Computational protocol: Tryptophan hydroxylase (TRH) loss of function mutations induce growth and behavioral defects in Daphnia magna

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Protocol publication

[…] Effects on reproduction were assessed followed established OECD guidelines with only minor modifications. The food study was conducted with the selected seven TRH mutated clones plus the wild type one (W). Two independent an almost identical experiments were performed. The first experiment included the seven studied mutated clones plus the wild type one and aimed to determine life-history effects. The second one was performed with clones TRHA−/+, TRHA−/−, TRHB−/− and the wild type one and aimed to study how consistent were measured life-history and behavioral effects. In the first and second experiment neonates (<24 h old) were exposed until their fifth and third brood, respectively, at low and high food ratio conditions (1 and 5 × 105 cells/ml of C. vulgaris, respectively) at 20 °C. Animals were exposed individually in 100 mL of ASTM hard water. Each treatment was replicated 10 times. The test medium was changed every other day. Measured life-history traits were: initial and body length of experimental animals across adult stages; juvenile and adult survival; age at first reproduction; clutch size and population growth rates (r), estimated from the age specific survival and reproduction rates according to the Lotka equation. Body length measurements were performed from the top of the head to the base of the spine using a Nikon stereoscope microscope (SMZ 150, Nikon, Barcelona, Spain) and the ImageJ software ( Clonal growth length curves across food ration levels were estimated from body size measurements of experimental animals by non linear least-squares fit to the von Bertalanffy function:1Lt=Lmax(1−e−K(t−t0))where Lt is body length, Lmax is asymptotic length, k is the growth rate, t is age and t0 is included to adjust the equation for the initial size of the organism and is defined as age at which the organisms would have had zero size. In each case, residuals of the least-squares models were examined to assess goodness-of-fit and possible bias. Despite that Daphnia growth is stepwise the von Bertalanffy function take into account age and initial length at birth length, which may help to correctly interpret observed differences in body size across the studied clones. In the SSRI study four clones TRHA−/+, TRHA−/− and TRHB−/− and the wild type one were exposed to 100 ng/L of fluoxetine (dissolved in water) at low food level (1 × 105 cells/ml of C. vulgaris) since our previous results showed that at low food levels effects of fluoxetine enhancing reproduction were the greatest–. Treatments included unexposed and exposed treatments for each clone replicated 10 times. Animals were exposed individually from birth until the third brood and their growth and reproduction responses measured. The remaining experimental conditions were similar to the “food study”. We selected 100 ng/L since it is an environmental relevant concentration of fluoxetine that can be found in surface waters. […]

Pipeline specifications

Software tools ImageJ, von Bertalanffy
Application Microscopic phenotype analysis
Organisms Daphnia magna, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus
Chemicals Fluoxetine, Serotonin, Tryptophan