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

<div><p>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 (<i>U</i>_<i>max</i>) 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 <i>U</i>_<i>max</i> of larvae and juveniles, an 18–24% faster <i>U</i>_<i>max</i> of adult males vs. females, and a 14–20% age-related reduction in <i>U</i>_<i>max</i> for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.</p></div

Protocols for evaluating maximal swimming speed (<i>U</i>_<i>max</i>) of each group of zebrafish.

<p>Protocols for evaluating maximal swimming speed (<i>U</i>_<i>max</i>) of each group of zebrafish.</p

Relationship between target and observed flow rates.

<p>Data points represent the average observed flow rate (between 5–30 s of the trial) versus the target or expected flow rate at each stage of a slow flow protocol (A) and a fast flow protocol (B). The solid line is the line of identity. Mean deviation (average of the differences between observed and target flow rates across entire trial) was 0.067 cm s⁻¹ for the slow flow protocol and 0.083cms⁻¹ for the fast flow protocol.</p

Standard length and maximal swimming speed (<i>U</i>_<i>max</i>) of larval and juvenile zebrafish.

<p>(A) Standard length (<i>SL</i>). The relationship between SL and age (dpf) was described by <i>y</i> = 0.258<i>x</i> + 1.377, <i>R</i>² = 0.920. (B) Absolute <i>U</i>_<i>max</i>. The slope of the relationship between <i>U</i>_<i>max</i> in cm s⁻¹ and age (dpf) for 14–28 dpf fish was 0.698, with 95% confidence intervals of 0.414 to 0.981. The slope of the relationship for 28–42 dpf fish was 0.344, with 95% confidence intervals of -0.173 to 0.862. (C) <i>U</i>_<i>max</i> relative to standard length. The slope of the relationship between <i>U</i>_<i>max</i> in cm s⁻¹ and age (dpf) for 14–28 dpf fish was 0.394, with 95% confidence intervals of 0.4186 to 0.603. The slope of the relationship for 28–42 dpf fish was -0.071, with 95% confidence intervals of -0.452 to 0.310. Values are mean ± SE for 5, 5, 5, 6, and 6 fish at 14, 21, 28, 35, and 42 dpf, respectively.</p

Design of the flume.

<p>(A) Schematic diagram of the flume. (B) Expanded view of the working section. Diagrams are not drawn to scale. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199712#pone.0199712.s001" target="_blank">S1 Appendix</a> for details on components and construction.</p

The relationship between flow rate and the PWM signal.

<p>(A) Slow flow rates evaluated with restricted flow through the flume circuit. Polynomial regression results: <i>y</i> = −8.430<i>x</i> − 1.042<i>x</i>² + 242.8, <i>R</i>² = 0.999. (B) Fast flow rates evaluated with no restriction to flow. Polynomial regression results: <i>y</i> = −1.374<i>x</i> − 0.021<i>x</i>² + 244.4, <i>R</i>² = 1.00.</p

Calibration of the flow meters.

<p>Linear regression between flow meter pulses per s and the measured flow rate. The filled circles represent data collected with no circuit restriction in order to maximize the range and magnitude of the flow rate for studies of adult fish. The cross symbols represent data collected with the circuit partially restricted in order to slow the flow rate into a range suitable for studying larvae and juveniles. (A) Regression results for meter 1: <i>y</i> = 2.44<i>x</i> + 0.79, <i>R</i>² = 0.999. (B) Regression results for meter 2: <i>y</i> = 2.38<i>x</i> + 1.55, <i>R</i>² = 0.999.</p

Summary of literature values for maximal swimming speed (<i>U</i>_<i>max</i>) of zebrafish.

<p>Summary of literature values for maximal swimming speed (<i>U</i>_<i>max</i>) of zebrafish.</p

Arduino wiring.

<p>Abbreviations: R1, 10 kΩ; S1, switch (for exiting current mode of operation); POT1, potentiometer (for manual control of flow); R2, 4.7 kΩ; S2 and S3, switches (for powering pumps); GND, ground; 3V3, 3.3 V output; P, pump; F flow meter; TP, temperature probe. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199712#pone.0199712.s002" target="_blank">S2 Appendix</a> for details on components.</p

Cyclin A1 expression levels in FSHD and other myopathies (microarrays).

<p><i>CCNA1</i> Human Exon 1.0 ST Array signal levels in FSHD (n = 4), healthy controls (n = 7), CAV3 (n = 4), DYSF (n = 4) and FHL1 (n = 3).</p