Siqi Meng

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.

In the context of global warming, the frequency of severe weather occurrences, such as unexpected cold spells and heat waves, will grow, as well as the intensity of these natural disasters. Lizards, as a large group of reptiles, are ectothermic. Their body temperatures are predominantly regulated by their environment and temperature variations directly impact their behavior and physiological activities. Frequent cold periods and heat waves can affect their biochemistry and physiology, and often their ability to maintain their body temperature. Mitochondria, as the center of energy metabolism, are crucial for maintaining body temperature, regulating metabolic rate, and preventing cellular oxidative damage. Here, we used RT-qPCR technology to investigate the expression patterns and their differences for the 13 mitochondrial PCGs in Sphenomorphus incognitus (Squamata:Scincidae), also known as the brown forest skink, under extreme temperature stress at 4 °C, 8 °C, 34 °C, and 38 °C for 24 h, compared to the control group at 25 °C. In southern China, for lizards, 4 °C is close to lethal, and 8 °C induces hibernation, while 34/38 °C is considered hot and environmentally realistic. Results showed that at a low temperature of 4 °C for 24 h, transcript levels of ATP8, ND1, ND4, COI, and ND4L significantly decreased, to values of 0.52 ± 0.08, 0.65 ± 0.04, 0.68 ± 0.10, 0.28 ± 0.02, and 0.35 ± 0.02, respectively, compared with controls. By contrast, transcript levels of COIII exhibited a significant increase, with a mean value of 1.86 ± 0.21. However, exposure to 8 °C for 24 h did not lead to an increase in transcript levels. Indeed, transcript levels of ATP6, ATP8, ND1, ND3, and ND4 were significantly downregulated, to 0.48 ± 0.11, 0.68 ± 0.07, 0.41 ± 0.08, 0.54 ± 0.10, and 0.52 ± 0.07, respectively, as compared with controls. Exposure to a hot environment of 34 °C for 24 h led to an increase in transcript levels of COI, COII, COIII, ND3, ND5, CYTB, and ATP6, with values that were 3.3 ± 0.24, 2.0 ± 0.2, 2.70 ± 1.06, 1.57 ± 0,08, 1.47 ± 0.13, 1.39 ± 0.56, and 1.86 ± 0.12, respectively, over controls. By contrast, ND4L exhibited a significant decrease (to 0.31 ± 0.01) compared with controls. When exposed to 38 °C, the transcript levels of the 13 PCGs significantly increased, ranging from a 2.04 ± 0.23 increase in ND1 to a 6.30 ± 0.96 rise in ND6. Under two different levels of cold and heat stress, the expression patterns of mitochondrial genes in S. incognitus vary, possibly associated with different strategies employed by this species in response to low and high temperatures, allowing for rapid compensatory adjustments in mitochondrial electron transport chain proteins in response to temperature changes. Furthermore, this underscores once again the significant role of mitochondrial function in determining thermal plasticity in reptiles.

Ecological factors related to climate extremes have a significant influence on the adaptability of organisms, especially for ectotherms such as reptiles that are sensitive to temperature change. Climate extremes can seriously affect the survival and internal physiology of lizards, sometimes even resulting in the loss of local populations or even complete extinction. Indeed, studies have shown that the expression levels of the nuclear genes and mitochondrial genomes of reptiles change under low-temperature stress. At present, the temperature adaptability of reptiles has rarely been studied at the mitochondrial genome level. In the present study, the mitochondrial genomes of three species of lizards, Calotes versicolor, Ateuchosaurus chinensis, and Hemidactylus bowringii, which live in regions of sympatry, were sequenced. We used RT-qPCR to explore the level of mitochondrial gene expression under low-temperature stress, as compared to a control temperature. Among the 13 protein-coding genes (PCGs), the steady-state transcript levels of ND4L, ND1, ATP6, and COII were reduced to levels of 0.61 ± 0.06, 0.50 ± 0.08, 0.44 ± 0.16, and 0.41 ± 0.09 in C. versicolor, respectively, compared with controls. The transcript levels of the ND3 and ND6 genes fell to levels of just 0.72 ± 0.05 and 0.67 ± 0.05 in H. bowringii, compared with controls. However, the transcript levels of ND3, ND5, ND6, ATP6, ATP8, Cytb, and COIII genes increased to 1.97 ± 0.15, 2.94 ± 0.43, 1.66 ± 0.07, 1.59 ± 0.17, 1.46 ± 0.04, 1.70 ± 0.16, and 1.83 ± 0.07 in A. chinensis. Therefore, the differences in mitochondrial gene expression may be internally related to the adaptative strategy of the three species under low-temperature stress, indicating that low-temperature environments have a greater impact on A. chinensis, with a small distribution area. In extreme environments, the regulatory trend of mitochondrial gene expression in reptiles is associated with their ability to adapt to extreme climates, which means differential mitochondrial genome expression can be used to explore the response of different lizards in the same region to low temperatures. Our experiment aims to provide one new research method to evaluate the potential extinction of reptile species in warm winter climates.