Characterization of PxZNF568, PxZNF93, and PxZNF266

The full-length sequence for PxZNF568 (LOC105380647) is composed of 2,752 base pairs (bp), which includes one intron and two exons. Its coding sequence (CDS) measures 900 bp, resulting in a protein that consists of 299 amino acids (aa). The calculated molecular weight and isoelectric point of PxZNF568 are 35.74 kDa and 9.90, respectively. Functional domain analysis predicts that PxZNF568 contains the ZnF_C2H2 zinc finger domain, as depicted in Additional File 1: Fig. S1 A.

Similarly, PxZNF93 (LOC119692916) also contains one intron and two exons, with a sequence length of 3,307 bp. The CDS for PxZNF93 is 2,340 bp, encoding a protein of 799 aa. This protein has a molecular weight of 92.23 kDa and an isoelectric point of 9.05, with predictions indicating the presence of the ZnF_C2H2 domain (Additional File 1: Fig. S1 B).

Finally, the full-length sequence of PxZNF266 (LOC105398739) spans 2,643 bp and includes three introns along with four exons. The CDS of PxZNF266 is 1,611 bp, leading to a protein comprising 537 aa. The molecular weight and isoelectric point calculated for PxZNF266 are 62.14 kDa and 8.70. Functional analyses suggest that PxZNF266 possesses both the ZnF_C2H2 and zf-AD domains (Additional File 1: Fig. S1 C).

Relative expression levels of PxZNF568, PxZNF93, and PxZNF266 showed significantly higher expression in HS compared to CS

To assess the expression profiles of the zinc finger proteins, we conducted quantitative PCR (qPCR) to compare the relative expression levels of PxZNF568, PxZNF93, and PxZNF266 between control strains (CS) and high-temperature strains (HS). All three genes showed significantly higher expression in HS compared to CS (Fig. 1).

Fig. 1

Relative expression level of PxZFP568 (A and B), PxZFP93 (C and D), and PxZFP266 (E and F) in CS and HS. Female: 1-day female adult; Male: 1-day male adult; CS:control strain; HS: hot-evolved strain. Independent-sample t-test was used. The relative expression level is represented as the mean ± SEM (n = 3). Asterisk (*) indicates P < 0.05; (**) indicates P < 0.01; (***) indicates P < 0.001

For PxZNF568, the relative expression levels in HS were substantially increased, being 5.29-fold higher in female adults and 4.92-fold higher in male adults compared to the CS group, with t-values of 34.31 (df = 4, P < 0.001) and 10.77 (df = 4, P < 0.001), respectively (Fig. 1 A, B).

In the case of PxZNF93, the expression in HS significantly increased by 2.79-fold in females and 3.14-fold in males compared to their CS counterparts, supported by t-values of 4.057 (df = 4, P = 0.02) for females and 4.838 (df = 4, P = 0.008) for males (Fig. 1 C, D).

Lastly, the expression of PxZNF266 indicated significant upregulation in HS, with a 1.54-fold increase in females and a 1.73-fold increase in males relative to the CS strain. The corresponding t-values were 9.777 (df = 4, P < 0.001) for females and 8.231 (df = 4, P = 0.001) for males (Fig. 1 E, F). These findings collectively suggest that PxZNF568, PxZNF93, and PxZNF266 are significantly upregulated in response to high-temperature conditions, indicating their crucial roles in the thermal adaptability of insects.

Enzymatic activity of CAT and total antioxidant capacity of HS were significantly greater than that of CS

To evaluate the antioxidant capacity differences between the control strains (CS) and high-temperature strains (HS), we measured the enzymatic activity of CAT along with the total antioxidant capacity. Antioxidant capacity of HS was significantly greater than that of CS (Fig. 2). Specifically, CAT activity in HS demonstrated substantial increases compared to both female and male CS adults, with t-values of 4.185 (df = 10, P = 0.001) and 7.050 (df = 10, P < 0.001), respectively (Fig. 2 A, B).

Fig. 2

Enzymatic activity of CAT (A and B) and total antioxidant capacity (C and D) in CS and HS. Female: 1-day female adult; Male: 1-day male adult; CAT: catalase; T-AOC: total antioxidant capacity; CS:control strain; HS: hot-evolved strain. Independent-sample t-test was used. Data are presented as mean value ± SEM (n = 6). Asterisk (*) indicates P < 0.05; (**) indicates P < 0.01; (***) indicates P < 0.001

Additionally, the total antioxidant capacity in HS showed marked enhancement compared to CS, with significant increases observed for female adults (t = 18.351, df = 10, P < 0.001) and male adults (t = 2.973, df = 10, P = 0.01) (Fig. 2 C, D). These findings confirm that high-temperature conditions lead to a significant upregulation of both CAT activity and overall antioxidant capacity.

Establishment of pure mutant strains for PxZNF568, PxZNF93, and PxZNF266

To generate mutant strains, 130 fresh eggs were microinjected with PxZNF568-SgRNA and Cas9. Out of these, 29.23% (38/130) successfully developed into adults (G0), with sequence alignment revealing that 7.89% (3/38) exhibited mutations. Subsequent mating with wild-type (WT) adults led to the analysis of the G1 generation, identifying a homozygous mutation of −5 bp, designated as PxZNF568-MU, in G5 (Fig. 3A).

Fig. 3

Identification of the mutant genotypes of PxZFP568 (A), PxZFP93 (B), and PxZFP266 (C). PAM (green): protospacer adjacent motif; sgRNA (red): Single guide RNA; WT: wild type (control strain); bp: base pairs

In a parallel experiment, 126 newly laid eggs were microinjected with PxZNF93-SgRNA and Cas9, resulting in a 24.60% (31/126) survival rate to adult (G0). Sequence analysis indicated that 6.45% (2/31) of G0 adults had mutations. Following mating and egg-laying, G1 was sequenced, revealing mutation types of −4 bp and −1 bp, leading to the establishment of two homozygous mutations, PxZNF93-MU-1 and PxZNF93-MU-4, in G4 (Fig. 3B and Additional File 1: Fig. S2 A).

For PxZNF266, a mixture of PxZNF266-SgRNA and Cas9 was injected into 118 eggs, resulting in 22.88% (27/118) developing into adults (G0). Sequencing indicated that 7.41% (2/27) of G0 had mutations. After mating and oviposition, the G1 generation was sequenced, identifying mutation types of −11 bp and + 1 bp. Consequently, two homozygous mutations were established in the G4 generation, designated as PxZNF266-MU-11 and PxZNF266-MU + 1 (Fig. 3C and Additional File 1: Fig. S2 B).

Survival rates of mutant strains were significantly lower than those of the WT in high temperature

We assessed the survival rates of both wild-type and mutant strains exposed to a temperature of 42 °C for various time intervals (1 h, 1.5 h, 2 h, 2.5 h, and 3 h). Survival rates of mutant strains were significantly lower than those of the WT (Fig. 4 and S3). In particular, the survival rate of female adults in the PxZNF568-MU strain was notably reduced at 2 h (t = 2.261, df = 10, P = 0.047) and 3 h (t = 4.029, df = 10, P = 0.007) (Fig. 4A), while male adults exhibited significant decreases at 2 h (t = 3.727, df = 10, P = 0.014) and 2.5 h (t = 13.328, df = 10, P < 0.001) (Fig. 4B).

Fig. 4

Effect of extremely high temperature on survival rates of WT, PxZFP568-MU, PxZFP93-MU-4, and PxZFP266-MU-11. H: hour; WT: wild type (control strain); MU: mutant strain. Independent-sample t-test was used. Data are presented as mean value ± SEM (n = 6). Asterisk (*) indicates P < 0.05; (**) indicates P < 0.01; (***) indicates P < 0.001

Comparing the PxZNF93-MU-4 strain to WT, female adults showed significantly reduced survival rates at 1.5 h (t = 2.985, df = 10, P = 0.027), 2 h (t = 3.997, df = 10, P = 0.002), 2.5 h (t = 2.777, df = 10, P = 0.028) (Fig. 4A), and 3 h (t = 2.535, df = 10, P = 0.029) (Fig. 4A), while male adults had significant reductions at 2 h (t = 3.312, df = 10, P = 0.021) and 2.5 h (t = 8.367, df = 10, P < 0.001) (Fig. 4B). Furthermore, the PxZNF93-MU-1 females demonstrated a significant decline in survival at 2 h (t = 2.834, df = 10, P = 0.017) and 2.5 h (t = 2.671, df = 10, P = 0.023) (Additional File 1: Fig. S3 A), while males showed similar reductions at 2 h (t = 4.108, df = 10, P = 0.009) and 2.5 h (t = 2.482, df = 10, P = 0.032) (Additional File 1: Fig. S3 B).

Additionally, female adults of PxZNF266-MU-11 exhibited lower survival rates at 1.5 h (t = 3.841, df = 10, P = 0.003), 2 h (t = 2.318, df = 10, P = 0.042), 2.5 h (t = 2.318, df = 10, P = 0.042), and 3 h (t = 2.683, df = 10, P = 0.029) (Fig. 4A), while male adults showed significant reductions at 1.5 h (t = 3.951, df = 10, P = 0.002), 2 h (t = 2.835, df = 10, P = 0.036), and 2.5 h (t = 17.112, df = 10, P < 0.001) (Fig. 4B). Lastly, the PxZNF93-MU-1 strain's females were significantly impacted at 1.5 h (t = 2.784, df = 10, P = 0.019), 2 h (t = 2.945, df = 10, P = 0.018), 2.5 h (t = 3.315, df = 10, P = 0.019), and 3 h (t = 3.070, df = 10, P = 0.011) (Additional File 1: Fig. S3 A), while males exhibited marked decreases at 1 h (t = 5.398, df = 10, P < 0.001), 1.5 h (t = 3.315, df = 10, P = 0.019), 2 h (t = 4.914, df = 10, P = 0.004), and 2.5 h (t = 14.546, df = 10, P < 0.001) (Additional File 1: Fig. S3 B).

Critical thermal maximum (CTMax) of mutant strains were significantly lower compared to the WT

The Critical Thermal Maximum (CTMax) for both WT and mutant strains was recorded at an initial temperature of 26 °C. As depicted in Fig. 5 and S4, the CTMax of mutant strains was significantly lower compared to the WT. Notably, the CTMax of females and males in the PxZNF568-MU strain were reduced significantly, with t-values of 8.782 (df = 38, P < 0.001) and 9.869 (df = 38, P < 0.001) respectively (Fig. 5A, B).

Fig. 5

Comparisons of CTMax of the WT, PxZFP568-MU, PxZFP93-MU-4, and PxZFP266-MU-11. CTMax: critical thermal maximum; Female: 1-day female adult; Male: 1-day male adult; WT: wild type (control strain); MU: mutant strain. Independent-sample t-test was used. Data are presented as mean value ± SEM (n = 20). Asterisk (***) indicates P < 0.001

Similarly, the CTMax for PxZNF93-MU-4 was significantly lower in both females (t = 14.036, df = 38, P < 0.001) and males (t = 7.138, df = 38, P < 0.001) compared to WT (Fig. 5A, B). Likewise, the CTMax values for PxZNF93-MU-1 exhibited significant reductions in both female (t = 5.469, df = 38, P < 0.001) and male (t = 6.816, df = 38, P < 0.001) adults relative to WT (Additional File 1: Fig. S4 A, B).

Lastly, in contrast to WT, the CTMax of females and males in the PxZNF266-MU-11 strain showed significant decreases, with t-values of 6.810 (df = 38, P < 0.001) and 5.397 (df = 38, P < 0.001), respectively (Fig. 5A, B). The CTMax of PxZNF266-MU + 1 also exhibited significant reductions in females (t = 3.130, df = 38, P = 0.003) and males (t = 7.730, df = 38, P < 0.001) compared to WT (Fig. S4A, B). ​

Population parameters of mutant strains were significantly lower compared to the WT

The developmental duration of the larval stage was significantly prolonged in mutant strains when compared to wild-type (WT) at both normal and elevated temperatures, with the exception of PxZFP93-MU-1 at normal temperature. Additionally, the fecundity of the mutant strains was considerably lower than that of WT across both temperature conditions. Oviposition rates were also significantly diminished in the mutant strains relative to WT, although PxZFP568-MU and PxZFP93-MU-4 exhibited similar oviposition rates as WT at high temperatures. The intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R0) for the mutant strains were consistently lower than WT values under both normal and high temperatures, except for PxZFP568-MU at normal temperature. Furthermore, the generation time (T) for the mutant strains was notably longer compared to WT at both temperature conditions, with the exceptions of PxZFP93-MU-4 and PxZFP93-MU-1, which displayed reversed trends under normal temperature conditions (Tables 1 and 2, Additional File 1: Table S1, and Additional File 1: Table S2).

Table 1 Developmental time, longevity, fecundity, oviposition, and population parameters on WT and mutant strains at normal temperatureTable 2 Developmental time, longevity, fecundity, oviposition, and population parameters on WT and mutant strains at high temperature

Survival rates at the immature stages, denoted as Sxj—the probability of neonates surviving to age x and stage j—varied across strains. Under normal temperature conditions, survival rates were recorded at 78.33% for WT, 60.83% for PxZNF568-MU, 69.17% for PxZNF93-MU-4, 37.50% for PxZNF93-MU-1, 60.00% for PxZNF266-MU-11, and 67.50% for PxZNF266-MU + 1. At high temperatures, these rates declined, recorded at 69.17%, 40.83%, 40.83%, 43.33%, 58.33%, and 40.83%, respectively (Fig. 6A-H and Additional File 1: Fig. S5 A-F).

Fig. 6

Age-stage survival rates (sxj) of WT and mutant strains of DBM at normal and high temperatures. Female: female adult; Male: male adult; WT: wild type (control strain); MU: mutant strain

The lx curve reflects changes in survival rates throughout the ages of the population. Notably, results indicated that mutant strains experienced a sharp decrease in survival during the immature stages resulting in a shortened overall lifespan at equivalent temperature conditions compared to WT. Conversely, the fxj curve, which illustrates the daily egg production per female of age x and stage j, showed that the highest daily fecundity for WT was 50.58 eggs. This was followed by the mutant strains, with PxZNF568-MU at 47.49 eggs, PxZNF93-MU-4 at 33.40 eggs, PxZNF93-MU-1 at 35.80 eggs, PxZNF266-MU-11 at 5.92 eggs, and PxZNF266-MU + 1 at 9.25 eggs at normal temperature. At high temperatures, these figures decreased, with WT producing 43.09 eggs, PxZNF568-MU achieving 43.54 eggs, PxZNF93-MU-4 reduced to 10.15 eggs, PxZNF93-MU-1 at 19.54 eggs, PxZNF266-MU-11 at 3.83 eggs, and PxZNF266-MU + 1 at 19.92 eggs.

Regarding the mx curve, which highlights the onset and duration of the reproductive phase, maximum daily oviposition rates occurred at varied times across strains. Specifically, these peaks were observed at 13 days for WT, 16 days for PxZNF568-MU, 12 days for PxZNF93-MU-4, and 16 days for PxZNF93-MU-1. For PxZNF266-MU-11 and PxZNF266-MU + 1, peak oviposition occurred at 13 days and 12 days, respectively, under normal temperature conditions, while the respective peak timings under high temperatures were 13 days, 14 days, 15 days, 13 days, 14 days, and 13 days (Fig. 7A-H and Additional File 1: Fig. S6 A-F).

Fig. 7

Age-specific survival rates (lx), female age-stage specific fecundity (fxj), and total population age specific fecundity (mx) of WT and mutant strains of DBM at normal and high temperatures. WT: wild type (control strain); MU: mutant strain

Different changes of enzymatic activity of CAT and total antioxidant capacity post-knockout

To investigate the roles of PxZNF568, PxZNF93, and PxZNF266 in antioxidant processes, we measured the enzymatic activity of CAT and total antioxidant capacity following knockout. The results indicated that CAT activity was significantly elevated in the mutant strains PxZNF568-MU, PxZNF93-MU-4, PxZNF93-MU-1, PxZNF266-MU-11, and PxZNF266 MU + 1 compared to WT, with statistical values as follows: t = 7.062 (df = 2.056, P = 0.016), t = 6.680 (df = 4, P = 0.003), t = 4.571 (df = 4, P = 0.010), t = 3.745 (df = 4, p = 0.002), and t = 4.447 (df = 4, P = 0.011). In contrast, the total antioxidant capacities of these mutant strains were significantly diminished, with statistical values of t = 6.811 (df = 4, P = 0.002), t = 11.227 (df = 4, P < 0.001), t = 14.619 (df = 4, P < 0.001), t = 9.827 (df = 4, P < 0.001), and t = 10.699 (df = 4, P < 0.001) (Fig. 8 and Additional File 1: Fig. S7).

Fig. 8

Enzymatic activity of CAT (A) and total antioxidant capacity (B) post-knockout. CAT: catalase; T-AOC: total antioxidant capacity; WT: wild type (control strain); MU: mutant strain. Independent-sample t-test was used. Data are presented as mean value ± SEM (n = 3). Asterisk (**) indicates P < 0.01; (***) indicates P < 0.001