An automated aquatic rack system for rearing marine invertebrates

Background One hundred years ago, marine organisms were the dominant systems for the study of developmental biology. The challenges in rearing these organisms outside of a marine setting ultimately contributed to a shift towards work on a smaller number of so-called model systems. Those animals are typically non-marine organisms with advantages afforded by short life cycles, high fecundity, and relative ease in laboratory culture. However, a full understanding of biodiversity, evolution, and anthropogenic effects on biological systems requires a broader survey of development in the animal kingdom. To this day, marine organisms remain relatively understudied, particularly the members of the Lophotrochozoa (Spiralia), which include well over one third of the metazoan phyla (such as the annelids, mollusks, flatworms) and exhibit a tremendous diversity of body plans and developmental modes. To facilitate studies of this group, we have previously described the development and culture of one lophotrochozoan representative, the slipper snail Crepidula atrasolea, which is easy to rear in recirculating marine aquaria. Lab-based culture and rearing of larger populations of animals remain a general challenge for many marine organisms, particularly for inland laboratories. Results Here, we describe the development of an automated marine aquatic rack system for the high-density culture of marine species, which is particularly well suited for rearing filter-feeding animals. Based on existing freshwater recirculating aquatic rack systems, our system is specific to the needs of marine organisms and incorporates robust filtration measures to eliminate wastes, reducing the need for regular water changes. In addition, this system incorporates sensors and associated equipment for automated assessment and adjustment of water quality. An automated feeding system permits precise delivery of liquid food (e.g., phytoplankton) throughout the day, mimicking real-life feeding conditions that contribute to increased growth rates and fecundity. Conclusion This automated system makes laboratory culture of marine animals feasible for both large and small research groups, significantly reducing the time, labor, and overall costs needed to rear these organisms.

1 x HPLC waste line adapter (part no. HPLC-LL-1/8-KIT; CPLabSafety, San Francisco, CA). Used as part of the food pump to connect the Teflon feeding line to very end of the Y connector. PTFE, Teflon tubing (feeding line; 6-8 feet long, as needed) 1/16" ID, 1/8" OD. (part no. 53XL53, Grainger Industrial Supply, Alsip, IL) Used as part of the food pump to deliver food to the sump compartment. Length should be kept to a minimum. 2 x 1/8" Barbed to male Luer lock connectors. (part no. 11525, Qosina, Ronkonkoma, NY) Used to attach the silicone tubing to the Luer lock Y connector and the airline tubing to the Luer lock one-way check valve, listed below. Note that a fine wire may be used to prevent the silicone tubing from slipping off the barbed connector by twisting this tightly around the tubing just below the flared, barbed end of the connector (like a fine twist tie). Do not over-tighten this wire, as it could cut through the silicone tubing. 1 x 1/8" Barbed to female Luer lock connector. (part no. 11524, Qosina, Ronkonkoma, NY) Used to attach the silicone tubing to the reservoir tubing. Note that a fine wire may be used to prevent the silicone tubing from slipping off the barbed connector by twisting this tightly around the tubing just below the flared, barbed end of the connector (like a fine twist tie). Do not overtighten this wire, as it could cut through the silicone tubing. 1 x Luer lock tubing (900 SPI 3/16 OD x 48" long, part no. 30526-28 Cole Parmer, Vernon Hills, IL). Used to draw food from the reservoir. Has both male and female ends. Cut off the female end, and use only the remaining tubing with the male end. Trim this tubing as short as possible so that the cut end just reaches the bottom of the reservoir.
Used to mount the peristaltic pump. A square opening must be machined in the lid of the box with screw holes for mounting the peristaltic pump. Another hole must be drilled to accommodate the cable gland. 1 x Machined ½" diameter, by 9 inches long aluminum rod and adjustable bracket. Bar has threaded end (1/4" -20). Used to hold the capacitive sensor that detects the presence of food in the food reservoir. Adjustable bracket has a clamping screw to secure its position (height) on the bar. (These parts mount directly onto the stir plate, which has a threaded hole on the back. (See Additional File 3F-H, Figure 5E).
Feeding system air injection parts (to purge the feeding line), Figure 5C,D: 1 x Air Pump (Uniclife part no. UL40, 4.0 watts, 4.0 L/min, 0.016 MPa pressure, dual twin outlet air pump, or equivalent, Uniclife, https://www.amazon.com/Uniclife-Aquarium-Outlets-Accessories-Adjustable/dp/B01EBXI7PG/ref=zg_bs_2975471011_2?_encoding=UTF8&psc=1&refRID=HH XH8MKDCFSMQQ7KKVQ7). Figure 5C. Used to purge the feeding line of residual food. (Use both pump channels joined with a barbed Y connector for maximum air pressure needed to purge the Teflon feeding line). 1 x Clear aquarium airline tubing (approx., 3/16" OD x 6 feet long). Used to supply air from the air pump to purge the feeding line. It is attached to a barbed Luer lock connector (see above). (This tubing is supplied as two pieces with the Uniclife air pump) 1 x Barbed dual-ended straight connector. Used to connect the two airline tubing pieces together (supplied with the Uniclife air pump) 1 x Barbed Y connector.
Used to connect both air pump, air channels together to increase air pressure (supplied with the Uniclife air pump. Cut two short 2 inch long pieces off of the clear airline tubing to connect this Y connector to the pump outlets) 1 x Luer lock one-way check valve, female to male (SAN plastic with silicone diaphragm, pack of 10, part no. UX-30505-92, Cole-Parmer, Vernon Hills, IL). Cracking pressure = 0.174psi (part no. 11582, Qosina, Ronkonkoma, NY). Cracking pressure = 0.217psi. Used as a key part of the food pump: As an alternative, a duckbill check valve can be used (part no. 80065, Qosina, Ronkonkoma, NY). Cracking pressure = 0.433 psi. These provide a good seal to prevent backflow. Used as part of the food pump to prevent liquid food from entering the purge airline. This part is connected to the Y connector (one can use two of these check valves in series, if desired, for extra security, though this does not appear to be necessary).
Feeding System Micro-controller: (to regulate automated feeding), Figure 5C: To build this microcontroller unit please see instructions, fritzing diagram ("Feeding controller2.fzz") and Arduino program sketch ("aquarium_feeding_controller9IRFinal copyl.int" program file). See Additional Files 4-5 and Figure 4C. 1 x Arduino Ethernet Shield (SunFounder Ethernet Shield W5100 for Arduino UNO R3 Mega product no. 2560 1280 A057, https://www.amazon.com/dp/B00HG82V1A). Used for an internet connection to relay various faults to the users. Note that there are different versions of the Wiznet ethernet chip. The Arduino program is designed to work with the W5100 chip, which is a slightly older chip. Some other chips are not compatible with the libraries used for compiling the Arduino program, and would require a slightly different script 1 x Project box, 5.3" x 3" x 1.9" (part no. MB-113, All Electronics, Van Nuys, CA, https://www.allelectronics.com/item/mb-113/abs-project-box-5.3-x-3-x-1.9/1.html). Used to house the relay module and outlets. Note that various openings will need to be machined to permit mounting of various components.
Mounting bracket for feeding system microcontroller unit, includes two 3D printed parts: (see Articulated arm base plate .stl files included in, Additional Files 7-8, and available on Henry Lab web site, www.life.illinois.edu/henry). Note that other arrangements can also be made for mounting the microcontroller. Figure 5C: 1 x Articulated Arm, all metal construction, Camvate 7" (147mm) articulated arm V1 black and red, with ¼-20 threads. (Camvate, https://www.amazon.com/dp/B017OTDZTM). Figure 5C. Used with the two 3D printed parts, detailed below, to position the microcontroller, see Additional Files 7-8. 1 x Plastic circular, beveled plate (has center tapped with ¼-20 hole for articulated arm) to be affixed to the control box by heavy duty double stick foam tape. (see "Articulated arm base plate v10.stl" file provided). 1 x Semi-circular mounting plate (has center taped ¼-20 hole for articulated arm). Two counterbored holes accept 10-32 cap screws for mounting this plate on the top of the beverage cooler using the unused door hinge threaded holes on the top of the mini refrigerator. Door is reversible so either left or right side can be used, but be aware of the arrangement of cables to best mount the arm. (See "Articulated arm base plate v10.stl" file provided). This part is mounted (glued into and secured with a screw-on retaining strap) to the base of the food line clamp.
Retaining straps (See "retaining strap v3.stl" file provided) See Additional Files 11-12. 2 x Water proof cable glands for cables to prevent water incursion. PG7 size, various suppliers. See above. Used to secure the cables and prevent water incursion into the box. 4 conductor cable (6 feet), various suppliers Used to connect the remote sensor to the microcontroller. 1 x Small water-proof box (BUD Industries, Inc., part number PN-1320) Used to protect the components of the IR sensor.

Peristaltic Food Pump
Stepper Motor Controller: (used to regulate food delivery), Figure  5C: To build this microcontroller unit please see instructions, fritzing diagram ("peristaltic_pump_controller.fzz") and Arduino program sketch ("stepper_motor_controller.int" program file). See Additional Files 13-14 and Figure 5C. Protein Skimmer (Used to remove uneaten food and dissolved organic material), Figure  3C-F: 1 x Plastic bulkhead connector single port, for 3/8" OD silicon tubing, with NPT threaded connector (5694T161, McMaster Carr, Elmhurst, IL) and ½" ID 3.32 O-ring and Teflon tape. Corresponding ¼" NPT threaded nut and ½" ID 3.32 O-ring and Teflon tape to seal connection. Hole for this fitting is drilled into side of Tunze collecting cup towards the bottom. Inner shoulder located in the central passage way of this of fitting is drilled out to allow for complete thru passage of the silicone tubing. Used as a continuous drain connection for the collecting cup silicone tubing. 1 x Plastic bulkhead connector dual ports, for 3/8" OD silicon tubing (To be installed in the side wall of the sump (Figure 3F). 5694T17, McMaster Carr, Elmhurst, IL). Inner shoulder located in the central passage way of this fitting is drilled out to allow for complete through passage of the silicone tubing. Used to allow for passage of the silicone skimmer drain line through the wall of the sump.
Custom motor control interface (regulates power to the protein skimmer via the Walchem 900 controller), Figure 5G: Fritzing diagram ("DC motor controller interface2.fzz") for assembling the circuit, and program for the ATtiny85 chip ("ATtiny_motor_controller copy.ino"). See files provided in Additional Files 15-16. Used to automatically turn off protein skimmer during feeding.
Fine insulated wire, size 30, solid strand, any 30 gauge wire wrapping wire should work (https://www.amazon.com/dp/B07WRTWWZW) 1x ATtiny85 operating at 1MHz (Sparkfun, COM-09378, https://www.sparkfun.com/products/9378). Note this is programmed using Arduino IDE and SparkFun Tiny AVR Programmer (see below). 24 gauge insulated hook up wire 2 x Rubber grommets (5/16" OD) Used to secure the cable, provide strain relief and prevent water incursion into the housing. 2 x small zip ties, various suppliers Used to secure the cables.
3D printed housing and cover plate to enclose the circuit (See 3D printer stl file: "control box v7.stl" provided). See Additional Files 17-18. Note that once installed the lid can be secured using a small amount of silicone cement.
Additional components needed for the rack system (e.g., sea salts, media for the various filters, and components needed to culture small animals, and to reduce splashing, evaporation and to regulate sea water level): Note, most of the standard components that make up the rack system provided by Iwaki Aquatics, are not listed here.
Instant Ocean Reef Crystals artificial sea salt (Spectrum Brands, Inc. Blacksburg, VA, UPC code 51378 01800). Used as the artificial sea salts to prepare the sea water, following the manufacturer's instructions. Seed Bacteria (Aquavitro, Seachem, Madison, GA) or Dr. Tim's One and Only Live Nitrifying Bacteria (Dr .Tim's Aquatics, LTD, Moorpark, CA). Used initially to seed the biofilter.
"Marine Buffer" (Seachem, Madison, GA). Used to raise the pH of the sea water, as needed.

Filters and filter media
Two Acurel Filter lifeguard media bags, 9" x 13" (prod. ID 42982 08033, Acurel, Cranbury, NJ). Placed inside the right-hand sump compartment and used to contain the crushed shell and coral, so that these course debris cannot escape and enter the water pump and filters. These bags should be securely tied shut once filled.
Crushed coral (Geo-Marine Argonite formula 15lb bag. Product ID 0847900120, CaribSea, White City, FL). Used as a source of calcium and as a buffer for the sea water. The substrate is placed inside two fine mesh bags, above, which go into the right sump compartment, so that the coarse debris cannot enter the water pump and filters.
Biological filter media consisting of a mix of 800 gm of Matrix (Seachem, Madison, GA) and 100 gm of de*Nitrate (Seachem, Madison, GA). Used as a substrate to support aerobic, anaerobic and facultative bacteria growth.
1-2 lbs of activated carbon pellets (Kent Reef Carbon Pellets, Kent Marine, Franklin, WI). Used to remove dissolved organic compounds from the sea water.

Automatic top off unit to add sea water, Figure 4H-I:
System automatic top off unit (ATK unit, Neptune Systems, Morgan Hill, CA). Used to automatically add sea water. Figure 4H.
Optical sensor with magnetic mount (OS-1-M, Neptune Systems, Morgan Hill, CA). Used to monitor the level of sea water inside the reservoir. Figure 4I.
Small nylon basket ("The People's Brew Basket", The Republic of Tea, Novato, CA). Used as a filter to prevent debris and ppt from entering the small ATK PMUP water pump. Figure 4I.
Clean 5 gallon plastic paint bucket with lid, various suppliers. Used as the reservoir for the sea water. Note that small holes need to be drilled through the lid to allow for the passage of the power cord, optical sensor cable and plastic tubing. Figure 4H.
Polycarbonate "Origami" cover for the large sump drain opening (See Additional File 3D, Figure 3B.

Culture tubes for animals:
Ultra-stiff mesh plastic canvas (for needle point). (#7 mesh = openings per inch). Used to create a curved barrier to keep culture tubes submerged inside the small tanks. Darice Canvas 3pc. Designer Ultra stiff Canvas 12"x18" (product ID 826709455). Darice, Inc. Strongsville, OH. Each piece can be cut with scissors to make two barriers (20cm x 22.5cm). Figure 1D, J.
2 x Sets of clamps/bolts/cables to secure the rack. Used to secure the top of the rack and prevent it from tipping over. Exact parts depend on where the rack is located and what supports are available to attach these parts. Figure 1B. 4 x Custom machined ½ inch thick, 4" x 4" square aluminum foot pads. Each has a central 1/8" deep by 1' -3/8" dia. recess for the leveling feet. Used to distribute the weight of the rack system. Figure 1C.