# Mitochondrial Gene Expression as a Novel Biomarker for Detecting and Discriminating Neurotoxic Pesticide Exposure in Ramulus phyllodeus (Chen & He, 2008)

## TL;DR

This study shows that changes in mitochondrial gene expression in a stick insect can detect and distinguish exposure to four neurotoxic pesticides.

## Contribution

The research introduces mitochondrial gene expression as a novel, compound-specific biomarker for neurotoxic pesticide exposure.

## Key findings

- Each pesticide induced a unique, non-overlapping pattern of mitochondrial gene upregulation.
- No gene was downregulated, suggesting activation of stress-response pathways rather than repression.
- Mitochondrial genes in R. phyllodeus serve as sensitive molecular indicators for pesticide exposure.

## Abstract

The application of synthetic pesticides remains a cornerstone of contemporary integrated pest management in agriculture; however, growing public and scientific concern surrounds the environmental persistence, non-target toxicity, and human health implications of pesticide residues. Chlorpyrifos, cyfluthrin, emamectin benzoate, and acetamiprid are four widely deployed neurotoxic insecticides that exhibit both contact and systemic toxicity. While their primary mechanisms involve disruption of neuronal signaling, emerging evidence indicates that secondary physiological consequences including impaired cellular energy metabolism and mitochondrial respiration are consistently observed following exposure. As central regulators of cellular bioenergetics, mitochondria harbor a compact, evolutionarily conserved genome encoding core subunits of the oxidative phosphorylation (OXPHOS) complexes. Their high metabolic activity, limited DNA repair capacity, and proximity to reactive oxygen species render them exceptionally vulnerable to xenobiotic stress. This functional and structural sensitivity prompts a critical question: can quantitative changes in mitochondrial gene expression serve as sensitive, mechanistically informed biomarkers for early detection and risk assessment of neurotoxic pesticide exposure?

This study investigates the mitochondrial transcriptomic responses of Ramulus phyllodeus (Chen & He, 2008); Phasmatodea: Phasmatidae) to acute exposure to four widely used neurotoxic insecticides: chlorpyrifos, cyfluthrin, emamectin benzoate, and acetamiprid. Using quantitative real-time PCR (qRT-PCR), we quantified transcriptional changes in 10 mitochondrial protein-coding genes, which showed significant transcriptional changes (p < 0.05) when the insect was exposed to four commonly used pesticides (each at a concentration of 5 μg/L) for 24 h. Exposure to chlorpyrifos induced significant upregulation of ND2 (2.08 ± 0.048) and ND5 (1.38 ± 0.15). Cyfluthrin triggered coordinated upregulation across seven genes: ND1 (1.71 ± 0.07), ND2 (2.33 ± 0.38), ND3 (1.74 ± 0.25), ND5 (1.65 ± 0.38), COX1 (2.91 ± 0.40), COX3 (1.69 ± 0.18), and Cytb (2.81 ± 0.53). Emamectin benzoate induced the upregulation of ND1 (1.98 ± 0.21), ND2 (3.04 ± 0.41), ND3 (1.82 ± 0.26), ND4 (2.79 ± 0.64), COX1 (2.36 ± 0.34), ATP6 (3.26 ± 0.61), and Cytb (2.39 ± 0.81). Acetamiprid induced more selective upregulation, affecting only ND1 (1.67 ± 0.18), ND4 (1.43 ± 0.16), and ND5 (1.66 ± 0.10). Critically, each insecticide elicited a distinct, non-overlapping transcriptional signature, defined by both the identity and magnitude of responsive genes, indicating compound-specific modulation of mitochondrial gene expression. Notably, no gene exhibited significant downregulation under any single-compound treatment, and all differentially expressed genes were upregulated exclusively in response to individual pesticides. This absence of transcriptional suppression suggests that these neurotoxicants converge on shared upstream stress-response pathways that preferentially activate mitochondrial biogenesis or compensatory transcription, rather than inducing global transcriptional repression. Collectively, these findings establish mitochondrial protein-coding genes in R. phyllodeus as sensitive, mechanistically grounded molecular sentinels for neurotoxic pesticide exposure. The compound-specific transcriptional profiles further suggest potential utility in multiplex detection strategies for environmental monitoring, enabling discrimination among individual residues.