Gene expression response of the non-target gastropod Physella acuta to Fenoxycarb, a juvenile hormone analog pesticide

Abstract

Pesticides are an environmental problem. The search for new pest control methods has focused on compounds with low or no toxic effects in non-target organisms. Analogs of the juvenile hormone (JH) interfere endocrine system of arthropods. However, the lack of effect on non-target species requires confirmation. This article analyzes the impact of Fenoxycarb, an analog of JH, on Physella acuta, an aquatic gastropod. For 1 week, animals were exposed to 0.01, 1, and 100 μg/L and the RNA was isolated to analyze the gene expression by retrotranscription and Real-Time PCR. Forty genes related to the endocrine system, the DNA repair mechanisms, the detoxification mechanisms, oxidative stress, the stress response, the nervous system, hypoxia, energy metabolism, the immune system, and apoptosis were analyzed. Three of the genes, AchE, HSP17.9, and ApA, showed responses to the presence of Fenoxycarb at 1 μg/L, with no statistically significant responses in the rest of the genes and at the remaining concentrations. From the results, it can be concluded that Fenoxycarb shows a weak response at the molecular level in P. acuta in the tested time and concentrations. However, Aplysianin-A, a gene related to immunity, was altered so the long-term effect could be relevant. Therefore, additional research is required to confirm the safety of Fenoxycarb in non-arthropod species in the long term.

Figure 1

HYPERLINK "sps:id::fig1||locator::gr1||MediaObject::0"Structure and conserved domains of the Physella acuta proteins code by the sequences identified. The characteristic motifs of each protein are shown. The domains have been defined according to the Conserved Domains Database (CCD) functional classification of proteins. The size is indicated by the numbers.

Figure 2

Transcript levels of endocrine-related sequences (estrogen receptor [ER], estrogen-related receptor [EER], membrane progestin receptor beta [MPR], estradiol 17-beta-dehydrogenase 8 [Hsd17b8], galanin receptor 2 [GalR2], and retinoic acid receptor [RXR]) in Physella acuta adults after in vivo exposure to Fenoxycarb for seven days at 19 °C. Transcriptional activity was quantified by RT-PCR using ribosomal protein L10 (rpL10), actin (Act), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes. The comparison was performed with the solvent-exposed controls. Whisker boxes are shown. Each box corresponds to 12 individuals. The horizontal line within the box indicates the median, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). No significant differences to control were observed in those genes (p < 0.05).

Figure 3

Transcriptional activity of genes related to DNA repair. The mRNA levels of poly(ADP-Ribose) polymerase I (PARP1), NFKB inhibitor Iκβ (IkBa), X-ray repair cross complementing 3 (XRCC3), RAD21 Cohesin Complex Component (RAD21), and RAD50 Double Strand Break Repair Protein (RAD50) in Physella acuta adults after in vivo exposure to Fenoxycarb for seven days at 19 °C are shown. RT-PCR was used to quantify the mRNA levels and ribosomal protein L10 (rpL10), actin (Act), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes. The comparison was performed with the solvent-exposed controls. Whisker boxes are shown. Each box corresponds to 12 individuals. The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). No significant differences were observed relative to the control (p < 0.05).

Figure 4

Transcriptional activity of genes related to the nervous system (acetylcholinesterase [AChE]), oxidative stress (catalase [Cat], copper-zinc superoxide dismutase [SOD CuZn], and manganese superoxide dismutase [SOD Mn]), and apoptosis (caspase 3 [Casp3] and apoptosis inducing factor 3 [AIF3]). Snails were exposed to Fenoxycarb for seven days at 19 °C. Quantification by RT-PCR was performed using ribosomal protein L10 (rpL10), actin (Act), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes. The comparison was performed with the solvent-exposed controls. Whisker boxes are shown. Each box corresponds to 12 individuals. The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant difference relative to the control (asterisk) is indicated (p < 0.05).

Figure 5

Transcriptional activity of stress (small heat shock protein 16.6 [sHSP16.6], small heat shock protein 17.9 [sHSP17.9], heat shock protein 60 [HSP60], heat shock cognate 70 4 [HSC70 (4)], heat shock protein 70 B2-like [HSP70B2], glucose regulated protein 78/binding immunoglobulin protein [Grp78/BiP], and heat shock protein 83 [HSP83]) and hypoxia-inducible factor-1 alpha [HIF1α]) genes in adult snails. The animals were exposed for one week to Fenoxycarb at 19 °C. Transcriptional activity was quantified by RT-PCR using ribosomal protein L10 (rpL10), actin (Act), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes. The comparison was performed with the solvent-exposed controls. Whisker boxes are shown. Each box corresponds to 12 individuals. The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant difference relative to the control (asterisk) is indicated (p < 0.05).

Figure 6

Transcriptional activity of genes related to epigenetic modulation (DNA methylase I [DNMT1], Lysine Acetyltransferase 6B [KAT6B], and histone deacetylase 1 [Hda1]), immunity (aplysianin-A [ApA]), and energy metabolism (glycogen phosphorylase L [PYGL] and oxysterol binding protein like 8 [OSBPL8]) in Physella acuta adults after in vivo exposure to Fenoxycarb for seven days at 19 °C. Transcriptional activity was quantified by RT-PCR using ribosomal protein L10 (rpL10), actin (Act), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes. The comparison was performed with the solvent-exposed controls. Whisker boxes are shown. Each box corresponds to 12 individuals. The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant difference relative to the controls (asterisk) is indicated (p < 0.05).