Evaluation of reference genes for reverse transcription-quantitative PCR assays in organs of zebrafish exposed to glyphosate-based herbicide, Roundup

Abstract

Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, β-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0.065, 0.65 and 6.5 mg N-(phosphonomethyl) glycine/l) for 7 days. Genes were ranked by geNorm, NormFinder, BestKeeper, Delta C t and RefFinder, and their best combinations were determined by geNorm and NormFinder programs. The two most stable ranked genes were specific to each organ: gill (β-act; rpl8); brain (rpl8; β-act); testis (ef1α; gapdh); and ovary (rpl8; hprt1). The cat transcript level was used to evaluate the effect of normalization with these reference genes. These are the first suitable reference genes described for the analysis of gene expression in organs of Roundup-exposed zebrafish, and will allow investigations of the molecular mechanisms of Roundup toxicity.

Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio)

“…Considering the analysis by tissues, some studies corroborate our findings. Jaramillo et al (2018) 16 found that the best RGs in the brain were rpl8 and actb1 ; the former was not investigated in our study, but the latter was also found by us as the best for that tissue in the non-transgenic group (Table 3 ), and the fourth best in the gh -transgenic group (Table 4 ). The results obtained by Lang et al (2016) 15 were compatible with our findings, where rpl13α, rpl7, actb2 and eef1a1 were the most suitable RGs in liver when exposed in different concentration groups to cadmium and subsequently infected with Aeromonas hydrophila .…”

“…In general, our analyses found the eef1a1 gene to be highly stable, occupying between the first and at least the third position in the rankings—except in the BestKeeper and NormFinder analysis in the gh -transgenic group—demonstrating to be one of the most specific and stable gene for analysis in zebrafish in both groups. Other studies have also examined the stability of RGs in non-transgenic zebrafish, showing that eef1a1 is a stable gene for qRT-PCR normalization 11 – 13 , 15 , 16 .…”

“…Besides that, the different results presented by each algorithm are expected due to the distinct statistical approach that was used to construct the rankings 44 . The discrepancies in the ranks of the most stable genes obtained by each software reflect the differences of the algorithms and procedures of each method and demonstrate the need to evaluate the RGs using several bioinformatic algorithms 16 , 23 .…”

“…The zebrafish ( Danio rerio ) is a model organism of increasing importance for developmental analyses, disease modeling and toxicological studies 7 – 9 . RGs have already been evaluated in wild zebrafish across developmental stages and considering differences of gender, tissues, chemical treatments and experimental protocols 1 , 10 – 16 . The genetic background of the animals is a factor that can affect the differential expression of various genes, including RGs 17 .…”

Synergistic action of glyphosate on novel pesticides against Culex pipiens L. (Diptera: Culicidae) mosquitoes under laboratory conditions

“…On the other side, glyphosate and its mimics inhibit the specific enzyme, 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), which prohibit the biosynthesizing of three aromatic amino acids (tyrosine, tryptophane, and phenylalanine) which are vital for the processing of plant growth (Ibrahim, 2015;Ibrahim, 2016a;Ibrahim, 2016b). However, negatively toxicological impacts of certain herbicides such as glyphosate, certain ALS-, and ACCase-inhibitors have been confirmed on different insects aquatic animals and these herbicides can affect the survival, ecology, behavior, physiology, reproductive, fecundity, and development of these organisms (Mona et al, 2013;Mohamed et al, 2016;Jaramillo et al, 2017;Kittle et al, 2018;Saba et al, 2018). Further, Recent studies were conducted to illustrate the dexterous effect of some herbicides on immature and adult stages of different mosquito species in the world.…”