# Zebrafish Larval Melanophores Respond to Electromagnetic Fields Exposure
## 2 Materials and Methods
### Zebrafish Maintenance and Treatments  
The simple devices and experimental sets presented above were made to optimize their use on zebrafish. To date, zebrafish have already been used to study the effects of magnetic fields [8; 9; 10]. Adult wild-type zebrafish (AB line) were maintained under standard rearing conditions in plastic tanks at 27-28 \({}^{\circ}\)C at the Department of Biological and Environmental Sciences and Technologies, University of Salento (Lecce, Italy). The fish were kept to a constant 14 h light: 10 h darkness photoperiod and fed thrice daily with commercial fish diet (ZEBRAFEED, Sporas I&D; Olhao, Portugal). Animal care and procedures were in accordance with the U.S. National Institutes of Health's Guide for the Care and Use of Laboratory Animals and European Community Directive 2010/63/EU. Fertilized eggs (diameter -0.6 mm) were collected within 1-2 h (not later than the 8- to 16-cell stage) of natural spawning and then transferred into Petri dishes (Corning, Milano, Italy) with deionized water added with commercial Instant Ocean (Blacksburg, VA, USA) sea salts (60 mg of Instant Ocean per liter of water; pH range 6.8-7.4) [11]. All experiments were performed in Petri dishes with a water column height of about 3 mm and a constant 14 h light:10 h darkness photoperiod for the entire duration of the test. With the fertilized eggs subjected to Brownian motion, the mechanical moment direction changed continually. Since, during the experiments, the fertilized eggs developed from embryos to larvae, we measured the effects of radiations during the early embryonic-to-larval development stages up to 5 dpf. \begin{table}
\begin{tabular}{c c c} \hline  & **Frequency** & **Intensity** \\ \hline Static (B\({}_{0}\)) magnetic field irradiation & 0.0 Hz & 40 mT \\ \hline Extremely low-frequency (VLF) magnetic field irradiation & 0.2 Hz & 40 mT \\ \hline Low-frequency (LF) magnetic field irradiation & 270 kHz & 470 \(\mu\)T \\ \hline Very-high-frequency (VHF) magnetic field irradiation & 100 MHz & 240 nT \\ \hline Ultra-high-frequency (UHF) magnetic field irradiation & 900 MHz & 240 nT \\ \hline \end{tabular}
\end{table} 
Table 1: Main radiation characteristics associated to the static, low-, and high- (radio) frequency magnetic fields used in this work. Within 1-2 h post-fertilization (hpf), developing embryos (diameter -0.9 mm) were separated into six groups. After 1-2 hpf, zebrafish embryos were exposed under the magnetic fields and radio frequencies for 5 days (-120 hpf). Specifically, five groups were subjected each to a single stress: static (0 Hz), extremely low-frequency (0.2 Hz), low-frequency (270 kHz), very-high-frequency (100 MHz), and ultra-high-frequency (900 MHz) field irradiation; the sixth group of untreated embryos was used as control (number of eggs \(n\) = 10 for each condition; number of experiments = 3). At the end of the experiments (length -3.9 mm for untreated larvae), larvae were anesthetized with tricaine methanesulfonate (MS-222; SIGMA-Aldrich Chemical Co., London, UK) and observed at low magnification (10\(\times\)) using a Nikon AZ100 stereomicroscope equipped with the Nikon NIS-Elements D suite for image capture. For our study, head and trunk pigmentation (morphology of melanophores) was analyzed By ImageJ software v 1.8.0 ([[https://imagej.net/Imagej](https://imagej.net/Imagej)]([https://imagej.net/Imagej](https://imagej.net/Imagej)); [PERSON], National Institutes of Health, Bethesda, MD, USA), the total count and area of melanophore were analyzed. Data are presented as mean \(\pm\)SE (standard error of the mean). The statistical analysis was performed using an unpaired \(t\)-test, considering \(p\) < 0.05 significant (* = \(p\) < 0.05; ** = \(p\) < 0.01; *** \(p\) < 0.001).