An open source microcontroller based flume for evaluating swimming performance of larval, juvenile, and adult zebrafish

Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s−1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18–24% faster Umax of adult males vs. females, and a 14–20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.


S1 Appendix: Flume components and assembly
-1.Short sections of 1 inch diameter polyvinyl chloride (PVC) tubing were used as couplers to join components.The use of these couplings allowed components to be easily disassembled for maintenance and cleaning while providing a degree of flexibility to the structure that was beneficial when loading fish into the working section.To prevent leaks, all threaded surfaces were wrapped with pipe thread sealant tape before assembly.

Modifications to components
The reservoir was made from a 7 l aquarium obtained from our zebrafish facility modified as follows: 1) a 1.875 inch hole was drilled in the bottom of the reservoir to accept the through hole pipe fitting, 2) two 0.75 inch holes were drilled in the upper sides of the reservoir as entry points for the return flow from the pumps.The polycarbonate tube working section was cut to length and a 0.25 inch hole was drilled through one wall of the tube near the downstream end as shown in Fig 1-B.

Pumps
Water circulation through the flume was provided by two industrial pumps that could be independently regulated via PWM.Each pump consisted of a magnetically driven spherical impeller that rotated at high speed on a ceramic bearing.The pumps should always contain water when operating as running them dry can result in irreversible damage to the bearing.The pumps were configured so that in the absence of PWM, they defaulted to their maximum speed.This results in an inverse relationship between the PWM signal and pump output.Be aware that for prolonged, sustained use at high outputs, the pumps will generate sufficient heat to alter the temperature of the flume water.We did not experience this problem with the relatively short duration protocols used in the present study.

Flow meters
Hall-effect flow meters were used to measure flow through the flume.We found these meters were highly linear with very little inter-meter variability (see Fig 3).In a preliminary design we used an infrared flow meter.This meter performed very well and is an acceptable substitution.However, we found that it was more sensitive to debris or waste that may be inadvertently circulating through the flume.

Frame
A frame for supporting the reservoir, working section, and pumps is illustrated in Fig S1-2 and a list of parts and potential suppliers are compiled in Table S1-2.The base was constructed out of T-slotted aluminum framing held together with inside corner brackets.Common laboratory aluminum rod, bench supports, and connectors were used to form supports for the reservoir and the working section as shown.The pumps were bolted directly into the aluminum framing.Alternatively, investigators can use their ingenuity and miscellaneous materials to construct a serviceable supporting structure for the flume.).H: Pump assembly (two assemblies required).Individual components were joined using lengths of 1 inch diameter PVC tubing (part no.4).To prevent leaks, all threaded surfaces were wrapped with pipe thread sealant tape before assembly.See Table S1 for a list all of the components illustrated here.S2.

Figure
Figure Fig S1-1 is an expanded view of the flume components and how they are assembled.A list of components and potential sources are compiled in TableS1-1.Short sections of 1 inch diameter polyvinyl chloride (PVC) tubing were used as couplers to join components.The use of these couplings allowed components to be easily disassembled for maintenance and cleaning while providing a degree of flexibility to the structure that was beneficial when loading fish into the working section.To prevent leaks, all threaded surfaces were wrapped with pipe thread sealant tape before assembly.
Figure S1-1.Expanded schematic diagram of the flume and assembly notes.A: Reservoir assembly.B: Working section assembly.C: Flow return assembly.D: Bubble trap assembly.E: Drain assembly.F: Fork assembly.G: Flow meter assembly (two assemblies required).H: Pump assembly (two assemblies required).Individual components were joined using lengths of 1 inch diameter PVC tubing (part no.4).To prevent leaks, all threaded surfaces were wrapped with pipe thread sealant tape before assembly.See TableS1for a list all of the components illustrated here.
Figure S1-2.Schematic diagram of frame for holding flume.Parts listed in TableS2.

Table S1
Cost is product of unit cost times quantity, rounded to nearest dollar.