A) A custom-made hypergravity centrifuge was used to perform the experiments. B) Gondola with the 96 well plate where the larvae were placed during the 5 days of treatment. C) Graphical representation of the centrifuge and gondola showing the variables and vectors used to calculate the value of the acceleration (artificial hypergravity) at the center of the gondola. The centrifuge radius, R, is defined as the distance from the center of rotation to the outer edge of the platter. The gondola arm length l was measured from the outer edge of the platter of the centrifuge to the end of the gondola. Both of them (R and l) were measured with a tape measure. The angle, θ, was calculated using taking an image and analyzing it using a program called Angulus (DPP v1 2020). r is the gondola radius, ν is the tangential velocity due to rotation, ac is the centripetal acceleration, and ω is the angular velocity.

Survival rates of control and hypergravity zebrafish larvae during 5 days of treatment.

Each data point shows the mean ± SE of six independent groups with a total number of 220 individuals per condition (control and hypergravity). The cumulative survival with different letters indicates a significant difference (P < 0.005) according to the Least significant difference (LSD) test.

Hatching rate of larvae at 2- and 3-days post fertilization treated with hypergravity compared with the control group.

Six biological replicates were made, with a total number of larvae of 220 and 220 in control and hypergravity, respectively. Data are presented as percentage ± standard error of the mean (SEM). Normality was evaluated with a Kolmogorov–Smirnov test, and Levene’s test was used to assess homoscedasticity of variances. No differences between groups were found according to Least significant difference (LSD) test.

Ethogram analysis consisted on identifying three locomotor characteristics: Position (A), movement frequency (B) and swimming behavior (C) of larvae of zebrafish at 5 days post fertilization (dpf). Bar graphs representing the percentage of individuals in control and hypergravity conditions. Six biological replicates were observed with a total number of 90 individuals in the hypergravity group and 90 in the control. The different colors represent the assessed features in each parameter. The data are presented as percentages of individuals. To evaluate significant differences, we performed a Chi-square test. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Teratology was observed at 5 days post fertilization (dpf) in zebrafish larvae exposed to ~3 g hypergravity from 0 to 5 dpf.

Positions of the larvae are shown in the figure: horizontal (A), vertical ascendant (B); horizontal lateral (C); and vertical descendent (D). Teratologies included four major types: body curvature (B), abnormal eye size (B), overall body deformation (C) and tail curvature (D).

Global DNA methylation in zebrafish larvae after 5 days of ~3 g hypergravity.

N = 10 larvae per group (control and hypergravity). The statistical analysis was conducted using a two-tailed t-test (p < 0.05), demonstrating a significant difference in DNA methylation levels between the control and hypergravity conditions. Each lollipop represents the percentage of methylation in control and hypergravity conditions.

Expression of three epigenetic-related genes in zebrafish larvae after 5 days of ~3 g hypergravity.

Data are shown as mean ± SEM of fold change Relative Expression = 2^(-ΔCt) using control values set at 1. N = 10 larvae per group (control and hypergravity). No significant differences were found.