Genes regulating wing patterning in Drosophila melanogaster show reduced expression under exposure of Daminozide, the fruit ripening retardant

Highlights

• Daminozide gets modified into its derivatives upon entry into the body of Drosophila melanogaster.

• Daminozide reduces the expression of wg, vg and dpp, the wing patterning genes of Drosophila melanogaster.

• In Silico study suggests it may bind with signaling proteins work in wing development.

Abstract

In our previous study we demonstrated that the fruit ripening retardant Daminozide or Alar causes change in life history traits, distortion of adult wing structure, DNA damage in brain cells and mutagenic effects in fruit fly Drosophila melanogaster. As a continuation of the previous study the present work is designed to explore the metabolic modification of Daminozide following ingestion, the effects of Daminozide on the expression of genes which are pivotal for wing development and molecular interactions of Daminozide with those proteins involved in wing patterning. We demonstrated through reporter gene construct assay using X-gal staining method and transgenic Drosophila melanogaster stocks that the vestigial, wingless and decapentaplegic genes in wing imaginal disc from 3^rd instar larvae exhibited reduced expression when exposed to Daminozide in compare to control larvae. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of those genes confirmed that exposure to Daminozide reduces the transcription level of those genes. In silico approach with molecular docking study revealed Daminozide may bind and interfere with the optimal functioning of expressed wing signaling proteins.

Introduction

Daminozide is used to prevent premature ripening of fruits and very popular among orchard owners and fruit merchants for commercial benefit. The chemical is synthesized from succinic anhydride and 1, 1-dimethylhydrazine (UDMH), a toxic component from rocket fuel. Daminozide is available in market as white powder and used as a spray on fruits following which it penetrates through skin and incorporated into pulp. It gets its entry in human body following consumption of treated fruits and upon digestion it is modified into its metabolic derivatives UDMH which is thousand times more carcinogenic (Gordon, 2011). Despite of these facts, the scientific approaches to address the issue of Daminozide toxicity in public health interest is surprisingly limited.

Previously, we demonstrated the neurotoxic effect of Daminozide in term of DNA degradation in brain cells and teratogenic alteration of adult wing structure in Drosophila melanogaster under Daminozide exposure (Roy et al., 2018). Moreover, we observed conspicuous change in foraging, locomotory and reproductive behavior of fly exposed to two different concentrations of Daminozide (unpublished data). All these observations made us curious to inquire whether observed distortions of wings were due to alteration in expression of genes that regulate wing development. For this study we selected three pivotal genes which are wing development regulators as the members of signaling cascade that express in larval imaginal disc and pattern the adult wing structure in Drosophila melanogaster. The genes are wingless (wg), vestigial (vg) and decapentaplegic (dpp). The wg is located on 2^nd chromosome (location: 27F1; Gene ID: 34009) and a conserved member of Wnt/Wg signaling pathway that expresses at several developmental stages in multiple tissues including the embryonic ectoderm (Bejsovec and Arias, 1991; Dougan and DiNardo, 1992; Bejsovec, 1993), head (Schmidt-Ott and Technau, 1992), midgut (Immerglück et al., 1990; Reuter et al., 1990; Thüringer and Bienz, 1993; Bienz, 1994), wing disc (Simcox et al., 1989; Cohen, 1990; Cohen et al., 1993; Phillips and Whittle, 1993; Williams et al., 1993) and leg disc (Baker, 1988; Campbell et al., 1993; Couso et al., 1993). Binding of the Wnt/Wg ligand to its coreceptors, Frizzled-2 (Fz2) and LRP/Arrow (Arr) initiates a sequence of cytoplasmic events that lead to the Dishevelled (Dsh)–mediated inactivation of the protein destruction complex, thereby allowing stabilized β-catenin/Arm to translocate to the nucleus, where it binds Tcf to direct the activation of Wnt/Wg-target genes (Bejsovec, 2006). In third instar wing imaginal disc the gene wg is expressed at the dorsal/ventral boundary, in two concentric rings and in a broad stripe in the dorsal region of the disc. The expression of wg in the disc regulates the patterning of the wing margin, blade, hinge, and notum of the adult fly. The second gene of our choice is vg, which is located on 2^nd chromosome in Drosophila genome (location 49E1; Gene ID: 36421), encodes a nuclear protein of 453 amino acids and most closely related to the mouse and human vestigial-like 1–3 (VGLL1 − 3) genes (Koontz et al., 2013). The gene wg acts as a co-factor of vg (Baena-López and García-Bellido, 2003). The activity of wg and Notch (N) in the wing margin leads to the expression of the vg (Kim et al., 1996; Williams et al., 1994; Zecca and Struhl, 2007). The expression of vestigial in the most proximal parts of the wing blade is regulated by the decapentaplegic (dpp) pathway and by vg itself (Kim et al., 1996; Zecca and Struhl, 2007). The product of vg interacts with the product of the gene scalloped (sd), a protein with DNA recognition motif (Campbell et al., 1992), to form a transcriptional activation complex (Halder and Carroll, 2001; Halder et al., 1998). The Vg-Sd complex is known to regulate the expression of downstream genes involved in wing development (Kim et al., 1996; Halder and Carroll, 2001; Halder et al., 1998; Klein and Martínez-Arias, 1998). The third gene of interest is another 2^nd chromosomal gene dpp, (location: 22F1-22F3; Gene ID: 33432), which activates a signaling cascade in wing disc by binding to its receptor Thickveins (tkv). The targets of this signaling are spalt(salm) and optomotorblind (omb) that are morphogens and involved in wing patterning (Restrepo et al., 2014).

As far as published literatures are concerned the studies on toxicity of some pollutants like cadmium (Guan et al., 2015) and ethylparaben (Xue et al., 2017) on gene expression in Drosophila melanogaster have been conducted previously, though the attempts are limited. Moreover, nobody has ever reported the effects of toxicant on developmentally relevant genes in Drosophila melanogaster. This is for first time ever we report the effect of Daminozide on the expression level of three genes from wing development signaling pathway in Drosophila melanogaster. Additionally, we provide data on metabolic modification of Daminozide in the body tissue of fruit fly and also predicted the probable interactions of Daminozide with other developmentally relevant proteins through in silico bioinformatics approach. The aim of our present study is to look into whether Daminozide could alter transcription and translation of the genes involved in wing development and also to see if Daminozide could affect the functioning of already expressed proteins encoded by those genes in subcellular environment.