A Semi-Automatic Dispenser for Solid and Liquid Food in Aquatic Facilities

bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A semi-automatic dispenser for solid and liquid food in aquatic facilities

Authors

• Raphaël Candelier 1 • Alex Bois 2 • Stéphane Tronche 2 • Jéremy Mahieu 2 • Abdelkrim Mannioui 2

Affiliations

1. Sorbonne Université, Centre National de la Recherche Scientifique, Laboratoire Jean Perrin, LJP, F-75005 Paris, France 2. Sorbonne Université, Institut de Biologie Paris-Seine (IBPS), Aquatic Facility, 75005, Paris, France

Corresponding author

Raphaël Candelier

Laboratoire Jean Perrin UMR 8237 - 4 place Jussieu, Case Courrier 114 - 75252 Paris Cedex 05

[email protected]

Telephone: +33 1 44 27 20 06

Fax: +33 1 44 27 47 16

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Abstract

We present a novel, low-footprint and low-cost semi-automatic system for delivering solid and liquid food to zebrafish, and more generally to aquatic animals raised in racks of tanks. It is composed of a portable main module equipped with a contactless reader that adjusts the quantity to deliver for each tank, and either a solid food module or a liquid food module. Solid food comprises virtually any kind of dry powder or grains below two millimeters in diameter, and, for liquid-mediated food, brine shrimps (Artemia salina) and rotifers (Rotifera) have been successfully tested. Real-world testing, feedback and validation have been performed in a zebrafish facility for several months. In comparison with manual feeding this system mitigates the appearance of musculoskeletal disorders among regularly-feeding staff, and let operators observe the animals’ behavior instead of being focused on quantities to deliver. We also tested the accuracy of both humans and our dispenser and found that the semi-automatic system is much more reliable, with respectively 7-fold and 84-fold drops in standard deviation for solid and liquid food. Introduction

Since the pioneering work of Streisinger et al. published in 1981 on cloning homozygous diploid zebrafish1, Danio Rerio has rapidly grown as a model system in many different fields including embryo development, tissue regeneration and neuroscience. In 2017, it has been estimated that more than 5 million zebrafish were used in more than 3.250 institutes spread across 100 countries2,3. It is more common in laboratories than other well-established aquatic vertebrates like xenopus4 and medaka5 and, for instance, it is the second most common animal species used for research in Great Britain6. As research on zebrafish has gained an impressive momentum in such a short time lapse, a large number of dedicated fish rooms have appeared. Other species like Danionella Translucida (now amenable to brain-wide functional imaging in adults with cellular resolution7) and killifish (whose short lifespan, fecundity and diapause of dried eggs make an ideal model for biogerontology8) also have a high potential for a rapid spread among research institutes in the future. Altogether, there has been and there will be a growing need for improving husbandry procedures in aquatic facilities, with very different scales ranging from a few hundred to hundreds of thousands animals.

Feeding is a fundamental task in any fish room, and though some research focus on improving the nutritional quality of the food9,10, only a few technical advances have been proposed on how food is actually delivered. It is traditionally performed manually with wash bottles for live food in liquid medium (e.g. Artemia nauplii, rotifers) and with various systems ranging from spoon-like tools to seed sowers for powders and granulates. Manual feeding raises serious issues though, with a clear lack of control over the delivered quantities and the appearance of musculoskeletal disorders among technicians. More specifically, wrist, elbow and shoulder tendinopathy are common among fish room staff, mainly because of the repetitive application of pressure on wash bottles. It may cause recurrent work stoppages, and in the most severe cases require steroid injections and surgery.

The only serious alternative to manual feeding is a fully-automated commercial solution, but it is extremely expensive, has a large footprint (which makes it difficult or impossible to install in small spaces, stand-alone racks or some fish rooms located in buildings that were not initially built for this purpose), processes very slowly (thus does not guarantee that microorganisms are still alive when delivered to the animals), delivers discrete quantities of food (which has limited accuracy), prevents access to the tanks during operation, and still requires food-filling and regular maintenance. In addition, breakdown – which is an inherent risk to every automated system – can have catastrophic consequences for both animals and the research associated.

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Here we present an intermediate solution between manual and fully-automated systems, keeping the assets of both approaches while eliminating most of their drawbacks. Our Semi-Automatic Food Dispenser (SeAFooD) is battery-powered and portable with a low footprint, delivers dry solid or liquid- mediated food in a modular manner, displaces all the weight of liquid in a self-supporting reservoir on caster wheels, requires no specific gesture for triggering and remains under the operation of a human agent at all times. We quantified that microorganisms have a high or perfect survival rate while going through the dispenser and that survivors are as motile as control. The dispenser can deliver either fixed quantities, operator-controlled quantities or obtain information on the number of individuals in each tank via near-field communication (NFC) and automatically deliver the exact amount of food. The latter mode (i) has an accuracy down to the single-animal scale or below, (ii) diminishes waste and improves water quality, (iii) allows for custom diets and (iv) let the operator focus on animal behavior during the feeding process. The whole system is low-cost and has been built with standard tools anyone can find in a FabLab (e.g. 3D printer, laser-cutter, soldering iron). Finally, it has been tested in a medium-scale zebrafish platform for several months and had a very positive impact on the staff health since all staff members observed a decrease in forearm pains during this period.

Materials and methods

General description. The dispenser is composed of three modules (Figure 1, A): a main module, a solid food module for dry powders and grains and a liquid food module for live microorganisms in water. The main module has an ergonomic handle, a trigger, fixation rails to attach the other modules and a microcontroller to interface a LCD screen, a rotary encoder (to select the mode of operation and navigate in the settings menu), a NFC read/write card, and a high-power LED (Figure 2, A). The trigger has been designed to fit on Tecniplast tanks, but it can be easily commuted to fit other types of tanks. The main module always acts as the master while other modules are slaves.

The solid food module has a removable reservoir (standard 50mL tube, Falcon ref. 352070) drilled at the tip and mounted on a sheath with a vibration coin motor (Figure 2, B). During normal handling only minute amounts of powder smear from the reservoir, but under vibration a regular flow of grains instantaneously establishes (Supplementary Movie 1). This phenomenon has been previously described11 and, in essence, relies on the fluidization of the granular bed by a constant injection of energy to overcome friction among grains. Vibrations are similar to those of video games paddles, and are neither unpleasant nor dangerous for the operator.

The liquid food reservoir (8L) is attached onto a custom skirt with caster wheels (Figure 2, C). The skirt comprises a battery, a pump and a magnetic stirrer. The latter is essential for ensuring homogeneity in the solution and Artemia nauplii survival during the whole feeding process. The reservoir can be closed with a lid or left open, and remains at atmospheric pressure at all times. The pump is triggered by a signal coming from the main module and sends the liquid to the module’s base for delivery (Figure 2, D).

Modules construction. Modules have been designed with Fusion 360 (Autodesk). Custom mechanical parts have been 3D-printed, mostly with PLA (on an Anet A8 printer) and for some parts in PETG (on a Prusa Mk3 printer). The lid of the liquid reservoir’s skirt has been laser-cut (5mm PPMA on a Full Spectrum Laser Hobby machine). Mechanical assembly has been realized with M3 metallic threaded inserts.

The electronics was custom-made and based on inexpensive, well-documented microcontrollers (Arduino Nano v3.1). For the liquid food module, a dedicated Printed Circuit Board (PCB) has been designed (Eagle, Autodesk) and manufactured (PCBWay) to reduce the footprint, avoid mistakes during

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soldering and ease mounting. The system is able to detect which module is mounted by means of a resistance specific to each module that creates a voltage divider (see Supplementary Materials).

Calibration. A dedicated setup has been developed for calibrating the solid and liquid food modules, comprising a rigid arm holding the dispenser and a scale (OHAUS PA2102C) as illustrated on Supplementary Figure 1. Both the dispenser and the scale where computer-controlled, and delivered amounts were recorded during series of activation (Supplementary Movies 1 and 2). The same system was used to determine the system’s accuracy with runs of 50 trials of random duration, similar to the human accuracy tests.

Fish room testing. The system has been tested in the aquatic facility of IBPS (Sorbonne Université, approx. 1.100 tanks, 15.000 fish). The experiments were made in agreement with the European Directive 210/63/EU on the protection of animals used for scientific purposes, and the French application decree Décret 2013-118. The aquatic facility has been approved by the French Service for animal protection and health, with the approval number A-75-05-25.

A simple, preliminary prototype for dispensing solid food with vibration, which was non-modular, without visual feedback and without NFC reader has been routinely used from Sept. 2017 to Sept. 2018. The final version of the system has been used on a daily basis since September 1st 2018.

The survival rates of rotifers (Rotifera) and brine shrimps (Artemia Salina) have been estimated with visual inspection under a binocular. Movies of rotifers and Artemia nauplii have been processed to extract individual trajectories (Figure 3, Supplementary Movie 3 and 4) with a novel cross-species tracking software (FastTrack) that will be published elsewhere.

Human accuracy tests. Human subjects were composed of two groups: staff of the fish facility who feed zebrafish more than twice a month (trained group, n=6) and people not working in a fish facility selected at random in the population (random group, n=27). Subject from the random group declared not to suffer from a musculoskeletal disorder. All subjects were aged between 18 and 62 and had no information about the setup or the purpose of the experiment beforehand, except that it would last approximatively 30 minutes. Experiments were not remunerated.

The tests were performed in a dedicated room containing only a table with the setup and a chair (Supplementary Figure 5). Subjects were instructed to enter the room alone, close the door, sit down and follow instructions on the screen. All steps of the protocol are detailed in the Supplementary Materials. The subjects had to perform successively four parts with 50 trials each, preceded by short training phases of three trials. Parts consisted in 1) pressing a button for a given duration with immediate visual feedback, 2) pressing a button for a given duration without visual feedback, 3) delivering given amounts of powder with a spoon and 4) delivering given amounts of liquid with a wash bottle.

The program controlling the screen, scale and button has been custom-made and written in C++ (Qt 5.8). The scale (OHAUS PA2102C) was controlled via a serial RS-232 connection and was blinded in a black box such that the subject could not see the LCD screen of the scale. The button was a red pushbutton (normally open switch without latch) mounted on a black plastic box containing a microcontroller (Arduino Nano v3.1) and linked to the computer via USB. The powder (Sucrose, Merck 84097-250G) was disposed in a beaker with a small spoon. The liquid (water with a blue dye, Indigo Carmine, Merck 57000-100G-F) was disposed in a 500mL wash bottle. A supplementary 250mL bottle was also provided in case the subject had to refill the wash bottle during the course of the experiment.

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Analysis. All data from the system calibration setup, the fish room tests and from accuracy tests have been processed with custom scripts in Matlab (R2018a, The MathWorks).

Results

Operation of the system in a fish room is presented in Supplementary Movies 1 and 2.

Live food survival. Live food is generally preferable to purely artificial diets12,13, as live feeds possess balanced nutritional profiles9, are visually and chemically attractive to fish, and are highly digestible14. It also contributes to the animal’s welfare in captivity with the ability to actively hunt and express natural feeding behaviors15. It is also mandatory according to the European regulation.

We estimated the survival rates of two standard live feeds, rotifers and Artemia nauplii. In control solutions, all animals were moving normally (Figure 3, B and E). After going through the liquid food module, all rotifers were moving in a similar fashion (Figure 3, B) and their observed survival rate was 100%. For Artemia it appeared that 5 to 10% of the nauplii died during delivery, probably crushed while passing through the pump. In addition, 20 to 25% of the nauplii were stunned and stopped moving for a few tenth of seconds before resuming normal motion. Trajectories of moving animals were very similar to control (Figure 3, E). We further quantified the motion of rotifers and Artemia nauplii before (control) and after delivery by plotting the Mean Square Displacement (MSD) over all trajectories as a function of time (Figure 3, C and F). This is a classical way of characterizing a diffusive process16: a straight line indicates diffusive motion, and the slope is equal to 4 times the diffusion coefficient. The coincidence of the MSD curves in both conditions for rotifers and brine shrimps indicate that the dispenser had no measurable effect on the dynamics of the motile micro-organisms, as compared to control.

In practice, Zebrafish ingest inert nauplii as well and no waste is left after a few minutes. Dead and stunned Artemia should in principle sink to the bottom of the tank but the tumult created by fish agitation in presence of food scatters the nauplii everywhere in the tank. Careful observation of fish behavior during feeding with the liquid food module make us suggest that such a 3:1 mixture of mobile and inert nauplii could be in fine beneficial to the fish, as they are forced to search for food in various places of the tanks and may adopt richer hunt strategies regarding competition with mates.

Comparison of human and machine accuracy. We developed a psychophysics setup (Supplementary Figure 5) to measure the average accuracy of humans in delivering powder with a spoon and liquid with a wash bottle, and pressing for a given amount of time on a button, with or without visual feedback. These four tasks encompass all reasonable scenarii for manual or manually-assisted delivery. In all tasks the subjects were asked to deliver random integer quantities between 1 and 50, corresponding to a number of animals in arbitrary units (see Supplementary Materials).

Experiments on humans revealed a generally poor accuracy (Figure 4, A-D). Surprisingly, we observed no difference between the performance of the trained and random groups, so we pooled all the data for analyses. Errors, quantified as the delivered amount minus the target amount, were symmetrical and Gaussian-distributed (Figure 4, G) with a dispersion that had little dependence on the target amount. The standard deviation of errors with liquid and solid were very high with 7.8 and 8.2 individuals respectively. These values can be considered too large for research-grade rearing; indeed, undernutrition may lead to developmental issues and fertility losses while overfeeding rapidly degrades water quality. Subjects performed slightly better in measuring time with a button, presumably because the mechanical action is reduced to its minimum, with a standard deviation of 5.0 individuals. Having a visual feedback of the elapsed time further improved accuracy, with a standard

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deviation of 2.8 individuals. Though this is presumably the best accuracy human subjects can achieve, transposed in a fish room that would require that the operator focuses on a screen while feeding.

In our system, NFC detection allows for the dispenser to know the number of animals in the tank and calculate the delivery time accordingly. The operator thus only manages the correct placement of the dispenser over the tank, and the accuracy is solely set by the dispenser’s own reproducibility. The latter is excellent (Figure 4, E-F) and the standard deviation of errors goes down to 1.2 individuals for the solid food module and as low as 0.3 individuals for the liquid food module (Figure 4, G), yielding respectively 7-fold and 84-fold decreases in standard deviation as compared to human performance for similar tasks.

For liquid feed the fluctuations in microorganism concentration are the main remaining source of inaccuracy, while for solid food errors come from minute irregularities in the flow rate. We also expect that for powders the reproducibility is highly sensitive to hygrometry. We also checked that the reservoir tube has to be tightly fixed to the sheath, unless large errors can appear (Supplementary Figure 5).

Feeding duration. We measured the average time to feed a complete rack via manual feeding (wash bottle for liquid, seed sower for solid food) and semi-automatic feeding in NFC mode (Supplementary Table 4). For unexperienced people, the semi-automatic dispenser slightly increased the feeding time (average +7.5%) with solid food and decreased the feeding time (average -21%) for liquid food. For trained staff, we observed a systematic increase of duration with the semi-automatic dispenser (average +56% for both solid and liquid food).

This discrepancy can be explained by the fact that trained staff have naturally developed stereotyped gestures over the years for optimizing the manual feeding time. These stereotyped gestures are not desirable in general, since they generate and aggravate musculoskeletal disorders. For trained staff, the longer time spent in front each rack with the semi-automatic dispenser is partly compensated by the consequent reduction of food-filling episodes: the liquid reservoir has a maximal volume equal to 16 wash bottles and the tubes containing solid food can be carried along and loaded very rapidly.

Discussion

Our semi-automatic dispenser is a new solution to the numerous issues raised by the pivotal but tedious task of feeding in fish rooms. It is more advanced and convenient than other semi-automatic solutions we are aware of (some being published17, but most are not): it is versatile, reliable, accurate, truly portable, easy to clean and do not soot over time. It has also several assets as compared to the commercial fully-automatic solution since it is low-cost, low-footprint and let the operator at the center of the feeding process such that discrepancies are immediately detected and corrected. Without the burden of constantly measuring quantities the operator can use the feeding time to perform routine visual inspection of fish health or check tanks labels. It is also readily accessible to untrained operators, such as students or seasonal staff, who can feed immediately and without any loss of accuracy.

In our opinion, one of the most important aspect of this work is the leap on the ground of musculoskeletal disorders. As soon as the system has been introduced in the fish room, all our staff members observed a relief in forearm pains during and after feeding. A staff member who was previously unable to feed due to repeated wrist tendinitis is now able to feed again without particular pain. These preliminary observations are still to be confirmed over time and with more users, but it is already very promising and we hope that this work will inspire other faculties and companies to invest

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in research for improving staff health. One possible way for future research is to relieve efforts on shoulder and elbow with an exoskeleton18,19.

Acknowledgements

We would like to thank Sylvie Authier, Édouard Mazoni and Jacques Gaudumet for their help and feedback during testing phases. This work has been financed by ANR JCJC program (ANR-16-CE16- 0017). We also thank the Satt Lutech (http://www.sattlutech.com) for endorsing the project, helping in patent writing and financing patent fees and prototype replication.

Author contributions

All authors have participated in drafting the system specifications. RC designed and built the modules, designed the human-testing setup, supervised these experiments, and analyzed the data. AB, ST and JM have conducted the tests in the fish room. RC and AM wrote the manuscript.

Author Disclosure Statement

All authors are inventors of a patent covering the present semi-automatic feeding system. The patent has been filed at the French Institut National de la Propriété Industrielle (INPI) and extended for international protection. Source materials including plans, electronic diagrams and Arduino microcode are available upon signature of a material transfer agreement.

References

1. Streisinger G, Walker C, Dower N, Knauber D, Singer F: Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 1981;291:293-296. 2. Kinth P, Mahesh G, Panwar Y: Mapping of Zebrafish Research: A Global Outlook. Zebrafish 2013;10(4):510-517 3. Lidster K, Readman GD, Prescott MJ, Owen SF: International Survey on the use and welfare of zebrafish Danio Rerio in research. Journal of Fish Biology 2017;90:1891-1905 4. Parisis N: Xenopus laevis as a Model System. Mater methods 2012;2:151 5. Shima A, Mitani H: Medaka as a research organism: past, present and future. Mechanisms of Development 2004;121(7):599-604 6. Home Office of Great Britain annual statistics 2015, https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/537708/sc ientific-procedures-living-animals-2015.pdf 7. Schulze L, Henninger J, Kadobianskyi M, Chaigne T, Faustino AI: Transparent Danionella Translucida as a genetically tractable vertebrate brain model. Nature Methods 2018;15:977- 983 8. Taylor CHV: Killifish as a Model Organism. Mater MEthods 2017;7:2156 9. Watanabe T, Kitajima C, Fujita S: Nutritional values of live organisms used in Japan for mass propagation of fish: A review. Aquaculture 1983; 34:115-143 10. Singh S, Gamlath S, Wakeling L: Nutritional aspects of food extrusion: a review. Int. J. of Food Scienceand Technology 2007;42:916-929 11. Matsusaka S, Yamamoto K, Masuda H: Micro-feeding of a fine powder using a vibrating capillary tube. Advanced Powder Technol. 1996;7(2):141:151 12. Lawrence C: The husbandry of zebrafish (Danio Rerio); a review. Aquaculture 2007;269;1-20

7 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

13. Carvalho AP, Araújo L, Santos MM: Rearing zebrafish (Danio Rerio) larvae withjout live food: evaluation of a commercial, a practical and a purified starter diet on larval perfromance. Aquaculture Research 2006;37(11):1107-1111 14. Cahu CL, Zambonino Infante JL: Substitution of live food by formulated diets marine fish larvae. Aquaculture 2001;200:161-180 15. Williams TD, Readman GD, Owen SF: Key issues concerning environmental enrichment for laboratory‐held fish species. Laboratory Animals 2009;43:1–14 16. Qian H, Sheetz MP, Elson EL: Single particle tracking: Analysis of diffusion and flow in two- dimensional systems. Biophys. J. 1991;60:910-921 17. Oltová J, Barton C, Certal AC, Argenton F, Varga ZM: 10th European Zebrafish Meeting 2017, Budapest: Husbandry Workshop Summary. Zebrafish 2018;15 18. Gorgey AS: Robotic exoskeletons: The current pros and cons. World J. Orthop. 2018;9(9):112- 119 19. Papadopoulos E, Patsianis G: Design of an Exoskeleton Mechanism for the Shoulder Joint. 12th IFToMM World Congress, Besançon (France), 2007

8 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure legends

Figure 1

The semi-automatic food dispenser. (A) Scheme of the system. The main module can host either the liquid food or solid food module to form a functional assembly. The solid food module directly hosts 50mL tubes of powder while the liquid food module has an external 8L reservoir mounted on caster wheels. (B) Picture of the liquid food assembly during delivering. (C) Picture of the solid food assembly during delivering. bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2

System parts and localization of relevant elements. (A) Main module. Inset: view of the back of the main module (B) Solid food module. (C) Zoom and partial inside view of the skirt of the liquid food reservoir. (D) Liquid food module. bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3

Estimating micro-organisms motility after passing through the dispenser. (A) Tracking of a movie of rotifers under a binocular. Inset: Blow-up of a single rotifer. Scale bars: 100µm. (B) Trajectories and (C) Mean Square Displacement (MSD) of moving rotifers in the control condition and just after delivery with the dispenser. (D) Tracking of a movie of Artemia nauplii under a binocular. Inset: Blow-up of a single nauplius. Scale bars: 250µm. (E) Trajectories and (F) MSD of moving Artemia nauplii in the control condition and just after delivery with the dispenser. bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4

Comparison of Human and dispenser accuracy. (A-D) Quantity delivered by humans as a function of the target quantity in the four tested conditions: powder (A), liquid (B), button (C) and button with visual feedback (D). Data points from all subjects (n=33) are shown in gray and data from one individual chosen at random are highlighted in color. (E-F) Quantity delivered by the dispenser as a function of the target quantity for powder (E) and liquid (F) media. Data points from an equal number of runs (n=33) are show in gray and one randomly chosen run is highlighted in color. (G) Probability density functions (pdf) of the difference between delivered and target quantities for the different conditions with Humans and dispenser. Inset: Standard deviations (std). Additional numbers on dotted lines indicate std ratios. bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary materials

A semi-automatic dispenser for solid and liquid food in aquatic facilities Raphaël Candelier, Alex Bois, Stéphane Tronche, Jéremy Mahieu, Abdelkrim Mannioui

Table of Contents 1 General information ...... 2 1.1 Sources of the prototype ...... 2 1.2 Sources of the psychophysics experiment ...... 2 1.3 Electronics ...... 2 1.4 Operation and settings ...... 3 1.4.1 General operation ...... 3 1.4.2 Menu tree ...... 4 1.4.3 EEPROM management ...... 4 1.4.4 Nomenclature of the NFC tags: number of animals and cleaning protocol ...... 5 2 Measuring the system’s accuracy ...... 6 2.1 Liquid food module ...... 6 2.2 Solid food module ...... 7 3 Feeding duration ...... 8 4 Measuring Human accuracy ...... 9 4.1 Rationale and setup ...... 9 4.2 Step-by-step description ...... 10 5 Supplementary Movies...... 14 5.1 Supplementary Movie 1 ...... 14 5.2 Supplementary Movie 2 ...... 14 5.3 Supplementary Movie 3 ...... 14 5.4 Supplementary Movie 4 ...... 14

1 General information

1.1 Sources of the prototype

A patent covering the present semi-automatic feeding system has been filed at the French Institut National de la Propriété Industrielle (INPI) and extended for international protection. Source materials including plans, electronic diagrams and Arduino microcode are available upon signature of a material transfer agreement.

1.2 Sources of the psychophysics experiment

The source code of the program used for human accuracy experiments is available under the terms of the CeCILL licence for French regulation and more generally the terms of the GNU General Public License.

All files are available at the following URL:

http://www.raphael.candelier.fr/Papers/SeAFooD/ReproPsycho.zip

1.3 Electronics

All parts of the system, including the electronics, have been designed to be accessible and customizable by non-specialists. All components have a through-hole packaging and only basic soldering equipment is required for assembly. For simplicity, we choose the Arduino Nano v.3.1 as microcontrollers for the main and liquid food modules. A PCB (Printed Circuit Board) has been designed for housing the electronic components of the liquid food module, in the form of a cape for the Arduino Nano.

Modules are automatically recognized when connected to the main module. A resistance is located in each module between the ID and GND pins, which forms a voltage divider with the internal resistance of the A0 analog input pin of the Arduino. According to Arduino specifications this internal resistance is between 20kΩ and 50kΩ, so the possible resistance ranges are listed below:

Resistance value Analog value range Usage 2.2 kΩ 43 - 102 10 kΩ 170 – 341 Solid food module 27 kΩ 358 – 588 100 kΩ 682 – 853 Liquid food module 250 kΩ 852 - 948 ∞ (open circuit) 1023 No module detected

Supplementary Table 1 - Ranges for the recognition resistance and associated analog values and usages. Grayed cells are reserved for future module development.

1.4 Operation and settings

1.4.1 General operation Both the liquid and solid food module operate at fixed flow rates. The control over delivered quantities is therefore performed by controlling the delivery times 푡퐿 and 푡푆, which are related to the number of animals 푛 by fixed coefficients 훼퐿 and 훼푆:

• For liquid food: 푡퐿 = 훼퐿. 푛, with by default 훼퐿 = 80 ms/fish • For solid food: 푡푆 = 훼푆. 푛 with by default 훼푆 = 35 ms/fish

Note that both 훼퐿 and 훼푆 have to be adapted to the food type, the feeding protocol and the fish diet: 훼퐿 depends linearly on the concentration in microorganisms (unless the concentration is very high and change the effective viscosity of the liquid medium, but this is not recommended) while 훼푆 depends on the type of solid food and the size of the hole on the delivery tube. The default value of 훼푆 has been optimized for grains 300-500µm in diameter (Gemma Wean 0.3) and a hole of 2.8mm. The coefficients can be changed in the settings menu and are kept in memory even when the device is turned off.

When a solid or liquid food module is mounted on the main module, the device automatically detects which module is mounted and chooses which coefficient 훼 to use accordingly.

The system can function in three different modes:

• Manual mode. The delivery time is exactly the time during which the trigger is active. The operator thus directly controls the delivered quantity by pulling the trigger for a short or long duration. • Fixed mode. In this mode the amount of delivered quantity is fixed for all tanks. As soon as the trigger is pulled, the device starts to deliver the fixed quantity, regardless of the state of the trigger until delivery is complete. When delivery is over, the system marks a pause of 1 second before being active again. The fixed quantity can be changed in the settings menu. This mode is particularly interesting for tanks with isolated fish (푛=1) as precise temporal control for one animal is difficult to achieve in manual mode. • NFC mode. In this mode the system has to read first a Near-Field Communication (NFC) tag before being active. Once the quantity to deliver is read, it is displayed on the screen and the trigger becomes active. As soon as the trigger is pulled, the device starts to deliver the required quantity, regardless of the state of the trigger until delivery is complete. These steps can be performed in a single movement (see Supplementary Movie 1 and Supplementary Movie 2) since reading the NFC tag is quasi-instantaneous and usually happens before the trigger touches the tank. Once delivery is complete, the system waits to read a different NFC tag before re-activating the trigger.

The operator can easily switch between these three modes with a rotary encoder. The latter is equipped with an RGB LED that shows a different color for each mode (by default: manual=OFF, fixed=cyan, NFC=yellow). The current mode is also displayed on the LCD screen.

Counters of the total number of deliveries (i.e. number of tanks) and the total number of fed animals are always displayed on the screen. It is reset to zeros when the device is switched off.

The system is also equipped with a white power LED that illuminates the tanks, allowing the operator i) to check that food is actually arriving in the tank and ii) to observe the behavior of the animals during feeding. The intensity of this LED can be different at rest and during delivery, and it can be turned off.

Many settings, including the coefficients 훼퐿 and 훼푆, colors and LED intensity can be accessed and modified via a menu interface on the main module. When the button on the rotary encoder is pressed for a one second the systems enters in the Menu mode (red color of the rotary encoder by default). Navigation in the menu is achieved with the following commands:

• Clockwise / counterclockwise rotation of the rotary encoder: change selection • Rotary encoder button: Enter, validation • Trigger: Cancel, go back or exit the menu

1.4.2 Menu tree

Manual Mode Fixed Value General NFC Version Version Reset to default Confirmation Time per unit Value (ms) Liquid Min Value Recognition range Max Value Time per unit Value (ms) Solid Min Value Recognition range Max Value Light at rest Value (%) Light during delivery Value (%) Manual Color Lights Fixed Color Colors NFC Color Delivery Color Menu Color

Supplementary Table 2: The menu tree, from left to right. Fields in italics are selectable. Color can be either: Red, Green, Blue, Magenta, Yellow, Cyan, White or None.

1.4.3 EEPROM management The Arduino Nano has an embed EEPROM memory of 1024 bytes. EEPROM is non-volatile, meaning that it can be used to store settings even when the device is powered off. The following table resumes how the 28 first bytes of EEPROM are managed to store settings:

Address bytes Description Possible values Default value 0 Version first number 0 – 255 1 1 Version second number 0 – 255 2 2 Version third number 0 - 255 2 3 Version day 0 - 255 17 4 Version month 0 - 255 01 5 Version year, after 2000 0 - 255 19 0: Manual, 1: Fixed, 6 Mode 0 2: NFC 7 Fixed value (only for the Fixed mode) 0 - 100 5 10 - 11 Liquid module detection lower value 0 - 1024 680 12 - 13 Liquid module detection higher value 0 - 1024 860

14 – 15 Solid module detection lower value 0 - 1024 170 16 - 17 Solid module detection higher value 0 - 1024 350 18 - 19 Coefficient 훼퐿, milliseconds per animal 0 – 10,000 80 20 – 21 Coefficient 훼푆, milliseconds per animal 0 – 10,000 35 22 LED intensity at rest (%) 0 – 100 1 23 LED intensity during delivery (%) 0 - 100 25 24 Color at rest, Manual mode 0 0: None, 1: White, 25 Color at rest, Fixed mode 4 2: Red, 3: Green, 26 Color at rest, NFC mode 7 4: Blue, 5: Magenta, 27 Color during delivery 1 6:Cyan, 7: Yellow 28 Color in the menu 5

Supplementary Table 3: Management of EEPROM memory in the main module.

1.4.4 Nomenclature of the NFC tags: number of animals and cleaning protocol Data has been written on the NFC tags with the free version of NFCTools and a compatible smartphone (Samsung Galaxy Young 2 SM-G130). The written data is always plain text.

For NFC tags used for feeding, only the number of animals in the tank is written on the tag. So when the main module reads a single number, it is interpreted as a number of animals and the delivery signal is set for the corresponding duration. For instance:

18 Sets delivery for 18 animals

Special diets can be programmed for some tanks only (by punctually changing the value on the NFC tag) or globally (by changing the coefficients 훼퐿 and 훼푆).

The cleaning procedure of the liquid food module is also defined on a separate NFC tag. When the module reads such a tag, it automatically triggers the cleaning. The protocols are composed of sequences of delivery with 10 second pauses in between. The syntax is as follows:

1x60 Triggers a continuous delivery phase lasting 60 seconds

3x60 Triggers a cleaning protocol composed of three 60 seconds-long delivery phases separated by 10 second pauses.

NFC tags can have many shape and materials, and we chose round 25mm-diameter stickers since they are cheaper (small price differences become significant with thousands of tanks). They do not go well in the cleaning machine, so in order to remove and replace them at will we glued it on transparent post-its (see Figure 1.B-C, Supplementary Movie 1 and Supplementary Movie 2) or on thin plastic film usually used for electrostatic stickers. As the information stored on the NFC tags can be re-written endlessly, it is thus easy to reuse it on clean tanks.

2 Measuring the system’s accuracy

We used the setup show in Supplementary Figure 1 to measure the system’s accuracy for both the solid and liquid food modules. The scale (OHAUS PA2102C) was connected to the computer via an USB to RS232 serial converter. For the solid food tests we used Gemma Wean 0.3 (Planktovie) in a 50mL tube with a 2.8mm drilled hole, and for the liquid food module we used tap water.

Supplementary Figure 1 – Left: Picture of the setup used to measure quantities delivered by the solid food module. Right: Picture of the setup used to measure quantities delivered by the liquid food module. 2.1 Liquid food module

Supplementary Figure 2 – Delivered quantity as a function of the delivering time for the liquid food module. Error bars: standard deviation.

2.2 Solid food module

Supplementary Figure 3 - Delivered quantity as a function of the delivering time for the solid food module. Error bars: standard deviation.

In contrast with the liquid food module, several factors can influence the accuracy of the solid food module. For instance, if the containing tube is not tightly attached to the sheath and is able to rotate during vibration the reproducibility is greatly hampered:

Supplementary Figure 4 – Cumulative quantity of food delivered during runs of 400 trials of 2.0 second of vibration. The containing tube is full (30g) at the beginning of each run and empty at the end. Tight (green) and loose (red) fixation of the containing tube inside the sheath are compared.

Also, controlling the degree of moisture in the powder is essential for accuracy. Water creates bonds between grains that may lead to large aggregates. The latter need a lot of energy to break, so the granular bed is vibration-fluidized with reduced efficiency and severe losses of efficiency can appear. So the food should be kept as dry as possible, which may be difficult in a fish room. We recommend to store the food in a dry place (room with controlled hygrometry or drying box with desiccant).

3 Feeding duration

Fish room staff Unexperienced people

Manual (seed sower) 45.6 82.7 Solid food Dispenser (NFC mode) 71.6 89.0

Manual (wash bottle) 41.2 112.5 Liquid food Dispenser (NFC mode) 64.2 88.9

Supplementary table 4 – Average feeding time (seconds) for rack of 600 fish with an average of 15.4 fish/tank. Fish room staff have fed manually for many years, and used the semi-automatic dispenser for 4 months before the test. Unexperienced operators were volunteer students who had never fed before. Students were shown typical quantities to deliver by trained staff, and they could practice on a few tanks to adjust delivered quantities just before the test. For each condition, 3 subjects were tested on two racks.

4 Measuring Human accuracy

4.1 Rationale and setup

Our system has a good accuracy over delivered quantities, which we could measure with the automated setup shown in Supplementary Figure 1. But to determine if there is a gain as compared to manual feeding, we had to measure the accuracy of humans in standard feeding tasks. We thus developed a psychophysics setup (Supplementary Figure 5) to measure the average accuracy and reproducibility of human subjects in delivering some liquid with a washer bottle and some powder with a spoon. We also tested human reproducibility on a time-measuring task during which the operator had to press a button for a given amount of time, with or without visual feedback on the elapsed time.

The button was fixed on a small black enclosure containing an Arduino Nano, which was connected to the computer via USB. The scale (OHAUS PA2102C) was connected to the computer via a USB to RS232 serial converter. The powder was sucrose (Merck 84097-250G) and the liquid was distilled water with a blue dye (Indigo carmine, Merck 57000-100G-F).

Subjects were always asked to deliver an integer quantity between 1 and 50. We used arbitrary units in all parts of the experiment, and the following coefficients (unknown to the subject) were used:

• One unit of time was 100ms for tasks with the button • One unit of mass was 60mg for the solid delivery task • One unit of mass was 400mg (400µL) for the liquid delivery task

Supplementary Figure 5: Setup for testing human accuracy in measuring amounts of powder, liquid and time. From left to right: screen, button, scale, powder recipient with spoon (pink), liquid wash bottle.

9 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

4.2 Step-by-step description

Step-by-step display (French) English translation

Bonjour Hello

Appuyez sur le bouton rouge pour commencer Press the red button to start

Wait for button press

Cette expérience va se dérouler en trois parties de This experiment is composed of three parts of durée égale. equal duration

Dans chaque partie vous pouvez utiliser In each part you can use either your left or n'importe quelle main et en changer à tout moment. right hand and change anytime.

Appuyez sur le bouton pour continuer Press the button to continue

Wait for button press

Dans la première partie vous allez utiliser In the first part you will only use uniquement ce bouton rouge. this red button.

Vous devrez appuyer sur le bouton pendant une You will have to press the button for a given durée donnée en essayant d'être le plus précis duration and try to be as accurate as possible: possible: pas trop court, pas trop long. not too short, not too long.

Le temps se déroule dans une unité arbitraire, Time is counted in an arbitrary unit, and we will et nous allons tout d'abord nous familiariser avec. first familiarize with it.

Appuyez sur le bouton pour continuer Press the button to continue

Wait for button press

Appuyez sur le bouton et restez appuyé Press and hold the button

Observez le compteur défiler Observe the counter jusqu'à ce que vous relachiez until you release the button

- -

When the button is pressed the – is replaced by numbers linearly increasing with time.

When the button is released, the program holds the display for one second.

Maintenant essayez de faire exactement 18 Now try to make exactly 18

- -

When the button is pressed the – is replaced by numbers linearly increasing with time.

When the button is released, the program holds the display for one second.

Pas facile, mais vous vous en sortez bien. Not easy, but you are doing well. Essayez de faire exactement 23 Try to make exactly 23

- -

When the button is pressed the – is replaced by numbers linearly increasing with time.

When the button is released, the program holds the display for one second.

10 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Maintenant essayez de faire exactement 7 Now try to make exactly 7

- -

When the button is pressed the – is replaced by numbers linearly increasing with time.

When the button is released, the program holds the display for one second.

Cet entrainement est terminé, This training is over, nous allons maintenant passer à la phase de test. We shall now pass onto the test phase.

La tâche reste la même, vous devez appuyer The task remains the same, you have to press pendant le temps indiqué, exactement. the button for the indicated time, exactly.

Appuyez sur le bouton pour commencer Press the button to start

Wait for button press

Vous devez atteindre exactement You have to reach exactly N N

- -

N is a random integer between 1 and 50, different from the previous one.

Repeat 50 times 50 Repeat When the button is pressed the – is replaced by numbers linearly increasing with time. When the button is released, the program holds the display for one second.

Maintenant nous allons augmenter un peu la difficulté Now we will increase a little bit the difficulty et vous ne verrez plus le compteur défiler. and you won’t see the counter anymore.

Le résultat s'affichera The result will appear only une fois que vous relacherez le bouton. once you release the button.

Essayez de faire exactement 25. Try to make exactly 25.

- -

When the button is pressed time is measured but not displayed. When the button is

released, the – is replaced by the elapsed time and display is held for 1.5 second.

Vous devez atteindre exactement You have to reach exactly N N

- -

N is a random integer between 1 and 50, different from the previous one.

Repeat 50 times 50 Repeat When the button is pressed time is measured but not displayed. When the button is released, the – is replaced by the elapsed time and display is held for 1.5 second.

La première partie est maintenant terminée. The first part is now over.

A partir de maintenant le bouton ne sert plus From now on the button will only be used qu'a avancer dans les instructions. to go forward in the instructions.

Appuyez sur le bouton pour continuer Press the button to continue

Wait for button press

Prenez la coupelle de pesée avec une croix rose Take the weighing dish with a pink cross et déposez-la sur la balance. and place it on the scale.

Appuyez sur le bouton quand la coupelle est en place Press the button when the dish is placed

11 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Wait for button press

Dans la deuxième partie vous devez verser In the second part you will have to verse une quantité donnée de poudre dans la coupelle: a given quantity of powder in the dish: pas moins, pas trop. not less, not more.

La poudre se trouve dans le petit becher rose. The powder is in the small pink beaker.

Vous devez utiliser la cuillère rose fournie, You shall use the pink spoon, et ne faire qu'un seul versement par consigne. and verse powder only once per order.

Vous ne devez pas toucher la table pendant You shall not touch the table during the l'expérience. experiment.

Appuyez sur le bouton pour continuer Press the button to continue

Wait for button press

Ici encore, la quantité de poudre est comptée Here again, the amount of powder is counted en unité arbitraire in arbitrary units

Déposez une demi-cuillère de poudre. Sprinkle half a spoon of powder. Il faut attendre que la balance se stabilise You have to wait for the scale to stabilize après chaque versement. after each trial.

- -

When the scale stabilizes with a positive weight difference the – is replaced by the

measured quantity and display is held for 1.5 second.

Maintenant essayez de faire exactement 18 Now try to make exactly 18

- -

When the scale stabilizes with a positive weight difference the – is replaced by the

measured quantity and display is held for 1.5 second.

Pas facile, mais vous vous en sortez bien. Not easy, but you are doing well. Essayez de faire exactement 23 Try to make exactly 23

- -

When the scale stabilizes with a positive weight difference the – is replaced by the

measured quantity and display is held for 1.5 second.

Vous devez atteindre exactement You have to reach exactly N N

- -

N is a random integer between 1 and 50, different from the previous one.

Repeat 50 times 50 Repeat When the scale stabilizes with a positive weight difference the – is replaced by the measured quantity and display is held for 1.5 second.

La deuxième partie est maintenant terminée. The second part is now over.

Veuillez enlever la coupelle de pesée Please take off the weighing dish et la mettre de côté, avec le becher rose. and place it aside, with the pink beaker.

Appuyez sur le bouton pour continuer Press the button to continue

Wait for button press

12 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Prenez le grand becher bleu en verre Take the large glass blue beaker et déposez-le sur la balance. and place it on the scale.

Appuyez sur le bouton quand le becher bleu Press the button when the blue beaker est en place is placed

Wait for button press

Dans la troisième partie vous devez verser In the third part you will have to verse une quantité donnée de liquide dans le becher: a given quantity of liquid in the beaker: pas moins, pas trop. not less, not more.

Comme précédemment, vous n'avez droit qu'à As previously, you are only allowed to un seul versement par consigne. deliver once per order.

Appuyez sur le bouton pour continuer Press the button to continue

Wait for button press

La quantité de liquide est mesurée en unité The amount of liquid is measured in arbitrary arbitraire. units.

Effectuez une pression sur la pissette bleue. Press once on the blue washer bottle. Il faut attendre que la balance se stabilize You have to wait for the scale to stabilize après chaque versement. after each dump.

- -

When the scale stabilizes with a positive weight difference the – is replaced by the

measured quantity and display is held for 1.5 second.

Maintenant essayez de faire exactement 18 Now try to make exactly 18

- -

When the scale stabilizes with a positive weight difference the – is replaced by the

measured quantity and display is held for 1.5 second.

Pas facile, mais vous vous en sortez bien. Not easy, but you are doing well. Essayez de faire exactement 23 Try to make exactly 23

- -

When the scale stabilizes with a positive weight difference the – is replaced by the

measured quantity and display is held for 1.5 second.

Vous devez atteindre exactement You have to reach exactly N N

- -

N is a random integer between 1 and 50, different from the previous one.

Repeat 50 times 50 Repeat When the scale stabilizes with a positive weight difference the – is replaced by the measured quantity and display is held for 1.5 second.

Cette expérience est maintenant terminée. This experiment is now over.

Merci d'y avoir participé. Thanks for participating.

Vous pouvez quitter la pièce. You can leave the room.

13 bioRxiv preprint doi: https://doi.org/10.1101/535062; this version posted February 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

5 Supplementary Movies

5.1 Supplementary Movie 1

Solid food module. 1) Mounting of the solid food module (blue and black) on the main module (cyan). 2) Calibration setup, during a calibration experiment. The system is computer-controlled and delivers for random, uniformly distributed durations while recordings are performed with a scale. 3) Close-up view of the hole upon delivery. 4) Delivery in a tank of a fish room, in NFC mode. 5) Workflow on successive tanks.

5.2 Supplementary Movie 2

Liquid food module. 1) Mounting of the liquid food module (blue and black) on the main module (cyan). 2) Calibration setup, during a calibration experiment. The system is computer-controlled and delivers for random, uniformly distributed durations while recordings are performed with a scale. 3) Close-up view of the tube end upon delivery. 4) Delivery in tanks of a fish room, in NFC mode. 5) Workflow on successive tanks.

5.3 Supplementary Movie 3

Tracking rotifers. 1) Motion of rotifers in the control condition. 2) Same movie, with tracking overlaid.

5.4 Supplementary Movie 4

Tracking Artemia nauplii. 1) Motion of brine shrimps in the control condition. 2) Same movie, with tracking overlaid.