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Identification and expression analysis of cathepsin B genes in Myzus persicae (Hemiptera: Aphididae) and their response to environmental stresses

Published online by Cambridge University Press:  31 March 2025

Jin-Shan Zhang Open the ORCID record for Jin-Shan Zhang [Opens in a new window] ,
Jian-Yu Meng ,
Lei Yang  and
Chang-Yu Zhang Open the ORCID record for Chang-Yu Zhang [Opens in a new window]
Jin-Shan Zhang
Affiliation:
Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Institute of Entomology, Guizhou University, Guiyang 550025, China
Jian-Yu Meng
Affiliation:
Guizhou Tobacco Science Research Institute, Guiyang 550081, China
Lei Yang
Affiliation:
China Tobacco Hunan Industrial Co., Ltd., Changsha 410007, China
Chang-Yu Zhang*
Affiliation:
Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Institute of Entomology, Guizhou University, Guiyang 550025, China
*
Corresponding author: Chang-Yu Zhang; Email: zcy1121@aliyun.com
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Abstract

Cathepsin B (CTSB) is a cysteine protease that is widely found in eukaryotes and plays a role in insect growth, development, digestion, metamorphosis, and immunity. In the present study, we examined the role of CTSB in response to environmental stresses in Myzus persicae Sulzer (Hemiptera: Aphididae). Six MpCTSB genes, namely MpCTSB-N, MpCTSB-16D1, MpCTSB-3098, MpCTSB-10270, MpCTSB-mp2, and MpCTSB-16, were identified and cloned from M. persicae. The putative proteins encoded by these genes contained three conserved active site residues, i.e. Cys, His, and Asn. A phylogenetic tree analysis revealed that the six MpCTSB proteins of M. persicae were highly homologous to other Hemipteran insects. Real-time polymerase chain reaction revealed that the MpCTSB genes were expressed at different stages of M. persicae and highly expressed in winged adults or first-instar nymphs. The expression of nearly all MpCTSB genes was significantly upregulated under different environmental stresses (38°C, 4°C, and ultraviolet-B). This study shows that MpCTSB plays an important role in the growth and development of M. persicae and its resistance to environmental stress.

Keywords

cysteine proteasesgene expressiongreen aphidtemperatureultraviolet-B

Information

Type
Research Paper
Information
Bulletin of Entomological Research , Volume 115 , Issue 3 , June 2025 , pp. 329 - 339
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Copyright
© The Author(s), 2025. Published by Cambridge University Press.

Introduction

Insects are ectotherms, and their survival, population dynamics, and geographical distribution are determined by their ability to adapt to extreme environments (Chown and Terblanche, Reference Chown and Terblanche2006; Filazzola et al., Reference Filazzola, Matter and MacIvor2021; González-Tokman et al., Reference González-Tokman, Córdoba-Aguilar, Dáttilo, Lira-Noriega, Sánchez-Guillén and Villalobos2020). Temperature is a major source of environmental pressure that influences the biological activities of insects, including growth, development, physiological metabolism, lifespan, and reproduction (Herrera et al., Reference Herrera, Medrano, Rey, Sanchez-Lafuente, Garcia, Guitian and Manzaneda2002; Ji et al., Reference Ji, Li, Zhou and Yin2021; Kamel et al., Reference Kamel, Osman, Mahmoud, Haggag, Aziz and Shebl2019; Zhu et al., Reference Zhu, Wang and Ma2019). For instance, Aulacorthum solani nymphs cannot survive at extreme temperatures of 2.5°C and 30°C (Seo et al., Reference Seo, Kim, Ahn, Kim, Kang and Jung2020). The development of Acyrthosiphon gossypii is significantly prolonged at 36°C, and its reproduction, survival, and intrinsic rate of increase are inhibited (Gao et al., Reference Gao, Feng, Perkins, Sharma and Lu2018). With continuous changes in the global climate, the destruction of the ozone layer has resulted in an increase in solar ultraviolet (UV) radiation, which exerts varying degrees of stress on the entire biosphere (Madronich et al., Reference Madronich, McKenzie, Caldwell and Björn1995; McKenzie et al., Reference McKenzie, Aucamp, Bais, Björn and Ilyas2007; Xu and Sullivan, Reference Xu and Sullivan2010). UV-B (280–315 nm) is the most prominent waveband affecting biological activity, and high doses of UV-B radiation can cause damage to DNA, proteins, and membrane lipids, resulting in injury to the organism or death (Culotta, Reference Culotta1994; Douki et al., Reference Douki, von Koschembahr and Cadet2017; Hideg et al., Reference Hideg, Jansen and Strid2013). However, throughout a long evolutionary history, a number of species have developed a series of stress-response mechanisms, including antioxidants, programmed cell death, heat shock protein, and detoxification genes, to defend against UV-B stress (Li et al., Reference Li, Yuan, Gou, Zhang, Zhang, Zhou and Liu2021; Meng et al., Reference Meng, Zhang and Lei2010; Yang et al., Reference Yang, Meng, Yao and Zhang2021a; Zhang et al., Reference Zhang, Wang, Liu, Wang and Xu2021).

Cathepsins are a class of protein hydrolases found in lysosomes and are widely distributed in animals and plants (Brix et al., Reference Brix, Dunkhorst, Mayer and Jordans2008; Wu et al., Reference Wu, Zou, Jia, Wang, Zhang, Guo and Gui2011). They can be divided into cysteine proteases (cathepsin B, F, C, H, K, L, O, S, V, and W), aspartic proteases (cathepsin D and E), and serine proteases (cathepsin A and G) based on their active sites (Bond and Butler, Reference Bond and Butler1987; Rawlings et al., Reference Rawlings, Waller, Barrett and Bateman2014; Turk et al., Reference Turk, Turk and Turk2000). Cathepsin B (CTSB) is a cysteine protease that belongs to the papain family with a catalytic triad containing three conserved active site residues, i.e. cysteine, histidine, and asparagine (Cavallo-Medved et al., Reference Cavallo-Medved, Moin and Sloane2011; Kirschke, Reference Kirschke1995). CTSB is involved in various processes in insects, including embryo development, digestion, apoptosis, tissue differentiation, and metamorphosis (Medina et al., Reference Medina, León and Vallejo1988; Ortego et al., Reference Ortego, p, Ruíz, Marco and Castañera1998; Wang et al., Reference Wang, Niu, Hu, Zhu, Wang, Han, Qian, Tian, Yuan and Lou2019; Yan et al., Reference Yan, Cheng, Li and Aksoy2002). In addition, environmental abiotic stress (temperature, UV, pH, drought, salt, and phytohormones) affects the transcription levels of CTSB (Han et al., Reference Han, Chae, Choi, Kim, Chae and Kim2009; Peng et al., Reference Peng, Zhang, Zheng, Liang, Li, Chen, Guo, Guo, Yoshina, Chen, Zhao, Wu, Liu, Mitani, Yu and Xue2017; Wen et al., Reference Wen, Jiang, Liu, Zhou, Sun, Shi, Zhu and Wu2021). For instance, low temperature stress induces the accumulation of HvCTSB transcriptional products in Hordeum vulgare (Martinez et al., Reference Martinez, Rubio-Somoza, Carbonero and Diaz2003). High temperature and H2O2 stress upregulate the expression of BmCTSB and BmCTSD in Bombyx mori, thereby resulting in reduced larval lifespan and earlier pupation (Kim et al., Reference Kim, Lee, Sohn, Kim, Choi, Kang and Jin2011; Wu et al., Reference Wu, Zou, Jia, Wang, Zhang, Guo and Gui2011). The apoptosis regulation and autophagy by TuCTSB in Tetranychus urticae follow UV-B exposure (Murata and Osakabe, Reference Murata and Osakabe2017). Aphis gossypii improves tolerance to high-temperature environments by increasing the expression of heat shock protein 70 (Hsp70) and AgCTSB genes (Liu et al., Reference Liu, Liu, Li and Lu2023).

The green peach aphid Myzus persicae Sulzer (Hemiptera: Aphididae) is an important agricultural pest that is distributed worldwide and can harm >400 plant species in 50 families, including Cruciferae, Solanaceae, Rosaceae, and Leguminosae (Bass et al., Reference Bass, Puinean, Zimmer, Denholm, Field, Foster, Gutbrod, Nauen, Slater and Williamson2014; Mathers et al., Reference Mathers, Chen, Kaithakottil, Legeai, Mugford, Baa-Puyoulet, Bretaudeau, Clavijo, Colella, Collin, Dalmay, Derrien, Feng, Gabaldón, Jordan, Julca, Kettles, Kowitwanich, Lavenier, Lenzi, Lopez-Gomollon, Loska, Mapleson, Maumus, Moxon, Price, Sugio, van Munster, Uzest, Waite, Jander, Tagu, Wilson, van Oosterhout, Swarbreck and Hogenhout2017). M. persicae is a pest with piercing–sucking mouthparts. It not only feeds directly on plant tissues to cause damage but also serves as a medium to spread a variety of plant viruses that cause various diseases (Brault et al., Reference Brault, Uzest, Monsion, Jacquot and Blanc2010; Pitino and Hogenhout, Reference Pitino and Hogenhout2013). M. persicae has been reported to be able to survive from 4°C to 39°C, with an optimum temperature for population growth of 26.7°C (Davis et al., Reference Davis, Radcliffe and Ragsdale2006). However, high temperature stress shortens the nymphal stage and adult longevity, and reduces the mean generation time and the net reproductive rate (Khurshid et al., Reference Khurshid, Inayat, Ali, Tamkeen, Tahir, Niaz, ul, Ghramh, Boamah, Zhang and Liu2022; Pan et al., Reference Pan, Shen, Fu, Lu, Ma and Liu2024). In contrast, low temperatures extend the nymphal development time, reduce the developmental rate, and impair reproductive capacity (Baral et al., Reference Baral, Thapa, Jha, Joshi and Aryal2022; Liu and Meng, Reference Liu and Meng1999; Saleesha et al., Reference Saleesha, Kennedy, Rajabaskar and Geethalakshmi2022). Additionally, UV-B stress reduces the survival rate of M. persicae, inhibits nymphal growth and development, and leads to a decrease in population numbers (Gidske, Reference Gidske2020). However, only a few studies have focused on the physiological role of CTSB in insect resistance to environmental stress. In this study, we identified six MpCTSB genes from M. persicae and used quantitative real-time polymerase chain reaction (qRT-PCR) to measure their expression levels at different developmental stages and following exposure to various temperatures and UV-B stress. The aim of this study is to provide insights into the adaptation mechanism of M. persicae in response to environmental stress and the physiological function of CTSB.

Materials and methods

Insects

The nymphs and adult M. persicae insects used in this study were collected from tobacco plants in the fields of Guizhou, China (E 107°0′, N 27°17′). They were reared on young tobacco leaves in an artificial climate chamber with a temperature of 25°C ± 1°C, relative humidity of 70–80%, and a 14L:10D photoperiod.

Identification and analysis of MpCTSB gene sequences

Total RNA was extracted from the wingless adults of M. persicae using the Easte® Super Total RNA Extraction Kit (Shanghai Promega, USA). RNA quality and concentration were assessed using 1% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). cDNA was synthesised from 50 pg to 5 μg of RNA samples using the HiFiScript cDNA Synthesis Kit (CoWin Biosciences, Beijing, China) (Yang et al., Reference Yang, Meng, Zhou and Zhang2022). Specific primers (Supplementary Table 1) were designed using Primer 6 (PREMIER Biosoft International, USA), and the open reading frames (ORFs) of six CTSB genes (MpCTSB-N, MpCTSB-16D1, MpCTSB-3098, MpCTSB-10270, MpCTSB-mp2, and MpCTSB-16) were determined by PCR. The PCR mixture (25 μl) consisted of 12.5 μl of 2 × Taq PCR StarMix (GenStar, China), 1.5 μl of cDNA template, 1.5 μl of forward and reverse primers (10 mmol/L), and 8 μl of ddH2O. The reaction programme was as follows: 95°C for 3 min; 35 cycles at 95°C for 30 s, 55–65°C (depending on the primers) for 30 s, and 72°C for 1 min; with a final extension at 72°C for 5 min. The PCR products were separated by 1% agarose gel electrophoresis, and the expected fragments were purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech, Nanjing, China). The purified products were ligated into the pMD™ 19-T vector (TaKaRa, Dalian, China) and then transformed into DH5α competent cells (TaKaRa, Dalian, China). Single colonies were collected for PCR detection, and the plasmid DNA was sequenced by Sangon Biotech (Shanghai, China).

ORFs were identified using the ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The amino acid sequence of the encoded proteins was predicted using DNAMAN 8.0 (Lynnon Biosoft, San Ramon, CA, USA) (Meng et al., Reference Meng, Yang, Wang, Cao and Zhang2022). The ExPASy Molecular Biology Server (https://web.expasy.org/protparam/) was used to predict the physicochemical properties of the proteins. The conserved structural domains and active sites of the proteins were analysed using the NCBI Conserved Domain Search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Wang et al., Reference Wang, Yang, Xiao, Li, Liu and Xiong2022). The homology of six MpCTSB amino acid sequences was searched by BlastP in the NCBI database, and MEGA 11.0 software (MEGA Limited, Auckland, New Zealand) was used to construct a phylogenetic tree of M. persicae and other aphids via the neighbour-joining algorithm (1000 replications and Poisson model) (Ruan et al., Reference Ruan, Meng, Yang, Zhou and Zhang2022).

Transcript expression of MpCTSB genes under different developmental stages

Samples of M. persicae at different developmental stages were collected from first- to second-instar nymphs (n = 100), third- to fourth-instar nymphs (n = 80), 1-day-old wingless adults, and 1-day-old winged adults (n = 50). Three biological replicates were performed for each sample. All samples were immediately frozen in liquid nitrogen and stored at −80°C.

Temperature and UV-B treatments

Temperature variation and UV-B exposure were as follows: 1-day-old wingless adults (n = 50) were exposed to 38°C and 4°C for 0 (control), 30, 60, 90, and 120 min (N = 4) in a constant-temperature climatic chamber (Meng et al., Reference Meng, Yang, Wang, Cao and Zhang2022). A UV light source (Nanjing Huaqiang Electronic Engineering Co., Ltd., Nanjing, China) was selected to irradiate M. persicae at a range of wavelengths from 280 to 315 nm (UV-B) and an irradiance of 300 μW/cm2. To remove the effects of other light sources, M. persicae were maintained in the dark for 2 h in advance (Yang et al., Reference Yang, Meng, Zhou and Zhang2022). The irradiated area was maintained with a temperature of 25°C ± 1°C. One-day-old wingless adults (n = 50) were then exposed to UV-B for 0 (control), 30, 60, 90, and 120 min (N = 4). All samples were immediately frozen in liquid nitrogen and stored at −80℃.

Quantitative real-time polymerase chain reaction analysis

The expression of the MpCTSB genes in M. persicae was measured at different developmental stages and at various temperatures and UV-B exposures using the CFX-96 real-time fluorescence quantitative PCR system (Bio-Rad Laboratories, Hercules, CA, USA) (Yang et al., Reference Yang, Meng, Zhou, Yao and Zhang2021b). The reaction mixture (10 μl) contained 5 μl of TB Green® Premix Ex Taq™ II, 1 μl of forward and reverse primers (10 μmol/L), 2 μl of ddH2O, and 1 μl of cDNA template. The reaction conditions were 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. All reactions were analysed with a melting curve between 60°C and 95°C to ensure the specificity and consistency of the amplification products. All primers used in qRT-PCR (Supplementary Table 1) were designed using Primer 6 (PREMIER Biosoft International, USA). β-Actin and 18S ribosomal RNA (18S rRNA) were used as internal reference genes (Kang et al., Reference Kang, Liu, Tian, Zhang, Guo and Liu2017). Relative expression levels of the genes were calculated using the 2−ΔΔCT method. Normalisation was performed using the geometric mean of two selected internal control genes (Livak and Schmittgen, Reference Livak and Schmittgen2001).

Statistical analysis

All data were analysed via a one-way analysis of variance (ANOVA) using IBM SPSS Statistics for Windows, version 27.0 (IBM Corporation, Armonk, NY, USA), and the data are expressed as mean ± SE. Tukey’s method was used for multiple comparisons. A homogeneity test was performed on the data before ANOVA, and a P-value of <0.05 was considered statistically significant. Gene expression bar charts were created using OriginPro 2024b software (OriginLab Corporation, Northampton, MA, USA).

Results

Cloning and characterisation of the MpCTSB genes

Six CTSB genes, MpCTSB-N, MpCTSB-16D1, MpCTSB-3098, MpCTSB-10270, MpCTSB-mp2, and MpCTSB-16 (GenBank accession nos. OR167102, OR167103, OR167104, OR167105, MZ945515, and MZ945516, respectively), were amplified and cloned from M. persicae. Sequencing results showed that the ORFs of the six genes were 1017, 1014, 1014, 1026, 1023, and 1023 bp in length, encoding 338, 337, 337, 341, 340, and 340 amino acids, respectively. The predicted molecular weights ranged from 37.91 to 34.15 kDa; theoretical isoelectric points were 5.23–8.60; and instability indexes were 23.55–33.98 (table 1). The results of signal IP and protein bioinformatic analysis revealed that all six MpCTSB proteins contained an N-terminal signal peptide of 15–22 amino acid residues, three conserved active site residues – Cys, His, and Asn, and two conserved domains – Propeptide_C1 and Peptidase_C1A_Cathepsin B (fig. 1). The multiple sequence alignment results showed a high degree of homology among the six MpCTSB amino acid sequences (fig. 2).

Figure 1.

Nucleotide sequences of six M. persicae CTSB genes and the predicted amino acid sequences of the CTSB proteins. The initiation (ATG) and termination (TAA) codons are shown in boldface. The open boxes in the amino acid sequences indicate the putative signal peptide of CTSB. The conserved domains Propeptide_C1 and Peptidase_C1A_Cathepsin B are represented by underscores and shadows, respectively. The active site residues Cys, His, and Asn are marked with a red frame.

Figure 2.

Sequence alignment of six MpCTSB genes from M. persicae. The conserved active site residues Cys, His, and Asn are marked with red triangles.

Table 1.

Characteristics of the MpCTSB genes of M. persicae

Phylogenetic analysis of the MpCTSB genes

To analyse the evolutionary relationship between six MpCTSB amino acid sequences and CTSBs of other insects, the phylogenetic trees of M. persicae and other aphids were constructed (fig. 3). The results indicated that MpCTSB-16D1 was classified as an independent branch, and the remaining five MpCTSBs were categorised into five branches with other homologous species.

Figure 3.

Phylogenetic tree analysis of CTSBs in M. persicae and other insects. The numbers on the branches are bootstrap values obtained from 1000 replicates. The sequence tags are represented by the species name and GenBank accession number. The MpCTSB sequences are labelled in red. Insect species include Myzus persicae, Astegopteryx spinocephala, Astegopteryx styracophila, Adelges cooleyi, Aphis citricidus, Acyrthosiphon pisum, Aphis gossypii, Aphis craccivora, Aulacorthum solani, Tuberaphis taiwana, Tuberaphis coreana, Tuberaphis styraci, Sipha flava, Rhopalosiphum maidis, Diuraphis noxia, Melanaphis sacchari, and Pemphigus obesinymphae.

Expression analysis of the CTSB genes in M. persicae

The six MpCTSB genes exhibited varying expression patterns at different developmental stages in M. persicae. The expressions of MpCTSB-16D1, MpCTSB-mp2, and MpCTSB-16 were the highest in winged adults and second in first-instar nymphs (F 5, 12 = 141.113, P < 0.01; F 5, 12 = 638.969, P < 0.01; F 5, 12 = 1574.255, P < 0.01). MpCTSB-N had the highest expression in winged adults and the second highest in third-instar nymph stages (F 5, 12 = 178.814, P < 0.01). The expression of MpCTSB-3098 and MpCTSB-10270 was the highest in first-instar nymphs (F 5, 12 = 161.716, P < 0.01; F 5, 12 = 133.580, P < 0.01) (fig. 4).

Figure 4.

Relative expression levels of six MpCTSB genes at different developmental stages in M. persicae. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test). Developmental stages: N1−N4, first- to fourth-instar nymph stage; WlAD, wingless adult; WdDA, winged adult.

When M. persicae was exposed to 38°C, the expression of the six MpCTSB genes varied. The expression of MpCTSB-N, MpCTSB-16D1, and MpCTSB-3098 increased firstly and then decreased over time, whereas the expression of MpCTSB-N and MpCTSB-3098 reached a peak at 30 min (F 4, 10 = 412.977, P < 0.01; F 4, 10 = 438.699, P < 0.01), and the expression of MpCTSB-16D1 peaked at 90 min (F 4, 10 = 70.020, P < 0.01). The expression of MpCTSB-mp2 showed an increasing trend and reached the highest level at 120 min (F 4, 10 = 141.978, P < 0.01). The overall expression trend of MpCTSB-10270 and MpCTSB-16 decreased with the extension of time, with the lowest expression observed at 60 and 30 min (F 4, 10 = 43.138, P < 0.01; F 4, 10 = 121.843, P < 0.01) (fig. 5).

Figure 5.

Relative expression levels of six MpCTSB genes in wingless adults of M. persicae exposed to 38°C at various times. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test).

The expression of the six MpCTSB genes in M. persicae initially increased and then decreased with exposure time at low temperature stress (4°C). The expression of MpCTSB-N, MpCTSB-3098, and MpCTSB-10270 was the highest at 30 min (F 4, 10 = 8.253, P < 0.01; F 4, 10 = 126.510, P < 0.01; F 4, 10 = 9.022, P < 0.01, respectively), the expression of MpCTSB-mp2 and MpCTSB-16 was the highest at 60 min (F 4, 10 = 30.615, P < 0.01; F 4, 10 = 85.278, P < 0.01), and the expression of MpCTSB-16D1 was the highest at 90 min (F 4, 10 = 16.529, P < 0.01) (fig. 6).

Figure 6.

Relative expression levels of six MpCTSB genes in wingless adults of M. persicae exposed to 4°C at various times. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test).

The expression of the six MpCTSB genes in M. persicae was significantly increased following UV-B irradiation of M. persicae, and the expression levels increased firstly and then decreased over time. MpCTSB-10270 was expressed at the highest level at 90 min (F 4, 10 = 442.364, P < 0.01), whereas the other genes reached peak expression at 60 min (F 4, 10 = 189.945, P < 0.01; F 4, 10 = 42.776, P < 0.01; F 4, 10 = 111.876, P < 0.01; F 4, 10 = 161.953, P < 0.01; F 4, 10 = 109.312, P < 0.01) (fig. 7).

Figure 7.

Relative expression levels of six MpCTSB genes in wingless adults of M. persicae exposed to UV-B at various times. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test).

Discussion

Cathepsin B has an indispensable role in many insects, including digestion, growth and development, tissue differentiation, embryo development, and metamorphosis (Cristofoletti et al., Reference Cristofoletti, Ribeiro, Deraison, Rahbé and Terra2003; Uchida et al., Reference Uchida, Nishizuka, Ohmori, Ueno, Eshita and Fukunaga2004; Wu et al., Reference Wu, Zou, Jia, Wang, Zhang, Guo and Gui2011). For example, during the embryonic development of Antheraea pernyi and Helicoverpa armigera, CTSB participates in the degradation of yolk proteins (Zhao et al., Reference Zhao, An, Wang, Dong, Du, Sueda and Kondo2005, Reference Zhao, Wang, Yagi, Yamamoto, Watabe and Takahashi1996). During larval–pupal and pupal–adult metamorphosis in B. mori, CTSB is abundantly expressed during the process of histolysis in the gut, silk gland, and fat body (Lee et al., Reference Lee, Kim, Choo, Yoon, Kang, Woo, Sohn, Roh, Gui, Je and Jin2009; Wang et al., Reference Wang, Liu, Xia, Zha, Chen and Jiang2008). In the present study, we found that the six MpCTSB genes expressed at different developmental stages in M. persicae but exhibited different expression level patterns. Similarly, they were generally characterised by higher expression levels in winged adults or first-instar nymphs. This phenomenon has been observed at different developmental stages for other insects. For example, the expression of CTSB was detected in various stages of A. gossypii and in the larvae, pupae, and adults of H. armigera (Liu et al., Reference Liu, Zhang, Gao, Zhu, Wang, Ji, Luo, Cui, Li and Zhang2022; Zhao et al., Reference Zhao, Wang, Xu, Schmid and Wieczorek2002). A study reported that CTSB is highly expressed in the nymph and adult stages of Diaphorina citri. Silencing the female DcCTSB genes via RNA interference (RNAi) significantly inhibited the expression of the Vitellogenin A1-like gene, thereby reducing the number of oocytes in the female ovaries (da et al., Reference da, Schneider, Vk, Carmona, Alves, Belasque-Júnior, Rosa, Hunter, Henrique-Silva and Soares-Costa2015; Ibanez et al., Reference Ibanez, Vieira Rocha, Dawson, El-Mohtar, Robertson, Stelinski and Soares-Costa2023). Thus, we hypothesise that these MpCTSB genes may participate in the individual development and reproduction regulation of M. persicae.

Changes in environmental factors can induce stress responses in M. persicae. For example, extreme high temperatures shorten the lifespan of M. persicae, increase mortality, and decrease the net reproductive rate (Saleesha et al., Reference Saleesha, Kennedy, Rajabaskar and Geethalakshmi2022). Under low-temperature conditions, nymphal development time is extended and adult reproductive capacity is suppressed (Baral et al., Reference Baral, Thapa, Jha, Joshi and Aryal2022). UV-B exposure leads to impaired nymphal growth and development, reduced survival rate, and decreased number of population (Gidske, Reference Gidske2020). In the present study, we found that exposing M. persicae to varying temperatures (4°C and 38°C) and UV-B stress significantly increased the expression of almost all MpCTSB genes, with the exception of MpCTSB-10270 and MpCTSB-16 at 38°C. Several studies have demonstrated a role for CTSB in the stress response of insects to biotic and abiotic stresses (Saikhedkar et al., Reference Saikhedkar, Summanwar, Joshi and Giri2015; Zhao et al., Reference Zhao, Zhang, Fu, Li, Lin and Yu2020). Wu et al. (Reference Wu, Zou, Jia, Wang, Zhang, Guo and Gui2011) showed that B. mori nuclear polyhedrosis virus (BmNPV) infection and high temperature stress in B. mori result in the overexpression of CTSB in the larval fat body and a shortening of the larval developmental period and early pupation. Zhao et al. (Reference Zhao, Zhang, Fu, Li, Lin and Yu2020) found that Cry41-related toxins significantly induce CTSB activity in M. persicae and promote apoptosis. In the heat tolerance study of A. gossypii, it was found that AgCTSB genes were significantly induced by high temperature stress (35°C). RNAi-mediated silencing of this gene can reduce the survival rate and fertility of A. gossypii (Liu et al., Reference Liu, Liu, Li and Lu2023). Therefore, we hypothesise that the impact of MpCTSBs on the adaptation of M. persicae to environmental stress is similar to that of CTSB in other insects. Additionally, with extended treatment time, MpCTSB gene expression increased first and then decreased at various times, suggesting that there are different thresholds for the protection ability of CTSB genes. Similar results were observed in studies on the response mechanisms to environmental stress in Liposcelis bostrychophila, M. persicae, Spodoptera frugiperda, and Ostrinia furnacalis (Miao et al., Reference Miao, Tu, Guo, He, Jing, Wang and Wei2020; Ruan et al., Reference Ruan, Meng, Yang, Zhou and Zhang2022; Yang et al., Reference Yang, Meng, Zhou, Yao and Zhang2021b, Reference Yang, Meng, Zhou and Zhang2022).

Notably, the expression of most MpCTSB genes peaked at 30 or 60 min of various environmental treatments. For example, the genes MpCTSB-N, MpCTSB-16D1, MpCTSB-3098, and MpCTSB-mp2 under high temperature stress, MpCTSB-N, MpCTSB-3098, MpCTSB-10270, MpCTSB-mp2, and MpCTSB-16 genes under low temperature stress (except for MpCTSB-16D1, which peaked at 90 min), and all six genes under UV-B stress showed similar trends. Insects under environmental stressors such as high temperature, low temperature, or UV-B radiation rapidly activate a series of molecular and cellular mechanisms, including the expression of heat shock proteins, antioxidant enzymes, and membrane protective proteins, to enhance their survival and adaptability (Garnas, Reference Garnas2018; Yang et al., Reference Yang, Meng, Yao and Zhang2021a). Particularly, heat shock proteins are rapidly upregulated in a short time and protect cells from damage by participating in protein folding, unfolding, and repair. For example, under UV-B stress, the expression levels of MpHsp40 and MpHsp70 genes in M. persicae showed strong upregulation from 30 to 120 min, and after RNAi silencing, the survival rate, fertility, and body development of M. persicae exposed to UV-B radiation significantly decreased (He et al., Reference He, Meng, Smagghe, Yang, Tang and Zhang2024; Yang et al., Reference Yang, Meng, Zhou and Zhang2022). The genes SfsHsps, SfHsp70s, and SfHsp90s in S. frugiperda also exhibited a trend of short-term upregulation under high and low temperature stress. Furthermore, after the injection of dsSfHsp90s, the mortality of S. frugiperda larvae significantly increased (Ruan et al., Reference Ruan, Zhou, Yang, Meng and Zhang2024; Yang et al., Reference Yang, Meng, Zhou, Yao and Zhang2021b; Zhou et al., Reference Zhou, Meng, Ruan and Zhang2023).

The high expression of certain insect genes is often associated with specific physiological functions or adaptive responses. Research on these genes not only advances our understanding of insect biology but also provides novel insights for precise pest management. This study found that the CTSB gene in M. persicae is typically highly expressed in winged adults or first-instar nymphs. Under various environmental stresses, including 38°C, 4°C, and UV-B exposure, the expression of most genes was significantly upregulated. These findings suggest that the high expression of MpCTSB may make it a vital target for understanding the adaptive mechanisms of the peach aphid to environmental stresses. In the future, RNAi technology should be used to silence MpCTSB, providing deeper insights into the physiological functions and roles of CTSB in M. persicae and offering a basis for developing specific insecticide targets for M. persicae (Kim et al., Reference Kim, Soumaila Issa, Cooper and Zhu2015). Furthermore, the protein characteristics of MpCTSB can provide information for the development of small-molecule inhibitors, such as specific inhibitors CLIK148 and CA074 targeting cathepsin L and CTSB synthesis (Katunuma et al., Reference Katunuma, Murata, Kakegawa, Matsui, Tsuzuki, Tsuge, Turk, Turk, Fukushima, Tada and Asao1999). Studies have reported that injection of E-64 and CLIK-148 into H. armigera larvae delays moulting in fifth- to sixth-instar larvae (Liu et al., Reference Liu, Shi, Zhang, Zhang and Xu2006). These inhibitors can block gene expression or protein function, thereby weakening M. persicae’s adaptation to adverse environments and enhancing the control effect of natural or artificial stresses on M. persicae populations (Saikhedkar et al., Reference Saikhedkar, Summanwar, Joshi and Giri2015).

In the present study, six M. persicae MpCTSB genes were identified and their expression patterns were determined at different developmental stages and under extreme temperature and UV-B irradiation conditions. Our results revealed the role of CTSB in response to abiotic stresses and provided insights into its physiological functions, thereby facilitating the study of insect adaptability to environmental stresses.

Supplementary material

The supplementary material for this article can be found at https://doi.org/10.1017/S0007485325000136

Acknowledgements

This research was funded by the Foundation of Guizhou Provincial Department of Education (QianJiaoHe KY [2021]057) and the Guizhou Tobacco Company Program (No. 201919, 2021XM09).

Competing interests

The authors declare no conflicts of interest.

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Figure 0

Figure 1. Nucleotide sequences of six M. persicae CTSB genes and the predicted amino acid sequences of the CTSB proteins. The initiation (ATG) and termination (TAA) codons are shown in boldface. The open boxes in the amino acid sequences indicate the putative signal peptide of CTSB. The conserved domains Propeptide_C1 and Peptidase_C1A_Cathepsin B are represented by underscores and shadows, respectively. The active site residues Cys, His, and Asn are marked with a red frame.

Figure 1

Figure 2. Sequence alignment of six MpCTSB genes from M. persicae. The conserved active site residues Cys, His, and Asn are marked with red triangles.

Figure 2

Table 1. Characteristics of the MpCTSB genes of M. persicae

Figure 3

Figure 3. Phylogenetic tree analysis of CTSBs in M. persicae and other insects. The numbers on the branches are bootstrap values obtained from 1000 replicates. The sequence tags are represented by the species name and GenBank accession number. The MpCTSB sequences are labelled in red. Insect species include Myzus persicae, Astegopteryx spinocephala, Astegopteryx styracophila, Adelges cooleyi, Aphis citricidus, Acyrthosiphon pisum, Aphis gossypii, Aphis craccivora, Aulacorthum solani, Tuberaphis taiwana, Tuberaphis coreana, Tuberaphis styraci, Sipha flava, Rhopalosiphum maidis, Diuraphis noxia, Melanaphis sacchari, and Pemphigus obesinymphae.

Figure 4

Figure 4. Relative expression levels of six MpCTSB genes at different developmental stages in M. persicae. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test). Developmental stages: N1−N4, first- to fourth-instar nymph stage; WlAD, wingless adult; WdDA, winged adult.

Figure 5

Figure 5. Relative expression levels of six MpCTSB genes in wingless adults of M. persicae exposed to 38°C at various times. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test).

Figure 6

Figure 6. Relative expression levels of six MpCTSB genes in wingless adults of M. persicae exposed to 4°C at various times. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test).

Figure 7

Figure 7. Relative expression levels of six MpCTSB genes in wingless adults of M. persicae exposed to UV-B at various times. Significant differences are indicated by letters at the top of the bars (P < 0.05, Tukey’s test).

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