To obtain insights into the NlSOD gene family, the protein sequences of N. lugens were retrieved from the publically available National Center for Biotechnology (NCBI) (http://www.ncbi.nlm.nih.gov/cdd/) and eight NlSOD genes were identified. For the phylogenetic relationship of the NlSOD gene family, 8 NlSOD, Drosophila melanogaster (3 DmSOD), Laodelphax striatellus (3 LsSOD), Scirpophaga incertulas (4 SiSOD), Spodoptera frugiperda (4 SfSOD) proteins sequences were downloaded from the NCBI online database. Furthermore, To avoid the possible loss of a single NlSOD protein due to the fact of a missing domain, a local BLASTP with a 1E-5 cutoff was performed. The Conserved Domain Database (CDD) of the NCBI (http://www.ncbi.nlm.nih.gov/cdd/) (accessed on 14 January 2023), SMART database (http://smart.embl-heidelberg.de/) (accessed on 18 January 2023) (Schultz et al., 1998), Inter Pro Scan program (https://www.ebi.ac.uk/interpro/) (accessed on 18 January 2023), and Scan Prosite (https://prosite.expasy.org/scanprosite/) (accessed on 20 January 2023) was used to confirm the NlSOD gene family-specific domains. Additionally, the physiochemical properties of the NlSOD proteins in the studied species were discovered using the ExPASy online server (http://web.expasy.org/protparam/) following the same protocol as the previous study of Sohail et al. (2022).

To obtain detailed knowledge regarding the evolutionary relationship of the NlSOD gene family in N. lugens with the counter species. Herein, we investigated the phylogenetic relationships of the NlSOD gene family members of N. lugens with the developed genome D. melanogaster and the other three insects species (L. striatellus, S. incertulas, and S. frugiperda). Firstly, all the mentioned insect’s SOD amino acid sequences were downloaded from NCBI and were then aligned through ClustalW software (version 2.1) (http://www.genome.jp/tools/clustalw/) (accessed on 13 February 2023) following the default parameters to examine the evolutionary relationships among the sequences and construct the maximum likelihood (ML) phylogenetic tree using MEGA7 (version 7.0) (Kumar et al., 2016). For the reliability of clades, bootstrap with 1,000 replicates was used.

Domain architecture of identified NlSOD proteins was performed by using the full-length protein sequence to CDD NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 23 February 2023) and downloaded hit data subjected to DOG software (version 2.0) (visualization of protein domain structure) for domains visualization and finally for better graphical representation the Microsoft PowerPoint 365 was used.

Furthermore, the conserved motifs of the NlSOD gene family were predicted using the MEME online server (Version 4.12.0) (http://meme-suite.org/) (accessed on 23 February 2023) with the default settings. The details of the top 5 predicted motifs were obtained from the MEME suite and then cross-checked with NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 23 February 2023). Finally, the motif distribution was drawn via Microsoft PowerPoint 365 software.

The obtained NlSOD protein sequences were implied in the online server “CELLO2GO” (http://cello.life.nctu.edu.tw/cello2go/alignment.php) to determine the predicted functions, such as molecular functions, biological processes, and cellular components. Finally, the GO classifications were recovered using GraphPad Prism (Version 9.4.1) (GraphPad Software, Inc., LA Jolla, CA, United States) was used for graphical representation.

The online server String (version 11.5) (https://string-db.org) designed by the Swiss Institute of Bioinformatics (accessed on 25 February 2023) was used for the protein-protein interaction (PPI), and the network was recovered using the NlSOD1 protein as our reference protein. The obtained results were based on the available sources in the string database, including text mining co-expression, determined, co-occurrence, intersection, curated databases, and fusion (Szklarczyk et al., 2015). The pathway enrichment analysis was also conducted by searching for NlSOD genes in the NCBI database’s online pathway enrichment tool.

The insects BPH and the rice verity Ninjing4 were used in the study, obtained from China National Rice Research Institute (Hangzhou, China). The Ninjing4 rice variety, known to be preferred because of no resistance to BPH infestations, was used for rearing. Initially, the BPH colonies were reared on the rice seedlings in cement tanks covered with fine mesh outdoors (i.e., in natural conditions) for 6 months (i.e., April to October) and overwintered in lab-controlled conditions.

First, we soaked the seeds for 24 h in a water dip plastic tray with a standard size of one-quarter (60 cm H_100 cm W_200 cm L) in lab standard conditions of 26°C ± 2°C with the 16 h L:8 h D in relative humidity of 80% ± 10% in the ecological laboratory of Yangzhou University. The germinated seeds were transferred to cement tanks covered with fine mesh in an outdoor natural environment and were grown until the six-leaf seedling stage. The seedlings were then transferred into plastic pots (dimensions = R ¼ 16 cm). Finally, the stress treatments proceeded at the tillering stage (40 ± 2, 40 ± 4, and 40 ± 8 days) following the same procedure as Ahmad et al. (2022a). Furthermore, the JGM technical grade of 61.7% used in this study was obtained from the Qianjiang Biochemistry Co., Ltd. (Haining, Zhejiang, China). Following the protocol in a previous report by Ahmad et al. (2022a), the two hundred parts per million (PPM) solution was Tween 20 obtained from the Sinopsin Group Chemical Reagent Company (Shanghai, China). The fungicide was then sprayed on the rice seedlings, following the procedure in a previous study (Ge et al., 2010).

The rice seeds were sown in standard cement tanks containing standard rice-growing soil at the Yangzhou University experimental field under natural outdoor conditions (Ge et al., 2013). Seedlings bearing six leaves were transplanted into plastic pots (R ¼ 8 cm) with four hills per pot, three plants per hill, and grown under natural conditions for experiments. Rice plants were pulled from the pots (at about 25 days), keeping the root system intact. The plants were gently washed without damaging the roots, then cultured in plastic containers (D ¼ 12 cm and H ¼ 25 cm) containing rice hydroponic liquid solution obtained from International Rice Research Institute (Los Banos, Laguna, Philippines). The pH of the hydroponic solution was maintained at 5.0 by daily adjustments with 1 mol/L HCl and 1 mol/L NaOH. The root systems were immersed in the hydroponic solution, and the solution was replaced every 3 days to ensure sufficient plant nutrition. Experimental rice plants (n ¼ 3) were transferred into a 1% glucose, sucrose, and trehalose hydroponic solution after a week following the method of Lin et al. (2018), and controls (n ¼ 3) were cultured in normal hydroponic solution (without sugar). Thirty-third-instar nymphs were transferred onto rice plants in the 1% glucose, sucrose, and trehalose hydroponic solution and covered with 60 mesh nylon net, and the BPH nymphs infestations for two, four and 8 days were collected and snap frozen in liquid nitrogen then and stored in −80°C degree till further use. Finally, the stored samples were then used for qRT-PCR analysis, and the specific primers used in the study are listed in (Supplementary Table S1).

BPH was introduced on the forty-day-old (40 ± 2) to plastic buckets having rice plants, and samples were collected at 2, 4, and 8 days after infestation. First, the BPH samples were snap-frozen in liquid nitrogen and then stored at −80°C degrees till further experiments. Furthermore, total RNA was extracted from the samples using an RNA extraction kit (Vazyme, Nanjing, China). First, the free DNA was removed using DNase I, and the concentration and purity were measured using a NanoDrop 1,000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL, United States). Also, the integrity was checked using 1.5% agarose gel electrophoresis. Finally, the resulting complementary DNA (cDNA) was used as a template for quantitative real-time PCR (qRT-PCR) analysis using SYBR Green real-time PCR master mix (Vazyme, Nanjing, China). The qRT-PCR assays were performed in triplicate using a real-time PCR system (Bio-Rad, Hercules, CA, United States) following the manufacturer’s protocol.

Furthermore, the 2 μL aliquots of cDNA were amplified by qPCR in 20 μL reaction volumes using the SYBR Premix Ex Taq™ II (TaKaRa, Dalian, China). The cDNAs were amplified at 95°C for 2 min, followed by 35 cycles of 10 s at 95°C, then for 30 s, and at 72°C for 30 s, with a final extension step of 72°C for 10 min in a CFX96 real-time PCR system (Bio-Rad Co., Ltd., CA, United States). The mRNA amounts of all genes were separately quantified with the stable expression of the constitutive reference gene, actin. The specific primers are listed in (Supplementary Table S1). After amplification, the target gene cycle threshold (Ct) values were normalized to the reference gene (actin) by the 2^−ΔΔCT method (Livak and Schmittgen, 2001). The data’s mean values of three biologically independent replicates were used for the final graphs.

The data presented in this paper were analyzed using the SPSS software (version 25.0, SPSS Inc., Chicago, IL, United States) for statistical analysis (ANOVA), statistical significance, and a 95% confidence interval (p ≤ 0.05). The data were analyzed and expressed as the mean ± standard deviation (SD) of three biologically independent replicates in all measured parameters, and finally, GraphPad Prism (Version 9.4.1) (GraphPad Software, Inc., LA Jolla, CA, United States) was used for graphical representation.

The present study retrieved 8 NlSOD genes, and the nomenclature was given (NlSOD1-NlSOD8) based on the three functional domains and chromosomal positions. Among the eight NlSOD gene family members, the NlSOD1 contains two functional domains, namely, Sod_Fe_N and Sod_Fe_C; the NlSOD4 and NlSOD7 exhibit single domains (Supplementary Figure S1). Moreover, the five members of the SOD genes family (NlSOD2, NlSOD3, NlSOD5, NlSOD6, and NlSOD8) were found with the Sod_Cu conserved domain. The accession number and annotations of the conserved domains are listed in supplementary data (Supplementary Table S2).

Following the deep scan, eight NlSOD genes were isolated from the NCBI online database; the detailed information is listed in Table 1. Among them, NlSOD1 and NlSOD4 resided in the mitochondrial, NlSOD2 extracellular, and NlSOD3 nuclear, whereas NlSOD5, NlSOD6, NlSOD7 and NlSOD8 resided in cytoplasmic. Other features of NlSOD proteins such as locus ID, chromosomal coordinates, molecular weight, chemical properties, and isoelectric point (PI) are tabulated.

To obtain the evolutionary relationship of the NlSOD gene family, 8 NlSOD proteins with 4 other insects’ species, including 3 DmSOD, 3 LsSOD, 4 SiSOD and 4 SfSOD protein sequences were aligned using ClustalX software. After alignment, an ML phylogenetic tree was constructed using MEGA7 software (version 7.0). Finally, the phylogenetic tree was displayed using subscription-based iTOL: Interactive Tree Of Life (https://itol.embl.de/) (Letunic and Bork, 2021). The phylogenetic analysis clustered the SOD proteins into three subgroups such as SODI, SODII, and SODIII, and the number of SOD proteins was counted in each subgroup, whereas subgroup I accounted for 2 proteins and subgroup II and subgroup III with 3 NlSOD proteins (Figure 1), following the same procedure of our previous report by Ahmad et al. (2022b).

A total of eighteen conserved motifs were discovered using the MEME online server (Version 5.4.1) (https://meme-suite.org/meme/tools/meme) (accessed on 26 February 2023) reported by Bailey et al. (2009), and they were found to be appropriate for explaining the NlSOD gene’s structure (Figure 2). Among the eight NlSOD genes, the NlSOD2 and NlSOD8 were counted with four motifs, followed by NlSOD5 with three motifs, and NlSOD1 and NlSOD3 exhibited two motifs. Finally, the three NlSOD genes such as NlSOD4, NlSOD6, and NlSOD7, were recorded for a single motif.

NlSOD genes GO analysis showed various key functional predictions. For instance, the biological, molecular, and cellular processes following the same as our previous study by Ahmad et al. (2022b). In the biological processes category, the NlSOD genes are mainly involved in the regulation of stress-responsive with a high percentage of 22% and 20% in the biosynthetic process, whereas the other functions such as cell pigmentation, cell differentiation, and other environmental stimuli via hormonal and metabolic modulation were also observed (Supplementary Figure S2). As stated in the molecular processes category, the NlSOD genes were also found to regulate oxidoreductase activity, ion binding, protein binding, and enzyme binding activity. A large number of NlSOD genes reside in the intercellular region by up to 26%, followed by cytoplasm, and plasma membrane, whereas some members are localized to extracellular space and plasma membrane.

KEGG pathway enrichment analysis is an improtant tool for understanding the molecular framework ignited by a cluster of genes. For instance, in our study, the NlSOD genes showed their involvement in different KEGG pathways. Amongst them, the FoxO signaling pathway was regulated by seven percent of NlSOD genes, followed by the MAPK signaling pathway, which accounted for five percent of genes in their regulatory machinery. The chemical carcinogenesis-reactive oxygen species signaling pathway, which is moderately influenced by three percent of NLSOD genes in response to JGM stress and serves as the first line of defense was also found in the analysis. Additionally, the longevity regulating pathway, which is key to insect lifespan, was also fine-tuned by NlSOD genes. A series of disease-related pathways such as prion, parkinson, and huntington could rely on the transcriptional of NlSOD genes. The KEGG pathway analysis recorded the lipid and peroxisomes pathways (Figure 3).

The detailed information of our reference protein NlSOD1 protein interactive partners were initially searched in the online server FlyBase (https://flybase.org/reports/FBgn0032350) (accessed on 27 February 2023) and was confirmed by STRING: functional protein association networks (https://cn.string-db.org/) and the integrative protein partners network was programmed. The detailed information and accessions are tabulated in (Supplementary Table S3). The NlSOD1 protein plays a crucial role in BPH growth and development and is also highly stress-responsive. Numerous other crucial proteins are also found to be highly interactive with our reference NlSOD1 protein. (Supplementary Figure S3). For instance, the SOD1 protein interacted highly with CAT, Prx5, PHGPx, GCLC, and CG15116. Almost similar binding was also observed for SOD2 protein with other crucial proteins such as TAM and MRPS29, which is extensively studied on the stress/diseases side. Finally, the SOD3 protein was found with high interaction with all the proteins named above, revealing the potential role of the NlSOD protein as crucial in the developmental processes and highly stress-responsive.

Tissue-specific expression analysis unfolded the potential role of NlSOD genes in the development of BPH (Figure 4). For instance, dominant expression was observed in the fifth instral stage of NlSOD6 up to 14 folds, followed by NlSOD2 in the fifth instral stage, 1 day after emergence (1A) and 2A up to six folds expression. Furthermore, the NlSOD4 showed moderate transcription in 2A, and NlSOD8 displayed the five folds expression in 1A. However, the lower transcription was recorded in NlSOD1, NlSOD5, and NlSOD7, suggesting that the NlSOD gene family has a potential role in the developmental stages of N. lugens (Figure 4). Notably, the lowest transcription was observed by overall NlSOD genes in the first to fourth instral stage, excluding NlSOD3 and NlSOD7, which have a moderate expression pattern in the first-fourth instral development stage. Among all the developmental stages, the NlSOD genes showed high expression in the fifth instral stage; for a detailed description and a graphical representation, see the appended data in the supplementary file (Supplementary Figure S4).

We have confirmed the tissues specific expression of NlSOD genes in four major tissues (head, midgut, ovary, and fat body) at 2 days after emergence (2DAE) by qRT-PCR. The results showed that most of the NlSOD genes have the dominant expression mostly in the head and ovary followed by the fat body and midgut (Figure 5). In particular, the high expression of NlSOD5 up to 10 folds, followed by NlSOD1 with five folds in the ovary. Moreover, the moderate transcriptional pattern was shown by most of the NlSOD genes, such as the NlSOD2, NlSOD3, NlSOD4, NlSOD6, and NlSOD8, with 1.5 folds in different tissues. A single NlSOD family member NlSOD7 displayed lower transcription in almost all tested tissues (Figure 5).

We performed qRT-PCR analysis to validate the NlSOD genes expressions in response to JGM spraying treatment (Figure 6). Our study revealed that NlSOD genes, such as NlSOD1 and NlSOD5, displayed upregulated expressions at 2 days (2D) and 4D time points. Followed by NlSOD2 and NlSOD8 had moderate transcription, and the rest of the four genes, such as NlSOD3, NlSOD4, NlSOD6, and NlSOD7, had the lowest transcription patterns. These results suggest that the JGM spraying application induces the NlSOD genes and enhances BPH’s immunity regulations against JGM.

The qRT-PCR analysis revealed the potential role of the NlSOD gene family. Among the 8 NlSOD genes, the NlSOD6 gene displayed dominant expression of fifteen folds at 24h, followed by NlSOD2 and NlSOD3 with five and four folds expressions under glucose, respectively. Whereas the rest of the genes, such as NlSOD1, NlSOD4, NlSOD5, and NlSOD6, displayed comparatively low transcription at 3 h and 6h, while NlSOD7 showed a moderate expression of 2.5 folds at 24 h (Figure 7).

Sugar transporters may offer a new potential target for controlling insect pests (Kikuta et al., 2010). Herein, the expression of NlSOD genes in BPH fed on rice plants treated with sucrose hydroponics was investigated. Similar to glucose, NlSOD genes were triggered under sucrose treatment at almost all the time points (Figure 8). Furthermore, the NlSOD6 displayed a dominant expression of 15 folds at 24 h. Followed by NlSOD2 and NlSOD8 of 5 folds expression at 24h, whereas moderate expression was observed by NlSOD5 and NlSOD7 of 3 folds at 24h, and the lowest transcription was observed by NlSOD3 of 1.3 folds at 3 h. Seven of the eight NlSOD genes showed an upregulated expression, and only a single gene, such as the NlSOD1, displayed a relatively downregulated expression at 3, 6 and 24 h. The results suggested that most of the NlSOD genes are highly responsive to sucrose and could work in concert with metabolism to tailor developmental and defense processes.

Gene expression analysis was conducted to understand the role of NlSOD genes of BPH fed with rice plants treated with trehalose hydroponic solution. Furthermore, the eight NlSOD genes showed upregulated expression patterns under trehalose hydroponic treatment. In which the NlSOD6 was observed with the dominant expression of 15 folds at 24 h. Followed by NlSOD8 of 6 folds expression at 24h, whereas the four NlSOD genes such as NlSOD1, NlSOD2, NlSOD4 and NlSOD7, showed moderate expression of 5 folds at 24 h. Contrastingly, the NlSOD5 and NlSOD3 were observed with the lowest transcription of 4 and 3 folds, respectively. The expression analysis of NlSOD genes under trehalose showed upregulated expression patterns and might finetuning the development of BPH and provide defense on a cellular basis.

A BLASTP search demonstrated that the LsSOD gene family and the NlSOD gene family were closely related, with the LsSOD genes having a higher proportion of matched sequences, indicating that the NlSOD gene is more diverged compared to other SOD genes. Moreover, the phylogenetic analysis also confirmed this relationship (Umasuthan et al., 2012). Both insects are members of the same insect family, the Hemiptera (Delphacidae), and share shorter N-terminal sequences, which strongly suggests their taxonomic association (Zelko et al., 2002). The phylogenetic tree analysis demonstrated that Mn-SOD and Cu/Zn-SOD may have shared a common ancestor and that Mn-SOD may have undergone more evolutionary change than Cu/Zn-SOD. Additionally, the Cu/Zn-SOD clade was divided into the Cu/Zn-SOD and Cu/Zn-SOD subgroups (Figure 1), with the latter showing greater divergence; these two protein sub-families may develop through gene replication as previously reported by Liu et al. (2015).

Members of the SOD gene family are highly conserved, and our results also uncover this high homology. The current study identified highly inducible SOD genes and shared close similarities with other insect species. Comparing the amino acid sequence of NlSOD with its counterparts from Lepidopteran insect species, including L. striatellus, S. incertulas, S. frugiperda, and D. melanogaster, the NlSOD showed high similarity with L. striatellus (Figure 1).

Domains are a protein’s distinct functional and/or structural units (Marchler-Bauer et al., 2015). Usually, they are responsible for a particular function or interaction, contributing to the overall role of a protein (Sigrist et al., 2010). The current study found that NlSOD proteins exhibit three functional domains (Supplementary Figure S1). The SOD_Fe_N functional domain is reportedly strongly associated with innate immunity, which is essential for survival organisms, for instance, in the survival and stress response in Chlamys farreri (Wang et al., 2018). Similarly, we also found the SOD_Fe_N, Sod_Fe_C, and Sod_Cu domains, which might help the BPH withstand the JGM and high sugar.

Additionally, usually, they are responsible for a particular function or interaction; because of these domains in NlSOD proteins, the NlSOD candidate proteins (Supplementary Figure S3) are found in high interactions with other functional proteins. Moreover, sequence motifs are short, recurring patterns in DNA that are presumed to have a biological function. Often, they indicate sequence-specific binding sites for proteins such as nucleases and transcription factors. Others are involved in important processes at the RNA level, including ribosome binding, mRNA processing (splicing, editing, polyadenylation), and transcription termination (D'Haeseleer, 2006). Herein, we also found the five motifs in the NlSOD genes (Figure 2) to explain the gene structure and influence their role in development and stress response, which needs further study.

In prior investigations, it was discovered that SOD genes were crucial not only for growth and development but also for stress response and other vital processes throughout various tissues and organs (Okado-Matsumoto and Fridovich, 2001; Zelko et al., 2002). In the present study, we found that the SOD was also distributed in all the tested tissues, such as in the head, midgut, ovary, and fat body (Figure 5). Additionally, the SOD genes showed transcriptional activity in the BPH egg, first to fifth instral nymph, 1 day after emergence (1A) and 2A (Figure 4), suggesting that this transcript may be involved in stress sensing and signaling transduction.

Moreover, there were alterations in the NlSOD gene expression levels throughout distinct tissues. However, the fact that the midgut and fat body exhibited the greatest expression implies that it may be contributing to development. The midgut includes the malpighian tubule, which plays an important role in detoxifying and eliminating toxins (Beyenbach et al., 2010). And fat body is one of the prime sites for antioxidant enzymes (Yamamoto et al., 2005b; Lee et al., 2005; Yamamoto et al., 2005). Our findings are consistent with those of Glossina morsitans (Munks et al., 2005) and Agrilus planipennis (Rajarapu et al., 2011), in which the midgut and fat body showed a substantial upregulation of SOD genes. The hindgut and fat body’s upregulation of SOD suggests that these two organs served as the primary sites for the production of antioxidant enzymes and provided resistance to oxidative stress.

Aerobic organisms constantly consume oxygen, which the mitochondria utilize to produce the energy required to sustain life (Atalay et al., 2020). Some of these oxygen molecules may convert into reactive oxygen species (ROS) during metabolic processes (Qureshi et al., 2021a). Exposure of tissues and cells to ultraviolet radiation and chemical agents generates ROS such as superoxide anion (O₂ ⁻) and hydrogen peroxide (H₂O₂) and causes oxidative damage to cells (Hermes-Lima and Zenteno-Savı́n, 2002; Fukai and Ushio-Fukai, 2011; Lee et al., 2019). SOD gene family is an essential antioxidant that catalyzes the dismutation of superoxide anions into molecular O₂ and H₂O₂, thereby protecting cellular components from extreme oxidative stress impairments (Valentine and Hart, 2003b; Fukai and Ushio-Fukai, 2011). Insects have developed tolerance to the insecticides JGM and sugars as a consequence of their widespread application (Matsumura et al., 2008). In the present study, the qRT-PCR analysis showed that most of the NlSOD gene expressions were induced after JGM treatment. For instance, the NlSOD1 and NlSOD5 displayed dominant expressions (Figure 6). Following that, under the sugar (glucose, sucrose and trehalose) treatment, the NlSOD6 and NlSOD2 were observed with high transcription (Figures 7–9) and might be involved in regulating BPH immune response to JGM and high sugar. However, the rest of the NlSOD genes displayed mixed expression patterns under the JGM and high sugar treatment and cannot be ruled out from functional studies. These genes may work redundantly and therefore rely on each other transcriptional changes. Interactive protein studies would be useful to confirm the statement of whether these amino acids have homodimeric or heterodimeric interactions. Additionally, our results provide a theoretical platform for pesticide regulatory bodies.