as described above.

Human breast cancer PDX dissociation and 3D in vitro culture

Since both triple-negative breast cancer (TNBC) and luminal B breast cancers (BMBC) can generate bone metastases [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF], we tested the capacity of our bone-like niche to accommodate primary TNBC and bone metastases from BMBC. TNBC PDX were established from a primary TNBC patient (HBCx-66) with residual disease after neoadjuvant chemotherapy [START_REF] Marangoni | Capecitabine Efficacy Is Correlated with TYMP and RB1 Expression in PDX Established from Triple-Negative Breast Cancers[END_REF], named HBCx-66 PDX. Meanwhile, BMBC PDX were established from fresh bone metastasis biopsies of estrogen receptor-positive breast cancer patients (HBCx-124 and HBCx-131), which progressed under endocrine treatment, named HBCx-124 PDX and HBCx-131 PDX. This was in accordance with institutional guidelines and the rules of the Ethics Committee (project authorization no. 02163.02). Additionally, all patients signed the Institut Curie's institutional consent form that authorizes the use of medical records and biological specimens. Following this, both TNBC and BMBC PDX were dissociated and cultivated in the bone scaffolds, a 2D dish, and a Matrigel matrix. This was performed using the following method.

Breast cancer PDX dissociation

Fresh breast cancer PDX tissues were chopped into small pieces (~1 mm) with scalpels and further dissociated using a digestion medium that contained mouse collagenase (1 mg/mL) and hyaluronidase (1000 U/mL) for 45-60 minutes at 37 C. Dissociated PDX cells were resuspended in the PDX culture medium (DMEM with 10% FBS, 1% P.S., 1% Pyruvate, 1% HEPES, 500 uL insulin, and 10 nM estrogen).

Falcon® 100 µm cell strainers were used to remove the undigested tissue and PDX-derived tumor spheres that were larger than 100 μm. Several steps of washing were performed to remove single dead cells from PDX-cell suspensions. Then, Countess® Cell Counting Chamber Slides were used to quantify the concentration and viability of PDX cells.

Breast cancer PDX cell co-cultures in bone scaffolds and Petri dishes

After dissociation, two million breast cancer PDX cells were seeded into the bone scaffolds (both control and differentiated conditions) by gently shaking. In a similar manner as the MSC 3D seeding, 2 mL PDX cells (1 million/mL) were loaded into each 15 mL tube containing a bone scaffold and gently agitated every 15 minutes for the first 30 minutes and then every 30 minutes for the next 1.5 hours. After seeding, an 8 mL PDX culture medium was added. The PDX culture medium was renewed every two days for the next eight days. Meanwhile, 0.5 million breast cancer PDX cells were also co-cultured with osteogenic-differentiated MSCs on 12-well plates, which acted as the 2D co-culture conditions. The PDX culture medium was not renewed, in order to avoid PDX cell detachment during the following eight days.

Breast cancer PDX cell cultures in Matrigel

In addition to being co-cultured in the bone scaffolds and 2D Petri dishes, the breast cancer PDX cells were cultured in a 3D Matrigel matrix to test the viability and proliferation proficiency of PDX cells in vitro. First, both glass-bottom multi-well plates and Nunc Lab-Tek II Chamber Slides, which were used for the 3D culture, were coated with a thick layer of the Matrigel matrix (>1 mm) and incubated for 20 minutes in an incubator. Then, 100μL breast cancer PDX cells were mixed in a final concentration of the Matrigel matrix and 1 million/mL were loaded onto the top of the thick Matrigel layer, which was then incubated for another 45 minutes before the PDX culture medium was added. After incubation, the PDX culture medium was gently loaded, and PDX cells were cultured for seven days without the medium being changed.

TNBC PDX cell drug testing

Drug sensitivity was tested according to different culture conditions. Dissociated HBCx-66 PDX cells were seeded in 96-well plates at a density of 4×10 4 cells per well. After 24 hours, serially diluted cisplatin (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 [μM], NDC 67457-425-51, Mylan) was added by medium replacement for the next 72 hours of treatment. TNBC PDX cell viability was quantified using the CellTiter-Glo assay (Promega, G7570), while the dose-response curve and IC50 of cisplatin (2.4 μM) for inhibiting HBCx-66PDX cells was plotted by Prism software (Fig. SI3).

Subsequently, HBCx-66 PDX cells' response to cisplatin in the bone scaffolds and Matrigel was characterized using the following method. First, HBCx-66 PDX cells were seeded into the bone scaffolds and Matrigel in parallel, as previously described. After two days of culture, different final concentrations of cisplatin (2.4 μM [IC50 in vitro], 5.0 μM, and 10.0 μM) was added into the PDX cell culture medium by medium replacement every two days for the next four days of drug treatment. Here, the colonization and proliferation proficiency of HBCx-66 PDX cells in the bone scaffolds and Matrigel were characterized using the EpCAM area and Ki67 index, after drug treatment. Meanwhile, CellTiter-Glo 3D assays (Promega, G9682) were also used to evaluate the viability of HBCx-66 PDX cells in Matrigel with cisplatin treatment.

Statistical analysis

Results were presented as the mean ± standard error of the mean (SEM) for at least three repeated individual experiments for each group. Statistical differences were determined using Student's t-test for independent samples.

Results

Bioengineering a 3D biomimetic bone scaffold

To provide cancer cells with a 3D structure mimicking in vivo conditions, trabecular bone structures were first defined from μCT scan images. Then, a cylinder (ϕ=10 mm, H=2 mm) that faithfully replicates the trabecular bone structure was produced by stereolithography 3D printing. A biocompatible and visible light transparent photoresist, presenting stiffness of ~2×10 6 kPa (similar to native bulk bone), was used.

The 3D printed scaffold replicates trabecular structures, with appropriate stiffness and pore sizes ranging from 100 to 500 μm, similarly to the native trabecular bone (Fig. 1) [START_REF] Mézière | Experimental observation of ultrasound fast and slow waves through threedimensional printed trabecular bone phantoms[END_REF].

Fig.1 Reconstruction of bone scaffold by 3D printing.

A trabecular part of a leg bone was scanned by using micro-CT scanning (a, b), and the CT images were converted and represented by using 3D modeling software (ImageJ, c). A randomly chosen cylinder zone with 10 mm in diameter and 2 mm in thickness (d) was assembled with pillar structures preserving the first layers melting of the bone structure during 3D printing and fiducials assisting 3D observation during single-photon confocal 3D scanning (e). The bone scaffold made of resin (DS-3000) was printed (f), cured by UV light, and characterized by using SEM (scanning electron microscopy) showing a smooth surface in XY plane and clear Z layer height (30 µm ) along Z axis (g, top view, XY plane).

MSC proliferate and colonize the 3D printed bone scaffold

After pre-treatment and surface coating with fibronectin to promote cell adhesion, bone scaffolds were seeded with primary MSCs (10 6 MSCs) and basic culture medium. This was considered as Week "0." In order to evaluate MSC proliferation, living MSCs within each scaffold were stained with Cell Trace TM Yellow after the first, second, and third week of culture, and were imaged using a multi-field approach (Fig. 2a).

From the top view of the bone scaffold (Fig. 2b), the living MSCs gradually distributed and colonized the entire scaffold surface during the three-week culture. Even when the initial cell distribution was not completely uniform at the top of the structure (Fig. 2b), the top surface of the bone scaffold showed even coverage of living MSCs after three weeks of culture. The side view (Fig. 2b) reveals that the living MSCs also colonized the scaffold deep center. Quantitatively, the proliferation index of MSCs was evaluated as the fold change of the total whole-depth Cell Trace TM signal, compared with first-week conditions. This showed a significantly increased total amount of MSCs in the bone scaffold during the three-week culture (Fig. 2c), with a proliferation index in the third week three times higher than the first week. We also evaluated MSC distribution along the Zaxis of the scaffold (SI Movies 1-3). Normalized Cell Trace TM intensity distribution along the Z-axis showed an increase in Cell Trace TM intensity over time, at each Z depth (Fig. 2d). Interestingly, these curves obtained in the first and third weeks have the same profile, which indicates that the MSC cells proliferate as rapidly in the deep center of the scaffold as on its surface, during the culture. These results suggest that MSCs can survive, proliferate, and colonize within the entire bone scaffold, and gradually become homogenously distributed across the scaffold surface over the course of the culture.

3D printed bone colonized with differentiated MSCs recapitulates bone features

In order to properly reconstruct a bone metastasis microenvironment, primary MSCs were induced to undergo osteogenic differentiation in bone scaffolds. Primary MSCs were seeded in bone scaffolds at Week "0" and then differentiated into osteoblastic cells by switching to a Dex-diff medium and BMP-diff medium at the end of the first and second weeks, respectively (see M&M and Fig. 3a). MSCs that were cultivated in the bone scaffolds with the basic culture medium for three weeks were considered a control experiment (Fig. 3a). In parallel, MSCs were also cultured and differentiated in a Petri dish, following the same procedure (Fig. SI4).

MSCs within the scaffolds were stained with Cell Trace TM Yellow, providing information about MSCs viability after osteogenic differentiation. The quantities of living MSCs, either in the whole scaffold or distributed along the Z-axis in the differentiated conditions, suggested that MSCs submitted to the differentiation procedure survived in, proliferated in, and colonized the whole 3D-printed bone scaffold (Fig. SI5).

The efficiency of osteogenic differentiation was thus evaluated by staining osteocalcin and collagen I, after which the IF value for each marker was characterized to quantify the osteogenic lineage commitment of MSCs and the extracellular-matrix deposition, respectively. 3D-reconstructed figures showed significantly increased signals of osteocalcin (Fig. 3b) and collagen I (Fig. 3d) in the differentiated conditions, compared with the control conditions. The distribution of osteocalcin and collagen I indexes along the Z-axis (see M&M) revealed that both indexes were higher in the differentiated conditions than in the control conditions (Fig. 3c,e). In particular, the MSCs' osteocalcin index was ~4 times higher on the top surface and ~40 times higher at a depth of 1,000 μm, relative to the control conditions. Meanwhile, the collagen I index (Fig. 3e) was 3 times higher on the top surface and ~20 times higher at a depth of 1,000 μm in the differentiated conditions, compared to the control conditions. Furthermore, in the differentiated conditions, both osteocalcin and the collagen I index showed homogenous distributions along the Z-axis, while in the control conditions, these markers were mainly present at the surface. Taken together, these results demonstrate that MSC cells in the 3D-printed bone scaffold can undergo differentiation into osteoblast-like cells, with homogenously distributed osteocalcin expression and extracellular collagen I matrix deposition along the scaffold Z-axis. We also illustrated that there are no significant differences between the MSCs' nuclei area in the control and differentiated conditions (Fig. 3f), to further confirm that osteogenic-induced MSCs express more osteocalcin and collagen I in differentiated conditions. in the differentiated conditions compared to that in the control conditions. Correspondingly, normalized osteocalcin index (osteocalcin intensity per cell, see material and methods) and collagen I index (collagen deposition per cell, see material and methods) distribution along Z-axis (total 0-1000μm) were quantified (see material and methods). Quantification showed distribution of MSCs osteocalcin index (c) and collagen I index (e) in 1000μm depth in the control and differentiated conditions. (f) Nuclei area was measured for both the differentiated and control conditions, and it showed that there was no significantly difference between them. (g) Bone related genes expression was measured via RT-qPCR after MSCs 3D differentiation. mRNA levels of bone sialoprotein (BSP), distal-less homeobox 5 (DLX5), parathyroid hormone receptor (PTHR), Runt-related transcription factor 2 (RUNX-2), and Osterix (OSX) of MSCs in both the differentiated and the control conditions were assessed. (h) Mineral depositions in both the differentiated and the control conditions were characterized by using Alizarin red s Staining method. Calcium deposition was observed in the differentiated conditions (red dots in the right panel) but not in the control conditions (left panel). All results were obtained from independent experiments n≥3, the bars of graphics showed means ± standard error of the mean (SEM). * = p<0.05, ** = p<0.001, *** = p<0.0001, ns = not-significant, using unpaired Student t-test, and values were normalized to control conditions.

In addition, when osteogenic differentiation was performed with MSCs grown in a 2D Petri dish, the MSC monolayer formed discontinuous, unstable clusters with more than 50% of MSCs detached from the surface (Fig. SI4), suggesting that the 3D-printed bone scaffold provides a more appropriate in vitro microenvironment to induce MSCs into osteogenic lineage.

In order to refine the above quantitative characterization of the osteogenic differentiation and to provide more evidence, we measured the expression levels of several genes involved in osteogenic differentiation (BSP, DLX5, PTHR, RUNX-2, and OSX mRNA species [START_REF] Deschaseaux | Regulation and function of immunosuppressive molecule human leukocyte antigen G5 in human bone tissue[END_REF]) (Fig. 3g). All showed significant increases upon differentiation, particularly OSX, which increased 85 times. These results were consistent with the observed MSC osteogenic differentiation in Petri dish [START_REF] Naji | Endocytosis of indium-tin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells[END_REF], indicating a bona fide osteogenic differentiation of MSCs in our bone scaffolds.

The ultimate stage of osteogenic differentiation leads to calcium deposition in the bone environment. In the bone scaffolds, mineralized calcium deposition was characterized using alizarin red S staining.

Representative staining images showed widely distributed calcium mineralization in the differentiated conditions (Fig. 3h), whereas no signal was detected in the control conditions. This observation strongly suggests that our differentiated conditions replicated some features of an in vitro biomimetic bone.

Biomimetic bone niche maintains TNBC cells colonization and proliferation as in vivo

Since triple negative breast cancer (TNBC) is known to generate bone metastasis [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF], we tested the capacity of our bone-like niche to accommodate primary TNBC cells. TNBC PDX were established from a primary TNBC patient with residual disease after neoadjuvant chemotherapy [START_REF] Marangoni | Capecitabine Efficacy Is Correlated with TYMP and RB1 Expression in PDX Established from Triple-Negative Breast Cancers[END_REF], named HBCx-66 PDX (Fig. 4a). HBCx-66 PDX cells were seeded into bone scaffolds, in which MSCs had been differentiated (Fig. 4a). As a control, similar experiments were performed using MSCs grown in a normal medium. In order to assess the advantage of our 3D bone niche, the same batch of HBCx-66 PDX cells was co-cultured with osteogenic-differentiated MSCs on a 2D Petri dish and in a 3D Matrigel without MSCs (Fig. 4a).

Fig.4 TNBC PDX colonization and proliferation in biomimetic bone scaffold. (a)

Triple-negative breast cancer tissue (TNBC) from primary site was sectioned into small pieces and implanted into female nude mice to initiate and propagate TNBC PDXs. After several generations, TNBC (here, HBCx-66 PDXs) PDX tissue was dissociated and seeded into bone scaffolds (both control and differentiated conditions) and cocultured for 8 days. As 3D in vitro control, the dissociated TNBC PDX cells were cultivated in Matrigel matrix for 7 days. (b) The colonization and proliferation capacity of HBCx-66 PDXs cells were characterized by staining with EpCAM (magenta) and Ki67 (green) markers respectively in both the control and differentiated conditions, and the 3D reconstructed images showed more EpCAM positive area and stronger Ki67 signal in differentiated conditions than in control conditions. (c) Quantification of normalized EpCAM area showed ~40% more of EpCAM positive area in the differentiated conditions than in control conditions. (d) quantified Ki67 index (defined as % of cancer nuclear area that is Ki67 positive, see Fig. SI2) and showed more than 2-fold increase of Ki67 positive PDX cells in the differentiated conditions than in the control conditions. (e) HBCx-66 PDX cells cultivated in Matrigel matrix were stained with EpCAM and Ki67 showing apparent EpCAM (magenta, left panel) and nuclei signals (blue, right panel) in HBCx-66 PDX cells but extremely rare Ki67 signal (green, middle panel). (f) Ki67 stained biopsy of HBCx-66 patient from the primary site of TNBC patients, and the statistical results from 62 TNBC patients showed a median Ki67 expression index (defined as % of cancer cells that are Ki67 positive) around 32%. The median values were shown by red bars and explicitly written above the points. All results were from independent experiments n≥3, the bars of graphics showed means ± standard error of the mean (SEM). * = p<0.05, ** = p<0.001, *** = p<0.0001, ns = not-significant, using unpaired Student t-test, and values were normalized to co-cultured control conditions.

After eight days of co-culture, HBCx-66 PDX cells in the bone scaffolds were specifically identified by their epithelial marker EpCAM, while the Ki67 marker was used for proliferation characterization. We clearly observed more EpCAM and Ki67 positively stained HBCx-66 PDX cells in the biomimetic differentiated conditions than in control conditions (Fig. 4b). The EpCAM area and Ki67 index were thus calculated (Fig. SI2, see MM), we found an approximately 40% increase in the EpCAM-positive area (Fig. 4c) and more than a 110 % increase of the Ki67 index (Fig. 4d) for the differentiated conditions, compared to the control conditions. These results show that the HBCx-66 PDX cells performed better colonization, survival, and cycling proficiency in MSC osteogenic-differentiated conditions than in the bone scaffold that contained only undifferentiated MSC cells. In order to further assess the biological relevance of our bone-metastasis model, we investigated how this Ki67 index compares with an in vivo situation. Ki67 expression levels were quantified in a series of TNBC formalin-fixed paraffin-embedded (FFPE) samples for 62 TNBC patients. The median histopathology Ki67 index (indicating that 32% of cancer cells are Ki67 positive) (Fig. 4f) is similar to our biomimetic bone median Ki67 index (indicating that 37% of cancer-nuclear areas are Ki67 positive). This similarity suggests that our 3D-differentiated conditions actually provide a bona fide biomimetic microenvironment to maintain TNBC primary cells' cycling propensity in vitro.

TNBC PDX cells were also co-cultured with osteogenic-differentiated MSCs on a Petri dish for eight days and stained for EpCAM, Ki67, and nuclei. Unlike in the bone scaffolds, HBCx-66 PDX cells were rarely observed on Petri dishes, indicating that co-culturing with 2D-differentiated MSCs is insufficient for HBCx-66 PDX cell proliferation and colonization (Fig. SI6). The culture of cancer cells in Matrigel has been reported to better replicate some in vivo cancer-cell features, compared to 2D culture. However, after seven days of culture in the Matrigel, HBCx-66 PDX cells expressed EpCAM but only rarely were Ki67positive signals observed (Fig. 4e).

Altogether, these results suggest that our bone scaffolds containing differentiated MSCs provided more biomimetic microenvironment than 2D cell culture, with a substantial increase in HBCx-66 PDX cell survival and colonization and provided improved microenvironment conditions, compared to Matrigel, by maintaining the high biological fidelity of HBCx-66 PDX proliferation during in vitro culture.

Biomimetic bone niche maintains bone-metastatic cells colonization and cycling propensity.

In order to further demonstrate the potential applications of the biomimetic bone niche to develop bonemetastasis models, we used cells derived from PDX that had been established directly from bone metastasis biopsies. In particular, these PDX had been established with bone metastasis samples from estrogen receptor-positive breast cancer patients, whose cancer had progressed under endocrine treatment. Following the same procedure that was outlined above, two types of bone-metastasis breast cancer (BMBC) PDX cells (HBCx-124 PDX and HBCx-131 PDX) were seeded and cultured in the bone scaffold, with both control and differentiated conditions, as well as in Matrigel (Fig. 5a).

Fig.5 BMBC PDX cells represent the expression of Ki67 in biomimetic bone scaffold. (a)

Luminal B breast cancer tissue from bone metastatic site (BMBC) was sectioned into small pieces and implanted into female nude mice to initiate and propagate BMBC PDXs. After several passages, dissociated BMBC PDX cells were seeded into both the control and differentiated conditions and then co-cultured for 8 days. The dissociated BMBC PDX cells were also cultivated in Matrigel matrix for 7 days as a 3D in vitro control. (b, e) Two types of BMBC PDX cells (HBCx-124 PDXs and HBCx-131 PDX) were separately co-cultured in both control and differentiated conditions for 8 days followed by staining with EpCAM and Ki67. The reconstruction images of IF staining showed the 3D distribution of EpCAM area (magenta) and Ki67 (green) for both control and differentiated conditions. And corresponding quantification showed that the normalized EpCAM area was increased 31% and the Ki67 index was 2 times higher for HBCx-131 PDXs (c,d). Meanwhile, there was marginal increase of EpCAM area for HBCx-124 PDXs in differentiated conditions (f), but there was 1.5 times higher Ki67 compared to the control conditions (g). (h) Ki67 stained biopsy of HBCx-124 patient from the bone metastatic site of BMBC patients, and the statistical result from 11 BMBC patients showed a median Ki67 expression level around 21%. The median values are shown by red bars and explicitly written above the points. Results were from independent experiments n≥3, the bars of graphics showed means ± standard error of the mean (SEM). * = p<0.05, ** = p<0.001, *** = p<0.0001, ns = not-significant, using unpaired Student t-test, and values were normalized to control conditions.

Quantitative analysis of the HBCx-131 PDX cells grown in the 3D bone scaffold showed a 30% increase in the normalized EpCAM area and an increase of 105% for the Ki67 index in the differentiated conditions, compared to the control conditions (Fig. 5b,c,d). In the case of HBCx-124 PDX cells, we observed a marginal increase in the normalized EpCAM area and a 45% increase of the Ki67 index in differentiated conditions, compared with the control (Fig. 5e,f,g). The Ki67 expression level was also quantified for 11 BMBC patient samples, which retuned a median Ki67 expression level of around 21% relatively close to the values of BMBC PDX cells grown in our differentiated bone niche. These results suggest that, as previously shown for TNBC cells, the biomimetic differentiated conditions helped BMBC cells colonization and to maintain cells' cycling proficiency in vitro.

Additionally, both BMBC PDX cells were co-cultured with osteogenic-differentiated MSCs on Petri dishes, and rare PDX cells clusters survived for eight days (data not shown). Both BMBC PDX cells were also embedded in Matrigel, where EpCAM and nuclear signals were detected but no Ki67-positive cells (green) were observed (data not shown).

All these results suggest that, as with TNBC cells, our bone-like scaffolds provide a better biomimetic microenvironment that maintains the biological fidelity of BMBC cells during in vitro culture.

Modulated chemo-resistance to cis-platin of TNBC cells in biomimetic bone niche

Although our prior results are relevant to define adequate conditions for studying the biology of the bone metastasis of breast cancer, testing and predicting drug sensitivity or performing drug screening present other important applications.

Cisplatin is a common chemotherapy used for advanced TNBC patients with mitigated results underlining the need for refined preclinical evaluations. Cisplatin was applied to HBCx-66 PDX cells to measure the cells' dose-response as a function of the biomimetic microenvironments. The viability of TNBC PDX cells was first quantified with a series of diluted cisplatin concentrations in a 96-well plate. A IC50 of 2.4 µM, which characterized the inhibition of TNBC PDX cells by cisplatin, was calculated from the dose-response curve (Fig. SI3).

The drug responses of HBCx-66 PDX cells to cisplatin in biomimetic 3D scaffolds were then measured (Fig. 6a, SI7a) using the following method. First, dissociated HBCx-66 PDX cells were seeded and co-cultured with MSCs in the scaffolds, in both control and differentiated conditions, and in Matrigel without MSCs for two days. Then, various concentrations of cisplatin were added (from 2.4 µM to 10 µM) by replacing the medium followed by four days of incubation. normalized EpCAM area was also used to characterize the colonization capacity of HBCx-66 PDXs cells in Matrigel matrix (values were normalized to the condition in Matrigel matrix without drug treatment). Followed by EpCAM staining, normalized HBCx-66 PDXs EpCAM area showed a significant and continuous reduction with higher cisplatin concentration. (f) The viability of HBCx-66 PDXs cells in Matrigel was detected via Celltiter-Glo 3D assay with drug treatments (values were normalized to the condition in Matrigel matrix without drug treatment), and results showed that the decrease of HBCx-66 PDX cells viability was consistently with the decrease of HBCx-66 EpCAM area under the same cisplatin treatment. The median values are shown by red bars and explicitly written above the points. Results were from independent experiments n≥3, the bars of graphics showed means ± standard error of the mean (SEM). * = p<0.05, ** = p<0.001, *** = p<0.0001, ns = not-significant, using unpaired Student t-test.

As previously, the colonization and proliferation of HBCx-66 PDX cells in the scaffold were characterized by EpCAM and Ki67 staining in the presence or absence of cisplatin. In contrast to the results obtained in the 96-well plate, the normalized EpCAM area and Ki67 index of HBCx-66 PDX cells co-cultured in the 3D scaffold in differentiated conditions did not significantly decrease with 2.4 µM of cisplatin (Fig. 6b,c). A decrease of the EpCAM-positive area and a significant decrease of the Ki67 index were observed with 5 µM cisplatin treatment. By increasing cisplatin to 10 µM, the EpCAM-positive area decreased by around 50%, compared with the conditions without cisplatin, and the Ki67 index decreased from 34% to 16%.

Meanwhile, although HBCx-66 PDX cells also presented a modulated drug response in control conditions, this was to a lesser extent (Fig. SI7b,c), which could have been caused by secreted factors of the MSCs' exposure to cisplatin [START_REF] Engler | Matrix Elasticity Directs Stem Cell Lineage Specification[END_REF].

In accordance with the results obtained in the 96-well plate, the EpCAM area of HBCx-66 PDX cells in the Matrigel matrix was reduced to 50% upon 2.4 µM of cisplatin, and continuously decreased with further increased cisplatin concentrations (Fig. 6d,e). Correspondingly, the viability of HBCx-66 PDX cells in the Matrigel matrix with cisplatin treatment, as estimated by CellTiter-Glo, showed a consistent decrease with increasing cisplatin concentrations (Fig. 6f).

These data indicate that the HBCx-66 PDX cells grown in biomimetic bone models exhibited modulated drug sensitivity to cisplatin, as 50% of HBCx-66 PDX cells were inhibited with 10 µM (equal to 3,000 ng/mL) cisplatin treatment, compared with 2.4 µM (equal to 720 ng/mL) in the 96-well plate or the Matrigel matrix. Such results are consistent with the clinical observations, which demonstrates that chemotherapy often failed to completely eliminate TNBC cells colonizing the bone, as evidenced by the clinical disease progression [START_REF] Gobbini | Time trends of overall survival among metastatic breast cancer patients in the real-life ESME cohort[END_REF]. Furthermore, the results with 10 µM cisplatin in biomimetic bone models is not significantly different from the concentration of cisplatin (2000 ng/mL) in patients' steady-state serum [START_REF] Hellmann | Preliminary safety, pharmacokinetics, and efficacy of regorafenib, cisplatin, and pemetrexed in patients with advanced nonsquamous non-small-cell lung cancers[END_REF], which suggests that biomimetic bone-metastasis models might access more physiological drug response data in vitro. We could thus expect the sensitivity to drugs in our biomimetic bone model to adequately reflect in vivo sensitivity.

Discussion

To address the biology of the prevalent bone localization of many metastatic cancers, we need an adequate environment that replicates the in vivo environment conditions of metastatic cell growth and expansion. With the objective of bioengineering such an appropriate in vitro model of breast cancer bone metastasis, we first successfully established a biomimetic bone model that replicates some key features of an in vitro bone niche, particularly in terms of the native microstructures and stiffness of the trabecular bone and the presence of osteogenic-differentiated MSCs, a collagen I matrix, and mineralized calcium deposition. Compared with previously reported 3D bone models based on porous hydrogel and silk [START_REF] Reagan | Investigating osteogenic differentiation in multiple myeloma using a novel 3D bone marrow niche model[END_REF][START_REF] Qiao | Engineering 3D approaches to model the dynamic microenvironments of cancer bone metastasis[END_REF], our bone niche provides mechanical properties closer to the in vivo situation [START_REF] Olechnowicz | Contributions of the Host Microenvironment to Cancer-Induced Bone Disease[END_REF][START_REF] Mézière | Experimental observation of ultrasound fast and slow waves through threedimensional printed trabecular bone phantoms[END_REF][START_REF] Marks | Bone cell biology: The regulation of development, structure, and function in the skeleton[END_REF][START_REF] Lo | Cell Movement Is Guided by the Rigidity of the Substrate[END_REF]. The scaffold stiffness is expected to enhance the adhesion of cells to the microstructure [START_REF] Marks | Bone cell biology: The regulation of development, structure, and function in the skeleton[END_REF], while the spatial constraint allows cells to grow in a highly constrained space, as it does in bulk bone [START_REF] Marks | Bone cell biology: The regulation of development, structure, and function in the skeleton[END_REF][START_REF] Griffith | Capturing complex 3D tissue physiology in vitro[END_REF]. Our scaffold microfabrication presents several advantages, compared with decellularized bone scaffold [START_REF] Villasante | Bioengineered human tumor within a bone niche[END_REF] or fragmented mouse bones preloaded with breast cancer cells [START_REF] Wang | Bone-in-culture array as a platform to model earlystage bone metastases and discover anti-metastasis therapies[END_REF]. As demonstrated here with the trabecular bone [START_REF] Mézière | Experimental observation of ultrasound fast and slow waves through threedimensional printed trabecular bone phantoms[END_REF], our model can faithfully replicate the bone microstructure and, if required, the geometrical parameters of the scaffold can be easily varied thanks to the 3D printing versatility. Regarding the model's biological applications, using 3D-printed scaffold remains advantageous because it allows experiments to be conducted in controlled and similar conditions. The 3D printed resist has demonstrated its compatibility with in situ optical characterization of cells a major asset compared to existing technologies [START_REF] Bourgine | In vitro biomimetic engineering of a human hematopoietic niche with functional properties[END_REF] and its cytocompatibility has been shown suitable for cultivating and differentiating primary MSCs and PDX tumor cells. Even if this bone model does not fully replicate the real bone niche's complexity, it possesses the potential to address a wide range of biological issues related to complex cellbone interactions.

Different tumor cell types are supposed to reproduce bone metastasis, each of which has its own pros and cons. Since PDX cells better maintain the important biological properties of parental tumors [START_REF] Siolas | Patient-derived tumor xenografts: Transforming clinical samples into mouse models[END_REF], these cells were chosen to validate our in vitro model of bone metastasis. To the best of our knowledge, this is the first time that TNBC and BMBC PDX were co-cultivated in such biomimetic bone models to reconstruct triple-negative and bone metastatic tumors in the bone microenvironment. Our 3D biomimetic bone model remarkably enhanced both the colonization properties and cycling proficiency of the TNBC PDX and BMBC PDX cells, compared to a 2D or 3D Matrigel culture. It is noteworthy that the PDX cells' cycling proficiency (Ki67 index) maintained a similar level to that observed in patient samples.

All these features align with the assumption that the bone microenvironment can inhibit apoptosis and promote tumor-cell proliferation [START_REF] Mundy | Metastasis to bone: causes, consequences and therapeutic opportunities[END_REF][START_REF] Kingsley | Molecular Biology of Bone Metastasis[END_REF][START_REF] Calvi | Osteoblastic cells regulate the haematopoietic stem cell niche[END_REF][START_REF] Saidak | Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor[END_REF]. Furthermore, we observed that TNBC PDX cells in biomimetic bone scaffolds showed a reduced drug response, compared with a conventional culture, which aligns with the known resistance of bone metastasis under the same treatment [START_REF] Zheng | Therapeutic Antibody Targeting Tumorand Bone Metastasis to Chemotherapy[END_REF]. These results illustrate that critical components, such as the biomimetic tumor-cell co-culture in a biomimetic environment, could affect the bioavailability and/or efficacy of cisplatin [START_REF] Griffith | Capturing complex 3D tissue physiology in vitro[END_REF][START_REF] Roodhart | Mesenchymal Stem Cells Induce Resistance to Chemotherapy through the Release of Platinum-Induced Fatty Acids[END_REF]. Since cisplatin presented a strongly repeatable and modulated drug response in our biomimetic bone model, different chemotherapy drugs for metastatic breast tumors could be further tested.

Finally, the next step for this research could be to directly seed bone metastatic cells into our bone model after biopsy, with no PDX stage, in order to get closer to clinics. Moreover, as it has recently been reported that the metastatic bone microenvironment could modulate cancer's response to immunotherapy [START_REF] Jiao | Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy[END_REF], our biomimetic bone model could also be used in the presence of immune cells to investigate how the bone microenvironment could modulate the tumor's response to immunotherapy.

Fig. 2

 2 Fig.2 MSCs proliferation and colonization in 3D printed bone scaffold. (a) Primary MSCs were seeded in 3D printed bone scaffolds at "0" week. At 1 st , 2 nd , and 3 rd week, MSCs were freshly stained with Cell Trace TM yellow respectively, and the living cell signals were collected by using the multi-field acquisition model of a confocal microscope. (b) Over-view of the Cell Trace TM stained MSCs (red) in the bone scaffold (gray) which were merged by 44 fields and reconstructed via image stacks (see materials and methods). Detailed MSCs distribution (in yellow box) during three weeks culture was shown in Movie SI1-3. (c) The proliferation rates of MSCs were validated as fold change of total whole-depth Cell Trace TM signal during three-week culture (see material and methods). (d) Normalized Cell Trace TM intensities (see material and methods) distribution along Z-axis (0-1200 μm) during 3D culture. The Cell Trace TM intensities at each depth position (averaged intensity of five adjacent frames from 0 to 1200 μm) were normalized to the Zdivided basal Cell Trace TM intensity and plotted along Z-axis. All results were obtained from independent experiments n≥3, the bars of graphics showed means ± standard error of the mean (SEM). * = p<0.05, **

Fig. 3

 3 Fig.3 MSCs differentiation, collagen matrix deposition, and mineralization recapitulate a biomimetic bone. (a) Procedures of MSCs differentiation in 3D printed bone scaffold. For the differentiated conditions, primary MSCs were seeded and cultured in a bone scaffold at "0" week, followed by adding Dex-diff/BMP-diff medium at the end of first/second week separately for osteogenic differentiation. MSCs cultured with basic culture medium in a bone scaffold were considered as control conditions. (b-f) IF characterization of MSCs by staining with osteogenic markers (osteocalcin (b) and collagen I (d)) in the differentiated and control conditions. Reconstructed images of IF staining showed increased quantity of osteocalcin (b) and collagen I deposition (d) in the differentiated conditions compared to that in the control conditions. Correspondingly, normalized osteocalcin index (osteocalcin intensity per cell, see material and methods) and collagen I index (collagen deposition per cell, see material and methods) distribution along Z-axis (total 0-1000μm) were quantified (see material and methods). Quantification showed distribution of MSCs osteocalcin index (c) and collagen I index (e) in 1000μm depth in the control and differentiated conditions. (f) Nuclei area was measured for both the differentiated and control conditions, and it showed that there was no significantly difference between them. (g) Bone related genes expression was measured via RT-qPCR after MSCs 3D differentiation. mRNA levels of bone sialoprotein

Fig. 6

 6 Fig.6 Biomimetic bone scaffolds promote chemo-resistance of TNBC PDX cells. (a) Drug response test of HBCx-66 PDX cells in bone scaffolds and Matrigel matrix. HBCx-66 PDX cells were seeded into both control (see Fig.SI6) and differentiated conditions and Matrigel matrix as previously described and cultivated 2 days followed by treatments with 2.4 μM (in vitro IC50), 5.0 μM, and 10.0 μM of cisplatin, separately, for 4 days. (b,c) normalized EpCAM area and Ki67 index were used to characterize the colonization and proliferation capacity of HBCx-66 PDX cells with and without cisplatin treatment in differentiated conditions (values were normalized to control conditions without drug treatment), respectively. And, more than 75 fields were measured and plotted in each condition. (d) Cisplatin treatment performed

Acknowledgments

This work was supported by the French RENATECH network. It was supported as part of the MultiFAB project funded by FEDER European Regional Funds and French Région Occitanie (grant agreement number 16007407/MP0011594) and by the HoliFAB project funded by the European Union's Horizon 2020 research and innovation program (grant agreement No 760927). This work has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d'excellence, Equipex, "Investissements d'avenir" program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX-31-34). This work has been initiated and funded by Institut Curie PIC3I METABONE.


Jacqui H Todd
email: jacqui.todd@plantandfood.co.nz

Robert M Simpson

Joanne Poulton

Emma I Barraclough

Kurt Villsen

Amber Brooks

Kate Richards

Dan Jones

Detecting invertebrate ecosystem service providers in orchards: traditional methods versus barcoding of environmental DNA in soil

Keywords: Detecting invertebrate ecosystem service providers species-specific primers, metabarcoding, environmental DNA, decomposition, natural enemies

come

Introduction

Invertebrate ecosystem service providers (IESP) are integral to the sustainable management of agro-ecosystems [START_REF] Saunders | Ecosystem services in agriculture: understanding the multifunctional role of invertebrates[END_REF]. The services provided by invertebrates include pollination, pest suppression and decomposition [START_REF] Beynon | The application of an ecosystem services framework to estimate the economic value of dung beetles to the UK cattle industry[END_REF][START_REF] Cross | Arthropod ecosystem services in apple orchards and their economic benefits[END_REF][START_REF] Minarro | Impact of insect pollinators in agriculture: importance and management of their biodiversity[END_REF], and are estimated to be worth billions of dollars per year to land managers worldwide [START_REF] Losey | The economic value of ecological services provided by insects[END_REF][START_REF] Sandhu | The future of farming: The value of ecosystem services in conventional and organic arable land. An experimental approach[END_REF]. However, management practices, such as the application of agrichemicals, can interrupt services through negative effects on IESP populations [START_REF] Atwood | Evidence for multitrophic effects of pesticide seed treatments on non targeted soil fauna[END_REF][START_REF] Chagnon | Risks of large-scale use of systemic insecticides to ecosystem functioning and services[END_REF], potentially resulting in increased production costs (e.g., through needing to control secondary pest outbreaks: [START_REF] Gallardo | Capturing the economic value of biological control in western tree fruit[END_REF]. Employing mitigation techniques to restore or protect populations and services (e.g., by adding protective shelters, alley-cropping, or ground-covers to increase populations of natural enemies and decomposers: [START_REF] Ashraf | Alley-cropping system can boost arthropod biodiversity and ecosystem functions in oil palm plantations[END_REF][START_REF] Horton | Diversity and phenology of predatory arthropods overwintering in cardboard bands placed in pear and apple orchards of central Washington state[END_REF][START_REF] Shields | Potential ecosystem service delivery by endemic plants in New Zealand vineyards: successes and prospects[END_REF] would consequently be highly beneficial. However, the invertebrate species providing the services often remain unidentified and unmonitored, at least partially because current invertebrate monitoring methods are slow and time consuming. For example, it may take many months to morphologically identify all individual invertebrates collected in a few traps placed in an orchard for a single week [START_REF] Todd | Invertebrate community richness in New Zealand kiwifruit orchards under organic or integrated pest management[END_REF]. Interruptions to services are, therefore, usually discovered too late (e.g., when the secondary pest outbreak occurs) and land managers are required to implement emergency measures, such as applying additional agrichemicals, rather than mitigation techniques.

Barcoding and metabarcoding of environmental DNA (eDNA) in soil samples (e.g., [START_REF] Decaens | DNA barcoding reveals diversity patterns of earthworm communities in remote tropical forests of French Guiana[END_REF][START_REF] Taberlet | Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies[END_REF] can produce information on invertebrate populations more quickly, and without removing viable individuals from the system, compared with traditional monitoring methods [START_REF] Oliverio | A DNA metabarcoding approach to characterize soil arthropod communities[END_REF][START_REF] Yang | Using metabarcoding to ask if easily collected soil and leaf-litter samples can be used as a general biodiversity indicator[END_REF]. This technology could be used, therefore, to detect changes in IESP populations in time for land managers to employ mitigation techniques to restore or protect those populations. This hypothesis is based on work that has shown that invertebrates contribute free DNA molecules in the form of secretions, eggs, faeces and decomposing bodies to the environment, and that this eDNA is detectable in soil [START_REF] Bohmann | Environmental DNA for wildlife biology and biodiversity monitoring[END_REF]. In water samples, these molecules are harder to detect when the species' population is small, and easier if the population increases [START_REF] Bohmann | Environmental DNA for wildlife biology and biodiversity monitoring[END_REF]. If this is also the case for soil samples, then it may be possible to use changes in the detectability of IESP populations to warn land managers of potential changes in ecosystem services provided by those populations. However, since it is not possible to extract DNA from all the soil in an agro-ecosystem, subsamples must be taken, and these may not contain DNA from all taxa present in that ecosystem. This subsampling error plus the differential deterioration of DNA from different sources, the influence of capture and extraction protocols on DNA yield, and the tendency of PCR primers to bind to some sequences more readily than others, may mean that some species may not be detected even when they are abundant in the environment [START_REF] Deiner | Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA[END_REF][START_REF] Deiner | Environmental DNA metabarcoding: Transforming how we survey animal and plant communities[END_REF]. Thus, comparing the results of barcoding and traditional sampling methods [START_REF] Deiner | Environmental DNA metabarcoding: Transforming how we survey animal and plant communities[END_REF] is a useful first step for testing this method as a tool for monitoring IESP populations.

Previous studies have identified a number of IESP in apple and kiwifruit orchards in New Zealand and the management practices that may affect their populations (Malone et al., 2017b;[START_REF] Todd | Relationships between management practices and ground-active invertebrate biodiversity in New Zealand kiwifruit orchards[END_REF]. The aims for this study were to: (1) develop specific primers for 26 IESP found in New Zealand kiwifruit and/or apple orchards; (2) test the ability of those specific primers to detect the IESP in soil samples to which the IESP had been added; (3) test the ability of generic primers for the mitochondrial cyctochrome c oxidase gene subunit I (COI) to detect the IESP in soil samples to which the IESP had been added; (4) compare the ability of traditional invertebrate trapping methods and metabarcoding of eDNA in soil to detect the IESP in orchards; and (5) detect any differences in IESP populations in relation to orchard management systems.

Methods

Development of IESP-specific primers

Focal IESP were selected from lists of taxa previously collected in New Zealand apple and kiwifruit orchards (Malone et al., 2017a;[START_REF] Todd | Invertebrate community richness in New Zealand kiwifruit orchards under organic or integrated pest management[END_REF]. The 26 selected taxa were either natural enemies of orchard pests or involved in decomposition processes (Table 1). Most of the IESPs primarily occur on or under the soil surface, with seven taxa that spend very little time in these habitats also included (Table 1). Specimens of each IESP were collected and identified using morphological taxonomic keys (e.g., [START_REF] Berry | Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae: Euphorinae), a new record for New Zealand[END_REF][START_REF] Eyles | Revision of New Zealand Bryocorinae and Phylinae (Insecta: Hemiptera: Miridae)[END_REF][START_REF] Herman | Phylogeny and reclassification of the genera of the rove-beetle subfamily Oxytelinae of the world (Coleoptera: Staphylinidae)[END_REF]. DNA was extracted from these specimens using the prepGEM® insect kit (ZyGem, Southampton, UK) following the manufacturer's instructions. COI and/or 28S ribosomal DNA (28S rDNA) sequences were amplified from these extracts by PCR using KAPA2G Robust (Kapa Biosystems, Wilmington, MA, USA) with buffer A. The PCR cycle used was 94°C for 5 minutes, 40 cycles of 94°C for 30 seconds, 44°C (COI) or 49°C (28S rDNA) for 30 seconds, and 72°C for 45 seconds, with a final extension phase of 72°C for 10 minutes. The primers used for COI PCRs were LCOI490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], hereafter referred to as "Folmer primers". For 28S rDNA PCRs, primers 500F (5′-CTTTGAAGAGAGAGTTCAAGAG-3′) and 501R (5′-TCGGAAGGAACCAGCTACTA-3′) [START_REF] Nadler | Molecular and morphometric evidence for separate species of Uncinaria (Nematoda: Ancylostomatidae) in California sea lions and northern fur seals: Hypothesis testing supplants verification[END_REF], targeting the D2/D3 region, were used. PCR amplicons were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA), and Sanger sequenced in both directions. Primer design and genetic data manipulation were performed using Geneious R10.0.3 (https://www.geneious.com). Specific primers for each IESP (Table 1) were designed from the COI and/or 28S rDNA sequences with specificity checked using National Center for Biotechnology Information (NCBI) primer-BLAST [START_REF] Ye | Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction[END_REF]. The target parameters for primers were 40-60% GT, Tm greater than 60°C but within 5°C for a pair of primers, and a product size between 100 and 200 bp, although it was not possible to achieve all target parameters for all primer pairs. Primer specificity was checked for cross-reactivity against IESP extracts from within the same invertebrate order and from at least one other order (Table 1) using the PCR conditions described above apart from the annealing temperatures which are given in Table 1.

Detecting IESP in soil samples "augmented" with IESP DNA

Approximately 1 L of soil was collected from eight kiwifruit orchards (Bay of Plenty, New Zealand) and eight apple orchards (Hawke's Bay, New Zealand), in September 2016 (Figure 1). Soil was collected haphazardly from within a 500 m 2 area in each orchard, using multiple soil cores 8 cm in diameter and 2 cm deep, bulked to form one sample (in a 1 L beaker) per orchard and frozen at -20°C. In May 2017 each sample was defrosted, sieved to 2 mm, and divided in half. To test the ability of the primers to detect the IESP in the presence and absence of other DNA, one half of each sample was sterilised through receiving a total exposure of 73-74 kGy gamma radiation at the MSD Animal Health Laboratory in Wellington, New Zealand (www.msd-animal-health.co.nz). The pH of the samples ranged from 5.1 to 6.6, the acidity of which is likely to promote the binding of extracellular DNA to the soil surface [START_REF] Young | Limitations and recommendations for successful DNA extraction from forensic soil samples: a review[END_REF]. Consequently, each sterilised sample was inoculated with 50 g of potting mix. We hypothesised that the potting mix was likely to contain bacterial DNA but very little invertebrate DNA because the amount of time the mix had been sealed in its bag allowed for bacterial degradation of extant invertebrate DNA: the bacterial DNA would be available to bind to the soil during DNA extraction and, thus, reduce the loss of the IESP DNA through surface absorption during extraction.

The sterilised and unsterilised halves of each sample were further divided into five subsamples (average weight of 50 g, range 30-70 g) to which were added known weights of up to six IESP to produce the "augmented" soil samples (Table 2; Figure 1). Each IESP was added on its own to at least one sterilised subsample, with the 20 IESP found in apple orchards added only to apple soil, and the 20 IESP found in kiwifruit orchards added only to kiwifruit soil. The IESP specimens that were added to the soil had been collected during the previous 6 months and stored in 95% ethanol before being morphologically identified. This storage medium has been shown to result in high DNA yield from insects [START_REF] Moreau | DNA preservation: a test of commonly used preservatives for insects[END_REF]. Weighed IESP (or fragments thereof) were ground in liquid nitrogen, mixed thoroughly into the appropriate soil subsample, and stored at -20°C for later DNA extraction.

Equipment that was specific to each IESP was used for handling, grinding and mixing to avoid cross-contamination.

DNA was extracted from a 10 g aliquot of each "augmented" soil subsample using DNeasy® PowerMax® Soil kit (Qiagen, Hilden) following the manufacturer's instructions, except that disintegration was 10 minutes at 1250 Hz in a Genogrinder (SPEX SamplePrep, Metucen, NJ, USA). Extracted DNA was then treated using DNA Clean and Concentrator™ (Zymo Research, Tustin, CA, USA). Amplification of the DNA was performed twice. Firstly, the specific IESP primers were used under PCR conditions described above. These primers were only screened against soils to which the relevant IESP had been added to ensure the primers could detect the target and to assess the likelihood of detecting false negatives. Secondly, the Folmer primers were used under PCR conditions described above, except that Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA, USA) was used, and the products pooled from three to five PCRs. Folmer primers were chosen for metabarcoding because the COI gene is the standard barcode for invertebrates, and has been shown to produce beta diversities from eDNA samples that are strongly correlated with those from traditional biodiversity measures [START_REF] Drummond | Evaluating a multigene environmental DNA approach for biodiversity assessment[END_REF]. In addition, the target sequence is long (710-bp;[START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], potentially enabling us to only detect DNA that had been deposited recently (i.e., by current IESP populations) and had not had time to degrade. PCR products amplified using the Folmer primers from each of the five sterilised and five unsterilised subsamples were then recombined for sequencing, resulting in one sterilised and one unsterilised sample per orchard.

The 32 samples produced using Folmer primers were sent to the Australian Genome Research Facility (AGRF, www.agrf.org.au) where barcoded Nextera transposon libraries were generated and the resulting libraries sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, USA) generating 300 bp paired end fragments (V3 chemistry).

Sequences obtained from AGRF were assessed for quality using Fast QC v1.91 and analysed using the Qiime2 v2018.2 workflow (Bolyen et al., 2018). Briefly, samples were errorcorrected and assigned to Amplicon Sequence Variants (ASVs) using DADA2 [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF], the phylogeny of ASVs and alpha and beta diversity of samples was assessed, and ASVs were assigned to a taxonomic group. Taxonomic assignment was conducted within Qiime2 using the scikit-learn Python library (https://scikit-learn.org/stable/index.html), using custom sequence databases. Custom databases were constructed from COI sequences for each IESP (either obtained during this project or from Genbank) as well as a customised library of almost 2000 COI sequences for New Zealand invertebrates (e.g., from [START_REF] Drummond | Evaluating a multigene environmental DNA approach for biodiversity assessment[END_REF] and other closely related invertebrate taxa available on Genbank. Taxonomy assignment was performed using a p-confidence threshold of 0.7 [START_REF] Wang | Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[END_REF].

Comparing traditional and metabarcoding methods for detecting IESP in orchards

Ten soil cores (8 cm diameter × 2 cm deep) were collected haphazardly from within a 100 m 2 area in each of ten kiwifruit orchards (Bay of Plenty, New Zealand) and ten apple orchards (Hawke's Bay, New Zealand), during February and March 2017 (Figure 1). Five of the kiwifruit orchards (K1-K5) were managed using an integrated pest management system (IPM), and the remaining five (K6-K10) were under organic management, whereas five of the apple orchards (A1-A5) were managed using an integrated fruit production system (IFP), with five (A6-A10) under organic management.

The ten soil cores were combined into a single sample per orchard, sieved to 2 mm, and stored at -20°C for later DNA extraction. DNA was initially extracted from two 10 g aliquots from each sample, but if the DNA quantity seemed low (i.e., below 10 ng µL -1 ), a further two aliquots were extracted. DNA extracts for each orchard were combined and treated using DNA Clean and Concentrator TM (Zymo Research, Irvine, CA, USA). PCRs with the Folmer primers were conducted as described above, and PCR products (one sample per orchard) were sent to AGRF for sequencing. The resulting sequences were analysed for presence of the focal IESP sequences using the Qiime2 v2018.6 workflow as described above. Full details of the bioinformatics workflows can be viewed on request at https://github.com/PlantandFoodResearch/bioinf-eDNA-ESP.

To compare the efficiency of the metabarcoding methodology with traditional methods of invertebrate sampling, five yellow pan traps, five flight-intercept traps, five pitfall traps, and five yellow sticky traps were placed into the same 100 m 2 area immediately following the collection of the soil samples from each orchard. Traps were deployed for 6 days. These traps were selected based on the results of previous surveys of invertebrate taxa in apple and kiwifruit orchards that showed this combination of traps was the most likely to collect all of the focal IESP if they were present (Malone et al., 2017a;[START_REF] Todd | Invertebrate community richness in New Zealand kiwifruit orchards under organic or integrated pest management[END_REF]. Invertebrates collected in the pan and intercept traps were transferred into containers containing 95% ethanol, pitfall traps contained monoethylene glycol to preserve captured invertebrates, and sticky traps were stored at -20°C until captured invertebrates could be identified.

Conventional morphological identification methods were used to determine the abundance of each of the focal IESP in each sample.

Statistical Analysis

Statistical analyses were carried out using R version 3.5.1 (R Development Core Team, 2018). For the "DNA-augmented" soil samples, the analysis investigated the effect of IESP identity and weight added to the soil sample, and their interaction, on the ability of the Folmer primers to detect each IESP. Binomial generalised linear models with a logit link function using the package mvabund [START_REF] Wang | mvabund: Statistical Methods for Analysing Multivariate Abundance Data[END_REF] were selected for this investigation.

For the samples collected in traps from the ten apple and ten kiwifruit orchards, Poisson generalised linear mixed models were used to investigate the effects of orchard management (IFP or organic in apple; IPM or organic in kiwifruit) on the abundance of each IESP. Means and 95% confidence intervals were obtained with least square means, and post hoc pairwise comparisons were carried out using the Tukey test.

Results

Development of IESP-specific primers

COI and/or 28S rDNA sequences were produced for each of the 26 IESP selected for this study (COI GenBank MK736030-47, 28S GenBank MK748223-40), and specific primers for each taxon were successfully developed from these sequences (Table 1). It was not possible to obtain COI sequences for Conoderus exsul (Sharp), Anthomyia punctipennis Weidemann and Akamptogonus novarae (Humbert & Saussure), and COI sequences for several of the other IESP were difficult to obtain with the Folmer primers. Consequently, 28S rDNA sequences and primers for these sequences were developed for several IESP (Table 1).

There was no correlation between the taxonomic identity of the IESP and the ease of obtaining a barcode for that IESP.

Detecting IESP in soil samples "augmented" with IESP DNA

The IESP-specific primers detected the IESP in 96% of the sterilised and 100% of the unsterilised soil subsamples to which each IESP was added (Table 3), with primers for COI and 28S working equally well. The only species the specific primers failed to detect was Ausejanus albisignatus (Knight), which was only added to a single sterile soil subsample because of a lack of specimens. The detection rates for the other taxon-specific primers were greater than 90%, except for those for A. punctipennis at 75%.

The sequencing of the PCR products from the combined sterilised soil samples (i.e., one sample per orchard) using the Folmer primers resulted in an average of 1233 ASVs (range 879 to 1538) per sample. For the combined unsterilised samples, an average of 1313 ASVs (range 292 to 2675) were produced. Matching these to the sequences for the IESP that had been added to the sterilised and unsterilised samples resulted in very few detections. Only 33% of the IESP that had been added to the sterilised soil samples were detected, and none of the added IESP were detected in the unsterilised samples (Table 3). In the sterilised samples, 13 IESP were not detected at all, and for the remaining 13 IESP, detection rates ranged from 7%, for Arcitalitrus spp., to 100% for Lonchoptera bifurcata (Fallen) and Tetramorium grassii Emery. In addition, the Folmer primers detected Trigonospila brevifacies (Hardy) in a sterilised sample to which it had not been added. This may indicate that the sterilisation procedure was not completely effective at removing all DNA from the soil. Further analysis detected an interaction effect between the identity of the IESP and the weight of the IESP added to the sterilised soil on the detection of the IESP using the Folmer primers in both the apple (Ptaxa:weight = 0.03) and kiwifruit (Ptaxa:weight = 0.05) orchards. Consequently, there does not appear to be a direct relationship between the detectability of each IESP and the amount of DNA in the soil.

Comparing traditional and metabarcoding methods for detecting IESP in orchards

Analysis of the sequences produced from the soil samples from each orchard (following removal of sequences with fewer than 10 reads) identified a total of 34,679 ASVs. Of those, 13,303 ASVs were found only in the kiwifruit orchards, and 19,443 ASVs were found only in apple orchards, leaving only 1,933 ASVs in common between the two orchard types.

Individual orchards contained 128-5,150 ASVs. Very few of the ASVs could be matched to the sequences for the focal IESP. Only three IESP were detected: T. brevifacies was detected in orchards A3, A6 and K8; Nylanderia sp(p). was detected in orchards A2 and A6; Armadillidium vulgare (Latreille) was detected in orchard A6. Additionally, some of the ASVs matched one other species in the customised COI library: Carpophilus davidsoni Dobson (a beetle that was not included in the list of IESP) was detected in orchard A4. Some of the remaining ASVs were similar enough to be classified with a group of dipteran sequences or Arthropoda sequences, but the majority were unassigned.

The morphological analysis of the invertebrates collected in the pan, intercept, pitfall and sticky traps revealed the presence of all the focal IESP in at least one orchard, and a range of 8 to 17 of the 20 IESP found in each orchard (Figure 2). This contrasts starkly with the metabarcoding results described above that found very few IESP in the soil collected from the same location within the orchards. The three IESP that were detected in the soil (i.e., T. brevifacies, Nylanderia sp(p)., and A. vulgare) were also collected in traps from the same orchards, except for orchards A3 and A6 where T. brevifacies was detected in the soil but not collected in traps.

The abundances of the IESP varied between orchards, with some species found occasionally (e.g., the predatory beetle Thyreocephalus orthodoxus (Olliff) was found in one apple orchard and five kiwifruit orchards, with a maximum of seven individuals collected from one kiwifruit orchard) and others found relatively frequently in all orchards (e.g., between 5 and 209 Sericoderus sp. beetles were collected from each of the 20 orchards) (Figure 2). The Poisson models indicated that that abundances of most of the IESP in the apple orchards were not affected by orchard management, with equal numbers collected from the IFP and organic orchards (Table 4). However, four IESP (natural enemies Aphelinus mali (Haldeman), Nylanderia sp(p)., Platygaster demades Walker, and detritivore Sericoderus sp.) were in greater abundance in IFP orchards than in organic orchards, and four other IESP (natural enemy A. albisignatus and detritivores A. vulgare, Cartodere spp. and Ephistemus globulus (Paykull)) were captured in greater numbers from the organic orchards (Table 4). In the kiwifruit orchards, a difference in abundance between the organic and IPM orchards was detected for 12 of the IESP (Table 5), with nine IESP in greater abundance in the organic orchards (natural enemies Anoteropsis hilaris (L. Koch), C. exsul, Phalangium opilio L., T. orthodoxus, and detritivores Arcitalitrus spp., Atomaria lewisi Reitter, Anotylus sp., L. bifurcata, Sericoderus sp.,), and three in greater abundance in the IPM orchards (natural enemies Micromus tasmaniae (Walker), Monomorium antarcticum (F. Smith), and T. brevifacies).

Discussion

The results of this study show that the development of specific primers for IESP may be a useful way to monitor these beneficial invertebrates using eDNA in soil samples. Detection probabilities for the primers developed for most of the 26 focal taxa were greater than 90% in sterilised soil samples, and 100% in unsterilised samples to which the DNA of the taxa had been added. These taxon-specific primers could be used to monitor these IESP in future studies, and to potentially detect changes in the ecosystem services they provide, without needing to remove viable individuals from the system. Use of these primers is reasonably inexpensive, especially when compared with the cost of metabarcoding, and PCR results give an immediate result regarding the presence (or absence) of the IESP. The next step will be to test these primers with soil samples taken directly from orchards.

In contrast, the sequences produced using the Folmer primers did not match the sequences of most of the IESP, even in the sterilised soil samples. This may have resulted from preferential amplification of other DNA in the samples by these primers, or because the PCR conditions were not favourable for amplifying the IESP DNA. Whatever the reason, these results suggest that the Folmer primers are not appropriate for monitoring these IESP in orchard soil samples. In addition, the finding that it was difficult, if not impossible, to obtain COI sequences from the IESP DNA extracts using the Folmer primers also indicates that these primers are not adequate for detecting these IESP. There are a number of other primers that may be better alternatives. While the COI gene is the traditional barcode sequence for invertebrates, recent studies have shown that ribosomal 18S [START_REF] Horton | Suitability of PCR primers for characterizing invertebrate communities from soil and leaf litter targeting metazoan 18S ribosomal or cytochrome oxidase I (COI) genes[END_REF] or 16S rDNA [START_REF] Clarke | Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias[END_REF] genes may be more reliable sequences for detecting invertebrates.

Even with the COI barcode, the best primers for detecting different taxa can vary because of sequence mismatches in the target annealing position [START_REF] Geller | Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys[END_REF]. Consequently, primers that are better able to detect the COI sequences for New Zealand's terrestrial invertebrates, potentially those developed by [START_REF] Geller | Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys[END_REF]Rennstam Rubbmark et al. (2018), are needed.

The interaction effect of IESP identity and the weight of the IESP added to the soil samples on the detectability of those taxa indicate that increasing the amount of DNA present in the soil does not increase detectability for all taxa. This is consistent with other studies that have shown that the Folmer primers have sequence biases [START_REF] Clarke | Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias[END_REF][START_REF] Pinol | Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods[END_REF], and are, therefore, more likely to detect some taxa than others. This is backed up by the finding that the IESP that were detected in the soil collected from the ten apple and ten kiwifruit orchards (i.e., T. brevifacies, Nylanderia sp(p). and A. vulgare) were not the most abundant IESP collected in the traps in the orchards in which they were detected. The detection of T. brevifacies in the soil of three orchards using the Folmer primers does at least indicate that metabarcoding of eDNA in soil can be used to detect taxa that are present but that do not live primarily in the soil or on the soil surface. Trigonospila brevifacies is a tachinid parasitoid of Lepidoptera and, therefore, in the larval stage occurs within lepidopteran hosts that feed on plant material, and the adult stage disperses through flight [START_REF] Munro | A retrospective analysis of the establishment and dispersal of the introduced Australian parasitoids Xanthopimpla rhopaloceros (Krieger) (Hymenoptera : Ichneumonidae) and Trigonospila brevifacies (Hardy) (Diptera : Tachinidae) within New Zealand[END_REF]. Thus, if more consistent primers can be produced for metabarcoding of invertebrate eDNA in soil samples, then it may be possible to use this method to monitor both ground-dwelling and plant-dwelling taxa. Finally, differences in the abundances of IESP in orchards with different management systems was not unexpected given the results of earlier surveys of the invertebrate communities in apple and kiwifruit orchards (Malone et al., 2017a;[START_REF] Todd | Invertebrate community richness in New Zealand kiwifruit orchards under organic or integrated pest management[END_REF]. For instance, greater abundances of A. albisignatus, A. vulgare and E. globulus in organic apple orchards, and greater abundances of A. mali and Nylanderia spp. in the IFP apple orchards were found in both this study and that by Malone et al. (2017a). In the kiwifruit orchards, nine IESP (four natural enemies and five detritivores) were collected in greater abundances from the organic orchards, whereas three natural enemies were collected in greater abundances from the IPM orchards. This adds to the finding of greater taxonomic richness in the organic orchards by [START_REF] Todd | Invertebrate community richness in New Zealand kiwifruit orchards under organic or integrated pest management[END_REF], although in that study there was no indication of differences in detritivore communities between the two orchard types. Further work is needed to determine if these differences translate into functional differences in the ecosystem services provided by these taxa. Initial investigations suggest that the difference in natural enemy taxa between organic and IPM kiwifruit orchards does not translate into a difference in parasitism rates of leafroller pests [START_REF] Todd | Effect of orchard management, neighbouring land-use and shelterbelt tree composition on the parasitism of pest leafroller (Lepidoptera: Tortricidae) larvae in kiwifruit orchard shelterbelts[END_REF].

Table 1: Invertebrate ecosystem service providers (IESP) selected for this project. Species were selected because they were involved in either decomposition (decomp.) or pest suppression (pest sup.) on apple or kiwifruit orchards or both. At least ten IESP were selected from those that spend most of their life cycle in soil and/or leaf litter (ground), and at least five were selected from those that occur primarily above ground (on plants). Primers were designed against sequences generated in this study for either 28S ribosomal DNA (28S rDNA) or mitochondrial cytochrome c oxidase gene subunit 1 (COI), except those for Forficula auricularia where a Genbank sequence was used. management; A1 -A5 under an integrated fruit production system; and the remaining kiwifruit and apple orchards were under organic management. IESP were collected from each orchard using pan, intercept, pitfall and sticky traps. Note that some of the IESP were specific to either apple or kiwifruit orchards (see Table 1 for more details).

IESP

Figure 1 :

 1 Figure 1: Sample collection and processing methods used in this study. IESP = invertebrate ecosystem service providers.

Figure 2 :

 2 Figure 2: Relative abundance of invertebrate ecosystem service providers (IESP) in (a) ten apple orchards and (b) ten kiwifruit orchards. Orchards K1 -K5 were under integrated pest

Table 2 :

 2 Quantity of invertebrate ecosystem service providers (IESP), or parts thereof, added to sterilised and unsterilised soil samples.

	Sterilised	No. of	Target	Actual weight	Unsterilised	No. of	Target	Actual weight
	subsample	IESP	weight	of each IESP	subsample	IESP	weight	of each IESP
	no.	added	(g)	added to soil	no.	added	(g)	added to soil
	1	1	0.1	0.059*-0.13	1	1	0.1	0.101-0.113
	2	1	0.01	0.006*-0.013	2	1	0.01	0.008*-0.013
	3	3	0.033	0.033-0.049	3	1	0.033	0.033-0.037
	4	5 #	0.02	0.020-0.034	4	1	0.02	0.020-0.026
	5	5	0.002	0.002-0.004	5	1	0.002	0.002-0.003

*Maximum weight available for one of the IESPs added to a subsample. # Six IESP were accidentally added to a subsample from one orchard, but each was added to the subsample at approximately 0.02 g.

Table 3 :

 3 Detection of invertebrate ecosystem service providers (IESP) added to sterilised 594 (Ster.) and unsterilised (Unst.) soil subsamples obtained from eight apple and eight kiwifruit

	595
	orchards.

Table 4 :

 4 Comparison of invertebrate ecosystem service provider (IESP) abundances in ten apple orchards under two different management systems: five organic and five integrated fruit production (IFP) orchards were sampled. Mean abundances and 95% confidence intervals (CI) have been back-transformed. Each IESP was modelled separately.

				Lower	Upper	Letter of
		Orchard		95%	95%	difference
	IESP	System	Mean	CI	CI	(alpha=0.05) z.ratio p.value
	Arcitalitrus spp.	IFP	0	-	-	-	0	1
		Organic	0	-	-	-
	Anoteropsis hilaris	IFP	1.6	0.8	3.2	-	0.005	0.9963
		Organic	0	-	-	-
	Sericoderus sp.	IFP	86.2 78.43 94.73	A	10.79	<.0001
		Organic	31.6 27.04 36.93	B
	Atomaria lewisi	IFP	0.6	0.19	1.86	A	-0.377 0.7064
		Organic	0.8	0.3	2.13	A
	Ephistemus globulus	IFP	0.2	0.03	1.42	A	-2.975 0.0029
		Organic	4.2	2.74	6.44	B
	Conoderus exsul	IFP	6.8	4.86	9.52	A	1.883	0.0597
		Organic	4	2.58	6.2	A
	Cartodere spp.	IFP	15 11.96 18.81	A	-2.158 0.0309
		Organic	20.8 17.16 25.21	B
	Thyreocephalus	IFP	0	-	-	-	-0.003 0.9979
	orthodoxus	Organic	0.2	0.03	1.42	-
	Forficula auricularia	IFP	0	-	-	-	-0.003 0.9978
		Organic	0.4	0.1	1.6	-
	Anthomyia	IFP	1.2	0.54	2.67	A	-0.989 0.3226
	punctipennis	Organic	2	1.08	3.72	A
	Lonchoptera bifurcata IFP	1.8	0.94	3.46	A	1.924	0.0543
		Organic	0.4	0.1	1.6	A
	Trigonospila	IFP	0.2	0.03	1.42	A	0	1
	brevifacies	Organic	0.2	0.03	1.42	A
	Ausejanus albisignatus IFP	0.8	0.3	2.13	A	-2.721 0.0065

Table 5 :

 5 Comparison of invertebrate ecosystem service provider (IESP) abundances in ten kiwifruit orchards under two different management systems: five organic and five integrated pest management (IPM) orchards were sampled. Mean abundances and 95% confidence intervals (CI) have been back-transformed. Each IESP was modelled separately.

				Lower	Upper	Letter of
		Orchard		95%	95%	difference
	IESP	System	Mean	CI	CI	(alpha=0.05) z.ratio p.value
	Arcitalitrus spp.	IPM	0.2	0.03	1.42	A	-5.373 <.0001
		Organic	43.6 38.18 49.79	B
	Anoteropsis hilaris	IPM	2.2	1.22	3.97	A	-2.846 0.0044
		Organic	6	4.2	8.58	B
	Scymnus loewii	IPM	0.2	0.03	1.42	A	-1.24	0.215
		Organic	0.8	0.3	2.13	A
	Sericoderus sp.	IPM	18.4	15 22.57	A	-7.388 <.0001
		Organic	45.8 40.24 52.13	B
	Atomaria lewisi	IPM	0.4	0.1	1.6	A	-2.464 0.0137
		Organic	2.6	1.51	4.48	B
	Conoderus exsul	IPM	4.2	2.74	6.44	A	-3.833 0.0001
		Organic	11.2	8.62 14.55	B
	Cartodere spp.	IPM	35.4 30.55 41.02	A	-1.48	0.1388
		Organic	41.2 35.94 47.23	A
	Thyreocephalus	IPM	0.4	0.1	1.6	A	-2.078 0.0377
	orthodoxus	Organic	2	1.08	3.72	B
	Anotylus sp.	IPM	4.4	2.9	6.68	A	-2.457 0.014
		Organic	8.4	6.21 11.37	B
	Forficula	IPM	0.2	0.03	1.42	-	0.003	0.9979
	auricularia	Organic	0	-	-	-
	Akamptogonus	IPM	0	-	-	-	-0.003 0.9978
	novarae	Organic	0.4	0.1	1.6	-
	Lonchoptera	IPM	2.6	1.51	4.48	A	-2.041 0.0413
	bifurcata	Organic	5.2	3.54	7.64	B
	Trigonospila	IPM	3.2	1.96	5.22	A	2.27	0.0232
	brevifacies	Organic	1	0.42	2.4	B

Acknowledgements

We thank the orchard managers for providing access to their orchards for sample collection, and Sophie Hunt and Frances MacDonald for assistance with processing samples in the laboratory. We are also grateful to Richard Newcomb, Anuar Morales-Rodriguez, Vincent Dubut, and the anonymous reviewers for their helpful critique of the article. This project was funded by The New Zealand Institute for Plant and Food Research Ltd.

Conflict of Interest

All contributing authors declare that they have no conflicting interests with the research described in this manuscript. 6 ( 6) 2 (2) 0 (5) 0 (2)

1 Specific primers designed for each IESP (see Table 1) were tested for their ability to detect the IESP in each soil subsample to which it had been added (Tot.Pos.Subs.).

2 Folmer primers were used to produce COI sequences that were then matched to sequences for each IESP. Tot.Pos.Comb. = total number of combined samples to which each IESP had been added.

* indicates where there were not enough specimens of the IESP to add to soil samples # these sequences matched the sequence for Aphelinus abdominalis but not A. mali ^ IESP identified in sample to which it was not added