Tryptophan hydroxylase (TRH) loss of function mutations in Daphnia deregulated growth, energetic, serotoninergic and arachidonic acid metabolic signalling pathways

Bruno Campos,Benjamin Piña,Claudia Rivetti,Roma Tauler,Carlos Barata

doi:10.1038/s41598-019-39987-5

Bruno Campos, Benjamin Piña + Show 3 more

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https://doi.org/10.1038/s41598-019-39987-5

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Abstract

Serotonin has a pivotal function regulating development, growth, reproduction and behavior in animals. In this paper, we studied the deregulatory effects of the deprivation of serotonin in Daphnia magna TRH CRISPR-Cas9 mutants. Bi-allelic in-del THR mutants and, to a lesser extent, mono-allelic ones grew less, reproduced later, and produced smaller clutches than wild type clones. Transcriptomic and functional gene analyses showed a down-regulation of growth/molting and energy metabolism signaling pathways in TRH mutants, while revealing marked differences between mono- and bi-allelic clones. Bi-allelic mutants, lacking serotonin, presented the serotonergic synapse and arachidonic acid metabolic pathways down-regulated while the tryptophan to kynurenine was upregulated, thus indicating a cross-talk between the serotonergic and arachidonic acid metabolic pathways. Finally, the effects on the insulin growth factor-mediated signaling pathway were marginal. These changes in functional and metabolic pathways are consistent with previously reported effects in D. magna exposed to pharmaceuticals that inhibited arachidonic metabolism or enhanced the levels of serotonin.

Highlights

Serotonin plays an important role regulating development, growth, reproduction and behavior in most organisms
Hierarchical clustering of the fluorescence values of each of the four individual-replicates per clone for the 333 down-regulated probes showed a clear separation of wild type individuals from mutated ones, and that TB− individuals were forming a distinct cluster, together with some probes from TA− individuals (Fig. 1A)
When looking to the commonly de-regulated genes across CRISPR-clone-specific we identified 15 genes belonging to different specific regulatory signals or metabolic pathways related with serotonin synthesis and re-uptake, arachidonic/prostaglandin metabolism, insulin like growth factor signaling and the tryptophan catabolic process to kynurenine

Summary

Introduction

Serotonin plays an important role regulating development, growth, reproduction and behavior in most organisms. Reproduction, growth and phototactic behavior of TRH KO clonal lineages showed the opposed phenotype of those exposed to SSRIs: KO individuals grew less, reproduced later with smaller clutchs and were more responsive to light than wild type clones[14] Studies in both Drosophila and in the worm C. elegans indicate a link between serotonin and insulin signalling pathways. Previous efforts to detect insulin immuno-reactivity in the central and peripheral neurological system of D. magna have been so far unsuccessful (Dircksen & Campos, unpublished data) Another proposed physiological role of serotonin is the regulation of the arachidonic acid and eicosanoid metabolism, which play vital roles in Daphnia reproduction and growth[20,21,22,23,24]. Genome sequence analyses as well as data from transcriptomic and metabolomics studies in D. pulex and D. magna suggest the presence of the genes encoding for phospholipase A2 and prostaglandin metabolic enzymes in daphniids[22]

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