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Zinc finger proteins facilitate adaptation of a global insect pest to climate change

BMC Biology volume 22, Article number: 303 (2024) Cite this article

Abstract

Background

Global climate change significantly impacts ecosystems, particularly through temperature fluctuations that affect insect physiology and behavior. As poikilotherms, insect pests such as the globally devastating diamondback moth (DBM), Plutella xylostella, are especially vulnerable to rising temperatures and extreme heat events, necessitating effective adaptive mechanisms.

Results

Here we demonstrate the roles of zinc finger proteins (ZFPs) in mediating thermal adaptability in DBM. We utilized a comprehensive approach involving cloning and bioinformatics analysis of three ZFPs, PxZNF568, PxZNF93, and PxZNF266, measurement of their expression levels in hot-evolved and control strains, and assessment of catalase enzymatic activity and total antioxidant capacity. We also employed CRISPR/Cas9 technology to create five stable homozygous knockout strains to elucidate ZFP functions in high-temperature tolerance. Survival rates under high-temperature stress and the critical thermal maxima (CTMax) of the knockout strains were significantly lower than the wild-type strain, and exhibited marked decreases in antioxidant capacity.

Conclusion

Findings reveal the importance of ZFPs in thermal adaptability of DBM, contributing critical insights for future pest management strategies in the context of a warming climate and laying the foundation for further exploration of ZFP functionality in agricultural pest control.

Background

Global climate change has profoundly altered numerous habitats, subjecting organisms to a diverse array of abiotic environmental stresses, including temperature fluctuations, drought, hypoxia, elevated CO2 levels, and increased UV radiation [1, 2]. Insects, being poikilotherms, are particularly susceptible to these climatic shifts [3]. In the context of global warming, both average temperatures and the frequency of extreme heat events are projected to rise [4]. As a critical abiotic stress factor, temperature exerts a significant influence on insect physiology and behavior [5].

Insects have evolved sophisticated mechanisms to perceive and respond to complex, dynamic temperature signals. These adaptations involve both phenotypic plasticity and genetic changes, enabling insects to maintain homeostasis in the face of environmental fluctuations [6, 7]. As global warming intensifies, the challenge of extreme high temperatures has become increasingly pressing [8].

Exposure to extreme high temperatures can trigger the generation of reactive oxygen species (ROS), including hydroxyl free radicals (·OH), hydrogen peroxide (H2O2), and superoxide anions (O2-) [9, 10]. Excessive ROS accumulation is detrimental to organisms, causing oxidative damage to DNA, proteins, and lipids. This oxidative stress can accelerate age-related diseases, cellular aging, and ultimately lead to cell death [11].

Zinc finger proteins (ZFPs) can reduce ROS levels, particularly H2O2, thereby protecting cells and enhancing organismal adaptation to oxidative challenges [12, 13]. For instance, Rhit, a member of the ZFP subfamily, protects against oxidative damage by decreasing cellular H2O2 production [14]. Similarly, ZFB1 defends mouse endothelial cells by reducing ROS expression [15], while ZNF667 safeguards rat myocardial H9C2 cells against oxidative stress through H2O2 elimination [16]. Given the established functions of ZFPs in mammals, it is plausible that these proteins may also contribute to temperature adaptation in insect pests.

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a globally significant migratory pest that causes extensive damage to cruciferous crops [17]. The economic impact of DBM-related damage and control measures is estimated at US$4–5 billion annually [18]. Notably, DBM has demonstrated considerable resilience and adaptability as it has achieved close to global distribution. This suggests potential to adapt to projected future climate conditions across various global regions maintaining its status as a major agricultural pest [19, 20]. Explanations for its resilience are poorly understood but need to be elucidated in order to understand and predict fundamental biological aspects such as distribution patterns and population dynamics as well as for developing effective pest management strategies and protecting natural enemies [21]. To facilitate the study of high-temperature adaptation in DBM, a hot-evolved strain (HS) has been selectively bred from a control strain (CS) under controlled climate chamber conditions [22]. However, the specific functions and mechanisms of ZFPs in mediating high-temperature adaptation in DBM remain unclear.

In this study, we conducted a comprehensive investigation of three zinc finger proteins in DBM: PxZNF568, PxZNF93, and PxZNF266, for which relative expression levels in hot-evolved strain (HS) were significantly higher than in control strains (CS). Our approach involved several key steps: i) Cloning and bioinformatics analysis of the three ZFPs; ii) Assessment of catalase (CAT) enzymatic activity and total antioxidant capacity in HS and CS, and iii) CRISPR/Cas9-mediated knockout of PxZNF568, PxZNF93, and PxZNF266 to elucidate their roles in high-temperature adaptability.​Our findings demonstrate that ZFPs play crucial roles in high-temperature adaptability of DBM. These results provide novel insights into potential pest control strategies in the context of global warming, offering a foundation for future research and practical applications in agricultural pest management.

Results

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
figure 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

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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
figure 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

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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
figure 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

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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
figure 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

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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
figure 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

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

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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
figure 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

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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
figure 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

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Discussion

Temperature is a crucial determinant of both the distribution and population dynamics of insects [23, 24], such as Plutella xylostella [19, 20]. Our research utilized CRISPR/Cas9 technology to knockout the zinc finger proteins PxZNF568, PxZNF93, and PxZNF266 in this species, revealing that these genes are integral to high-temperature adaptation, primarily through enhancing antioxidant capacities by mitigating the reactive oxygen species (ROS) generated under thermal stress.

PxZNF568, PxZNF93, and PxZNF266 belong to the zinc finger protein family and share the essential ZnF_C2H2 functional domain, suggesting they may collectively engage in the same mechanisms to reduce ROS levels induced by abiotic stresses [25]. Since the identification of the first zinc finger protein, TFIIIA, in Xenopus oocytes in 1983 [26], numerous studies have elucidated the significance of zinc finger proteins. C2H2-type zinc finger proteins are particularly abundant and have been thoroughly studied in eukaryotes, constituting approximately 7% of the total gene count—around 0.8% in yeast and 3% in the Diptera and mammals [27,28,29]. C2H2-type ZPFs comprises a conserved sequence of 25–30 amino acids, characterized by two cysteine and two histidine residues, following the general motif: X-X-C-X1–5-C-X12-H-X3–6-H (where X represents any amino acid and the subscripts denote specific residue counts) [29, 30].

Notably, the relative expression levels of PxZNF568, PxZNF93, and PxZNF266 were significantly elevated in HS strains compared to control strains (CS), indicating that P. xylostella may increase the expression of these genes in response to high temperatures. A similar pattern was observed in female Spodoptera frugiperda, where ZFPs experienced significant upregulation under heat stress [31]. The RNA-seq of Psylliodes chrysocephala revealed that, among other genes, many ZFPs changed in expression in summer diapause, which conferred resistance to high temperatures in a beetle [32]. In Apis cerana, the expression of AcZFP41 was markedly higher at 45 °C, peaking after four hours [33, 34]. Furthermore, we observed that the enzymatic activity of CAT and the total antioxidant capacity of HS were significantly greater than those in CS. In addition to ZFPs, ROS generated by high temperatures can be countered by other enzymes such as catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), and superoxide dismutase (SOD), which collectively help protect against oxidative damage [9, 35, 36].

While RNA interference (RNAi) displays limited efficiency in Lepidoptera compared to other insect orders [37], the CRISPR/Cas9 system has emerged as a valuable tool for investigating gene functions in DBM [38, 39]. This study successfully employed this system to knock out PxZNF568, PxZNF93, and PxZNF266, resulting in the establishment of five stable homozygous mutant strains (PxZNF568-MU, PxZNF93-MU-4, PxZNF93-MU-1, PxZNF266-MU-11, PxZNF266-MU + 1). Assessments of survival under extreme high-temperature conditions (42 °C for durations of 1 to 3 h) revealed a significant decrease in survival rates across all mutant strains compared to wild type (WT). Additionally, the critical thermal maximum (CTMax) measurements indicated that mutant strains had significantly lower tolerance levels than WT.

These results underscore the essential roles of PxZNF568, PxZNF93, and PxZNF266 in maintaining a functional redox state, thereby safeguarding DBM from oxidative damage under extreme thermal conditions. The involvement of ZFPs in mitigating oxidative stress and enhancing resilience to environmental pressures has been well-documented, particularly in plants [40,41,42,43,44]. In rice (Oryza sativa), for example, the upregulation of OsZFP350 has been shown to enhance germination rate under abiotic stress while mitigating heat stress during root development [40]. Additionally, the expression of Zat12 and Zat7 increased in response to heat shock, with transgenic plants expressing these ZFPs exhibiting enhanced tolerance to oxidative stress [43].

We documented life table parameters for both WT and mutant strains at varying temperatures, revealing the intrinsic rate of increase (r), net reproductive rate (R0), and finite rate of increase (λ) between the two groups. At high temperatures, the mutant strains exhibited significantly lower life history parameters (e.g., r values of PxZNF568-MU: 0.13 ± 0.01, PxZNF93-MU-4: 0.05 ± 0.01, PxZFP266-MU-11: 0.14 ± 0.01), indicating that the absence of PxZNF568, PxZNF93, and PxZNF266 negatively impacts the capacity of DBM to tolerate thermal stress.

Interestingly, while CAT activity was significantly higher in mutant strains compared to WT, total antioxidant capacity was diminished, highlighting a potential compensatory mechanism that could be at play. ROS are unavoidable byproducts of various metabolic processes, with the potential to inflict damage on cellular structures [45,46,47,48]. To counteract ROS, cells employ multiple antioxidant pathways—both enzymatic and non-enzymatic—maintaining a delicate balance between ROS production and clearance to uphold cellular homeostasis [49]. Consistent with our findings, AcZFP41-silenced honeybees also demonstrated enhanced enzyme activity in CAT, SOD, POD, and GST, further supporting the notion that ZFPs are vital for managing oxidative stress [33].

Conclusions

In conclusion, our findings reveal that P. xylostella adapts to elevated temperatures by upregulating zinc finger proteins (ZFPs), which enhance its antioxidant capacity. These insights offer a significant contribution to pest management, particularly in addressing the challenges posed by climate-induced pest outbreaks. The functional role of ZFPs, such as PxZNF568, PxZNF93, and PxZNF266, as transcriptional regulators of temperature-responsive genes, underscores their potential as targets for innovative control strategies. Moreover, the parallels with AcZFP41 in honeybees, where ZFP expression is induced by oxidative stress from pesticides, highlight the broader implications of targeting ZFP pathways for pest mitigation [33]. Leveraging this knowledge, future strategies could focus on disrupting ZFP-mediated stress adaptation to suppress P. xylostella populations more effectively under global warming conditions. Such approaches could advance sustainable pest management practices while reducing reliance on chemical pesticides.

While our study highlights distinct phenotypic effects from individual ZFP mutations, the potential for functional redundancy among these genes remains a crucial question. Generating double and triple mutants will be essential to determine whether simultaneous disruption produces additive or synergistic effects, offering further clarity on their collective role in thermal adaptation. These investigations will not only refine our understanding of ZFP functions but also pave the way for the development of sustainable pest management solutions in the face of global warming.

Methods

Insect

A control strain of P. xylostella (CS), and hot-evolved strain (HS) were used in this study. The CS was provided by the Institute of Zoology, Chinese Academy of Sciences in 2012, and reared in a climate chamber at 26 °C. Larvae were fed on an artificial diet in plastic disposable 90 mm Petri dishes [50]. Pupae were transferred into new disposable paper cups until emergence. Newly emerged adults were reared with 10% honey solution for mating and oviposition. The heat-evolved strain (HS) was kept at 27 °C for 12 h (dark) and exposed to 32 °C for 12 h (light) over the course of one year [22]. The HS was maintained at 26 °C for two generations prior to experimental studies to eliminate environmental effects [51].

Total RNA isolation and cDNA synthesis

Total RNA was extracted from the 1-day adults of P. xylostella using an Animal Tissue RNA Extraction Kit (Promega, Madison, WI, USA) as described in the supplier's instructions. The concentration of RNA was assessed by Nano Drop 2000 spectrophotometry (Nano Drop, Wilmington, DE, USA). Then, the cDNAs were synthesized by Fast King gDNA Dispelling RT Super Mix (Tiangen, Beijing, China) according to the following reactions: 4 μL of 5 × FastKing-RT SuperMix, 50 ng-2 μg Total RNA, and RNase-Free ddH2O to make up 20 μL total volume. The reaction mixture was incubated for 42 °C for 15 min and 95 °C for 3 min.

Gene clone and sequence analysis

The sequence of PxZNF568, PxZNF93, and PxZNF266' was obtained from the ilPluXylo3.1 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_932276165.1/) [52]. Gene-specific primers (Table 3) for the coding sequences (CDSs) of PxZNF568, PxZNF93, and PxZNF266 were designed by Primer 3 Plus (https://www.primer3plus.com/). The CDSs of the three genes were amplified using PCR in a 50-μL reaction mixture (Vazyme, Nanjing, China) including 25 μL of 2 × Phanta Max Buffer, 2 μL of cDNA template, 2 μL of Forward Primer (10 μM), 2 μL of Reverse Primer (10 μM), 1 μL of dNTP Mix, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 17 μL of ddH2O. The amplification program was set as follows: 95 °C for 3 min; 34 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s; and a final extension step at 72 °C for 5 min. The PCR fragment was purified with a Gel Extraction Kit (Omega, Norcross, GA, USA) and subcloned into a pESI-Blunt Simple Vector (YEASEN, Shanghai, China). Three positive clones were sent to Biosune Biotech Company (Fuzhou, China) for final sequencing.

Table 3 Primers used in this study
Full size table

Protein-coding sequences were predicted by Translate Server (https://web.expasy.org/translate/). The molecular weight and isoelectric point were predicted by Expasy (https://web.expasy.org/protparam/). The signal peptides were predicted by SignalP 6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/). The functional domains were predicted with SMART (http://smart.embl-heidelberg.de/).

Relative expression level of PxZNF568, PxZNF93, and PxZNF266 in CS and HS

To compare the relative expression level of PxZNF568, PxZNF93, and PxZNF266 between CS and HS, we performed real-time quantitative PCR (qPCR). The specific primers (Table 3) of PxZNF568, PxZNF93, and PxZNF266 were designed by INTEGRATED DNA TECHNOLOGIES (https://sg.idtdna.com/Primerquest/Home/Index). The internal reference gene was the RPL32of DBM to normalize target gene expression and correct for sample-to-sample variation [22]. Three biological replicates with three technical replicates were used for each sample. Total RNA was extracted for 1-day female and male adults using the same method as in Sect. 2.2. The qPCR was performed with a 20-μL reaction system (Vazyme, Nanjing, China) containing 7.15 μL of Nuclease Free Water, 10 μL of 2 × Taq Pro Universal qPCR Master Mix, 0.4 μL of Forward Primer (10 μM), 0.4 μL of Reverse Primer (10 μM), and 2 μL of cDNA (500 ng μL–1). The qPCR was conducted on an ABI Prism 7500 Fast Detection System (Applied Biosystems, Carlsbad, CA, USA) followed by the conditions: 95 °C for 30 s; 40 cycles of 95 °C for 10 s and 60 °C for 30 s; melt curve step at 95 °C for 15 s; 60 °C for 1 min; and 95 °C for 15 s. The relative expression level was calculated using the comparative 2–∆∆CT method [53].

Determination of enzymatic activity of catalase (CAT) and total antioxidant capacity of CS and HS

To verify the relationship between high-temperature adaptability and antioxidant capacity in the DBM, we determined enzymatic activity of CAT and total antioxidant capacity of CS and HS. 1-day female and male post emergence adults were divided into six biological replicates respectively. All methods were performed according to the manufacturer’s instructions. The enzymatic activity of CAT was investigated using a catalase assay kit (Mlbin, Shanghai, China). Total antioxidant capacity was measured using a total antioxidant capacity assay kit (Mlbin, Shanghai, China).

Single guide RNA (sgRNA) design and synthesize

The sgRNA design was based on protospacer adjacent motif (PAM) (5’-NGG-3′) principle, with potential off-target effects, predicted using Cas-OFFinder (http://www.rgenome.net/cas-offinder/). The sgRNA recognition site of PxZNF568, PxZNF93, and PxZNF266 were both screened in exon 1. The specific primers (Table 3) of PxZNF568, PxZNF93, and PxZNF266 were used to amplify the transcription templates with the same method as in Sect. 3. The sgRNA of PxZNF568, PxZNF93, and PxZNF266 were transcribed using a HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA) following the manufacturer’s instructions and purified using phenol: chloroform extraction.

sgRNA/Cas9 protein microinjection

A reaction mixture containing 1 μL of 10 × Reaction Buffer, 300 ng μL–1 of sgRNA, 200 ng μL–1 of Cas9 protein (GenCrispr, Nanjing, China), and Nuclease-Free Water to made up 10 μL total volume, was incubated at 37 °C for 20 min to form a stable ribonucleoprotein (RNP) complex. Fresh eggs within 15 min were microinjected with an Olympus SZX16 microinjection system (Olympus, Japan).

Mutation screening

A series of continuing crossing was performed to screen stable homozygous lines of PxZNF568, PxZNF93, and PxZNF266 [54]. The microinjected eggs were considered as generation 0 (G0).Virgin G0 adults were crossed with virgin WT adult (not microinjected) to lay transgenic lines designated as generation 1 (G1). All genomic DNAs of crossed G0 were extracted with the TiANamp Genomic DNA Kit (Tiangen, Beijing, China) and genotyped by sequencing under the same methods as in Sect. 2.3. The sequence chromatograms contained double peaks in the nucleotide positions near the sgRNA target site of G0 was considered to carry the mutant alleles. Virgin G1 adults were sibling mated to generate G2. All sibling mated G1 individuals were genotyped by sequencing. G2 individuals containing the same allelic mutation were sibling crossed to get G3. G3 individuals containing homozygous mutations were retained to establish homozygous lines (mutant, hereafter called MU). If heterozygotes were obtained, in-crossing continued until homozygous mutations were established.

Response to extreme high temperature

DBM strains used in this assay were WT and mutant adults. Six biological replicates were used for each sample. Each sample was respectively exposed to 42 °C in the climate chamber with 1 h, 1.5 h, 2 h, 2.5 h, and 3 h. For example, the first group of 1-day female and male adults were exposed to a temperature of 42 °C for 1 h. Here were five different time intervals and we need to perform this experiment for 5 times. After heat shock, each sample was placed in another climate chamber for 24 h at 26 °C and meanwhile counted the survival rate. DBM was regarded to be alive if any movement of a limb including antennae, feet, and mouthpart was discernible.

Response to elevated temperature

In order to simulate the continuous warming process of the real temperature change in the field, the critical high temperature (CTMax) when the DBM can not crawl or fly after spasms during the gradual heating process was used as an index to measure its heat tolerance. DBM strains used in this assay were WT and mutant adults. Twenty adults were used in this study. The starting temperature and the heating rates were 26 ℃ and 0.25 ℃ min–1. The adults were put into the heating device and the temperature when DBM reached the critical state during the heating process was recorded.

Age-stage-specific sex life table

The life tables (development time, survival, and reproduction) of WT and mutant strains at normal and high temperature were investigated and compared. 120 fresh eggs of each strain were placed in plastic disposable 30 mm Petri dishes. The newly 1st instar larvae were counted and transferred to plastic disposable 60 mm Petri dishes with enough artificial diet. The survival and development times of each developmental stage were recorded daily. One pair of newly emerged female and male adults was placed in a disposable 25 mL plastic cup and 10% honey solution was provided. Eggs laid by each female adult were recorded daily until its death. Survivorship, fecundity, oviposition period, and total life span were also recorded daily.

The data of life tables were analyzed using TWOSEX-MSChar computer program (http://140.120.197.173/ecology/prod2.htm). The age-stage survival rate (Sxj), the age-specific survival rate (lx), the age-specific fecundity (mx), the age-stage-specific fecundity (fxj), the mean generation time (T), the intrinsic rate of increase (r), the finite rate of increase (λ), and the net reproductive rate (R0) were calculated. The formulae used were as follows:

$${l}_{x}=\sum_{j=1}^{m}sxj$$
$${m}_{x}=\frac{\sum_{j=1}^{m}{{s}_{xj}f}_{xj}}{\sum_{j=1}^{m}{s}_{xj}}$$
$${R}_{0}=\sum_{x=0}^{\infty }{{l}_{x}m}_{x}$$
$$\sum_{x=0}^\infty{{e^{-r(x+1)}l}_xm}_x=1$$
$$\lambda ={e}^{r}$$
$$T=({l}_{n}{R}_{0})/r$$

The variances and standard errors of parameters were subjected to 100,000 re-samplings using the bootstrap technique. Graphs were made using SigmaPlot 12.0.

Determination of enzymatic activity of CAT and total antioxidant capacity after knockout

To investigate whether knockout of PxZNF568, PxZNF93, and PxZNF266 in P. xylostella had an effect on the antioxidant capacity, we measured antioxidant capacity of WT and mutant strains. The methods was the same as in Sect. 5.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

DBM:

Diamondback moth

ZFPs:

Zinc finger proteins

CTMax:

Critical thermal maxima

ROS:

Reactive oxygen species

·OH:

Hydroxyl free radicals

H2O2 :

Hydrogen peroxide

O2- :

Superoxide anions

HS:

Hot-evolved strain

CS:

Control strain

CAT:

Catalase

CDS:

Coding sequence

bp:

Base pairs

aa:

Amino acids

qPCR:

Quantitative PCR

WT:

Wild-type

MU:

Mutant strain

r :

Intrinsic rate of increase

λ :

Finite rate of increase

R 0 :

Net reproductive rate

T :

Generation time

POD:

Peroxidase

GST:

Glutathione S-transferase

SOD:

Superoxide dismutase

RNAi:

RNA interference

s xj :

Age-stage survival rates

l x :

Age-specific survival rates

f xj :

Female age-stage specific fecundity

m x :

Total population age specific fecundity

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Acknowledgements

Not applicable.

Funding

This work was supported by the Fujian Natural Science Fund for Distinguished Young Scholars (2022J06013), the central government-guided local science and technology development projects (2022L3087), and the Youth Teachers Visit and Study Program at the Haixia Institute of Science and Technology (KFXH23021).

Author information

Author notes

  1. Tianpu Li and Jiao Guo contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Ecological Pest Control of Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Tianpu Li, Jiao Guo, Guilei Hu, Fang Cao, Haiyin Su, Mengdi Shen, Huimin Wang, Minsheng You, Geoff M. Gurr & Shijun You

  2. Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou, 350002, China

    Tianpu Li, Jiao Guo, Guilei Hu, Fang Cao, Haiyin Su, Mengdi Shen, Huimin Wang, Minsheng You, Geoff M. Gurr & Shijun You

  3. Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Tianpu Li, Jiao Guo, Guilei Hu, Fang Cao, Haiyin Su, Mengdi Shen, Huimin Wang, Minsheng You & Shijun You

  4. Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture and Rural Affairs, Fuzhou, 350002, China

    Tianpu Li, Jiao Guo, Guilei Hu, Fang Cao, Haiyin Su, Mengdi Shen, Huimin Wang, Minsheng You & Shijun You

  5. Key Laboratory of Green Control of Insect Pests of Fujian Province, Fuzhou, 350002, China

    Tianpu Li, Jiao Guo, Guilei Hu, Fang Cao, Haiyin Su, Mengdi Shen, Huimin Wang, Minsheng You & Shijun You

  6. Haixia Lnstitute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Yuanyuan Liu

  7. Gulbali Institute, Charles Sturt University, Orange, NSW, 2800, Australia

    Geoff M. Gurr

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T.P.L.: Methodology; Writing-original draft preparation; Data curation; Formal analysis. J.G.: Methodology; data curation; formal analysis; writing–original draft. G.L.H.: Methodology; Investigation; Data curation. F.C., H.Y.S., M.D.S., and H.M.W.: Methodology; writing–review and editing. M.S.Y. and Y.Y.L.: Reviewed manuscript for significant intellectual content and scholarly contribution to interpretation of results; Supervision. G.M.G.: Conceptualization; Writing-review and editing; Supervision. S.J.Y.: Conceptualization; Writing-review and editing; Supervision; Funding acquisition; Project administration. All authors reviewed the manuscript.

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Correspondence to Geoff M. Gurr or Shijun You.

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Supplementary Information

12915_2024_2109_MOESM1_ESM.docx

Additional file 1: Fig. S1 Functional domains of PxZFP568 (A), PxZFP93 (B), and PxZFP266 (C). Fig. S2 Identification of the mutant genotypes of PxZFP93 (A) and PxZFP266 (B). Fig. S3 Effects of extremely high temperature on survival rates of the WT, PxZFP93-MU-1, and PxZFP266-MU+1. Fig. S4 Comparisons of CTMax of WT, PxZFP93-MU-1, and PxZFP266-MU+1. Table S1 Developmental time, longevity, fecundity, oviposition, and population parameters on WT and mutant strains at normal temperature. Table S2 Developmental time, longevity, fecundity, oviposition, and population parameters on WT and mutant strains at high temperature. Fig. S5 Age-stage survival rates (sxj) of WT and mutant strains of DBM at normal and high temperatures. Fig. S6 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. Fig. S7 Enzymatic activity of CAT (A) and total antioxidant capacity (B) post-knockout.

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Li, T., Guo, J., Hu, G. et al. Zinc finger proteins facilitate adaptation of a global insect pest to climate change. BMC Biol 22, 303 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-024-02109-3

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BMC Biology

ISSN: 1741-7007

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