Short-Term Evolutionary Features and Circadian Clock-Modulated Gene Expression Analysis of Piezo, nanchung, and αTubulin at 67C in a Romanian Population of Drosophila suzukii

Abstract

Drosophila suzukii is a successful invasive insect species responsible for agricultural losses. The key to its prowess is the ability to swiftly adapt to new environments through various genetic mechanisms, including fast accommodation of mutations and gene expression fine-tuning. Piezo and nanchung (nan) genes are linked to circadian clock-related behaviors and, therefore, are expected to readily respond to stress stimuli. Herein, we compared the DNA sequences of Piezo, nan, and αTubulin at 67C, a highly conserved housekeeping gene, in ICDPP-ams-1, a Romanian local population of D. suzukii, and two well-annotated reference populations from the United States of America and Japan. Our results imply that short-term evolutionary accumulated single nucleotide and indel variants are overrepresented within introns, a propensity evaluated through the mutation accumulation tendency (MAT) original parameter. Piezo and nan gene expression under photoperiodicity changes challenges were assessed in a series of experiments on three groups of individuals from ICDPP-ams-1. We found that both genes are upregulated in females if their customary circadian rhythm is affected, a trend seemingly reverting if, after an initial perturbation, the circadian clock is reset to its initial timing. In conclusion, we found that both highly conserved and adaptability-related genes are rapidly evolving and that Piezo and nan have a fast functional reaction to circadian clock changes by modifying their gene expression profiles.

Keywords: Drosophila suzukii; adaptability; circadian clock; gene expression; short-term evolution.

Figure 1

Graphical summary of the experiment investigating the influence of the LD cycle on the expression of Piezo and nan genes. P and F1 indicate the parental and, respectively, their first-generation descendants that were constantly exposed to a 12 h:12 h LD cycle. A selection of F1 descendants (F2) was subjected for a period of 13 days to either the same LD cycle as their parents or to experimental modifications to the LD cycle, supposing a single or two LD cycle switches after an initial eight-day interval of a 12 h:12 h LD cycle. After the termination of experimental monitoring, we processed genomic DNA material from females and performed qRT-PCR analysis on target genes. Created in BioRender.com (accessed on 16 April 2025).

Figure 2

The original incubator built for this experiment includes (1) a flexible lid, (2) a temperature controller, (3) timer-controlled electrical outlets, (4) a metal mesh, (5) a heating plate, and (6) a temperature sensor.

Figure 3

Graphical representation of multiple alignments performed by using gene and transcript nucleotide sequences corresponding to ICDPP-ams-1 (ams1), CBGP_Dsuzu_IsoJpt1.0 (Jpt), and Dsuz_RU_1.0 (RU) populations. From top to bottom, each multiple alignment provides an overview of the consensus sequence (selected coordinate values descriptive for the multiple alignment are represented above), identity fraction, with green coloring being indicated the regions containing identical nucleotides in all five sequences, the gene sequence from ams1, Jpt and RU, respectively, and the reference transcript sequences from Jpt and, lastly, RU. Regardless of the designated gene, Piezo (a), nan (b), or αTub (c), the continuous horizontal lines represented within the transcript sequences uncover the regions corresponding to the specific introns of the respective gene. The vertical black lines (representative for isolated single nucleotide variants) or rectangles (representative for successive single nucleotide variants or broader indels) from the inside of genes and transcripts visuals point to conflictual sites or subregions containing a difference in at least one sequence as compared with the other ones. Created in BioRender.com (accessed on 16 April 2025).

Figure 4

The phylogenetic trees showing the evolutionary history of Piezo, nan, and αTub gene structure from ICDPP-ams-1 (“gene name”_ams1), Dsuz_RU_1.0 (“gene name”_RU), and CBGP_Dsuzu_IsoJpt1.0 (“gene name”_Jpt) populations of D. suzukii. Regardless of the gene in question, when comparing the Romanian population with the other two, the minimum evolutionary distances are calculated between the ams1 and RU populations. The variant of each gene pertaining to ICDPP-ams-1 is highlighted with a green rectangle. Created in BioRender.com (accessed on 16 April 2025).

Figure 5

Graphical representations of log₂FC values calculated for qRT-PCR cumulative data and statistical summary obtained by implementing the Mann–Whitney U test. Individual graphs for both Piezo (a) and nan (b) genes were generated in qDATA. Asterisks indicate statistically significant differences: p value < 0.05 (*), p value < 0.01 (**), and p value < 0.0001 (****). Differences labeled ns are not statistically significant. Blue bars are representative of gene overexpression, and red bars for gene downregulation. Created in BioRender.com (accessed on 16 April 2025).