Genome-wide identification of the cytochrome P450 superfamily in Trichoplusia ni and in-silico expression of resistance against HDAC inhibitors

Abstract

The cabbage looper, Trichoplusia ni, is a serious pest of cruciferous crops such as cabbage, cauliflower, collard/spring greens, and horseradish, with a major financial impact. Despite its significant financial importance, there has been little study on the characterization of Cytochrome P450 (CYP450) genes, which are critical components in the control of metabolic activities. This study discovers and investigates 19 CYP450 genes in T. ni that are involved in important metabolic processes such as fatty acid metabolism, resistance mechanisms, phytotoxin response, and insecticide detoxification. We identified important CYP450 genes that play critical roles in conferring resistance to HDAC inhibitor insecticides, particularly sulforaphane and Trichostatin A, using in silico gene expression profiling. The findings reveal insight into the importance of identified genes in detoxifying chemical treatments, which may contribute to the advancement of resistance in cabbage looper populations. Notably, CYP4C1-1, CYP4C1-like-1, and CYP4C1-2emerge as key genes, significantly contributing to resistance and detoxification processes under the effect of HDAC inhibitor-induced chemical stressors. This study provides insight on the genetic mechanisms behind resistance in T. ni, providing important information on potential targets for pest control. These initiatives will pave the way for the development of innovative and effective control measures for cabbage looper populations in agricultural areas.

Introduction

The cabbage looper, Trichoplusia ni, a lepidopteran pest belonging to the family Noctuidae, exhibits a widespread global distribution, characterized by its high polyphagous and significant implications as an agricultural pest¹. T. ni has demonstrated adaptive resistance to a spectrum of chemical insecticides²and it manifests as an herbivorous insect with a host range extending to over 160 plant species spanning 36 families³. The geographic prevalence of T. ni encompasses all continents where crucifer crops are cultivated. T. ni serves as an model lepidopteran model for investigations into fundamental aspects of insect physiology⁴biochemistry⁵and molecular biology⁶. Widely employed in research, T. ni has contributed substantially to studies on insect-plant interactions⁷insect chemical ecology, insect-microbe interactions, and insect diseases, owing to its status as a highly polyphagous noctuid generalist⁸. Furthermore, T. ni has played a pivotal role in assessing the impact of environmental contaminants on agricultural ecosystems, functioning as an herbivorous insect, and describing tritrophic interactions among host plants, insects, and microorganisms⁹.

Cytochromes P450 (CYP450) are a widely distributed class of enzymes that were discovered around 50 years ago and can be distinguished by their significant structure and wide range of catalytic activity. These hemoproteins, which are encoded by a superfamily of genes, regulate the conversion of a wide range of substrates and catalyze a wide range of captivating chemical processes¹⁰. CYP450, a polygenic enzyme superfamily, has a significant impact on processes involved in growth, development, nutrition, and xenobiotic detoxification¹¹. CYP450 enzymes have been implicated in the biosynthesis of potentially deleterious compounds within meticulously investigated contexts. For instance, their involvement has been observed in the synthesis of organophosphate inhibitors targeting cholinesterase, specifically derived from phosphorothioate insecticides exhibiting reduced lethality¹². CYP450 is the principal enzyme family responsible for the detoxification of numerous pesticides, as well as exogenous and endogenous compounds, in insect metabolism. Moreover, the enzymatic system under consideration exerts a pivotal influence on the synthesis and catabolism of diverse endogenous compounds. Additionally, this system plays a pivotal role in the synthesis and metabolism of various endogenous substances, including ecdysone hormone, juvenile hormone, and fatty acids¹³.

In plants, CYP450 enzyme produces various defensive compounds, plant hormones, fatty acids, and lignin¹⁴while in insects, these produce various compounds, but their most well-known function is the detoxification of plant allelochemicals, phytotoxins, and insecticides (natural and synthetic)¹⁵. CYP450 enzymes facilitate insect adaptations across a broad range of ecological niches, habitats, and diets. Their diverse functions empower insects to thrive in various environmental conditions¹⁶. Specific CYP450 enzymes are selectively expressed in pheromone glands, where they play pivotal roles in pheromone biosynthesis and signaling, shaping behavior through chemical communication¹⁷.

In accordance with previous investigations¹⁴the CYP450 enzyme has a diverse function in plant biology, contributing to the manufacture of defense chemicals, plant hormones, fatty acids, and lignin. In the context of insects, these enzymes play an important role in the synthesis of a range of substances, with a major emphasis on the detoxification of plant allelochemicals, phytotoxins, and both natural and manufactured insecticides¹⁵. The pivotal role of CYP450 enzymes in facilitating insect adaptations is underscored by their participation across diverse ecological niches, habitats, and nutritional contexts. The substantial involvement observed in the context of studying the adaptability of insects to flourish across diverse environmental conditions¹⁶. Notably, certain CYP450 enzymes are selectively expressed in pheromone glands, where they play critical roles in both pheromone manufacturing and signaling. Insect behavior is ultimately influenced by their involvement in chemical communication¹⁷.

Numerous studies have demonstrated that the CYP450 enzyme family plays a critical role in the development of resistance to insecticides. This enzyme family’s significance depends on its molecular variability, broad substrate selection, and substantial catalytic adaptability¹⁸. Particularly, the CYP450 super family has recently been discovered in a range of insect species, including B. mori (84)¹⁹, R. ferrugineus (77)²⁰, D. melanogaster (85)²¹, B. tabaci (24)²², and L. decemlineata (96)²³. These studies have demonstrated that these discovered supergenes are involved in activities like xenobiotic metabolism, insecticide detoxification, resistance mechanisms, and responses to a variety of endogenous chemicals. The discovery of the CYP450 supergene family in the T. ni genome represents a significant approach in entomological research, with broad implications for agricultural pest control. Understanding the identities and activities of these genes is critical for understanding the molecular resistance mechanisms of the cabbage looper. This knowledge is used to create adapted pest management measures, which lead to more sustainable practices. Furthermore, the genetic information lays the basis for understanding T. ni’s adaptability and host plant occupancy, revealing insights into its wider evolutionary and ecological context. The finding of CYP450 genes in this species is critical for developing integrated pest control and a better understanding of its agroecosystem resistance.

Material and method

Database finding, sequence retrieval and physiochemical properties of CYP450 genes

The peptide sequence of Trichoplusia ni, sourced from the Trichoplusia ni genome database (http://www.tnibase.org/), was systematically examined within the PF00067 domain. In order to enhance the precision of identifying cytochrome P450 genes in the cabbage looper, this specific peptide sequence underwent a BLAST²⁴ search against a reference sequence of Drosophila melanogaster, employing a stringent E-value threshold of 1e-52. Essential genomic information, including chromosomal location, gene position, strand orientation, as well as both genomic and coding sequences (CDS) of Trichoplusia ni, was retrieved from the Trichoplusia ni genome database. Furthermore, crucial physicochemical attributes such as molecular weight (Mw), grand average of hydropathy (GRAVY), instability Index (II), isoelectric point (pI), and aliphatic index (Ai) were meticulously calculated using Protparam, an online computational tool accessible at (https://web.expasy.org/protparam/).

Phylogenetic analysis of cytochrome P450 genes

The investigation involved the derivation of the peptide sequence of T. ni and two other insect species (B. mori, andS. frugiperda,). This peptide sequence data was employed to construct a phylogenetic tree, facilitating an analysis of evolutionary relationships among diverse insects possessing identified genes within the CYP450 supergene family. The alignment of peptide sequences was meticulously executed to generate a phylogenetic tree²⁵, employing the Maximum Likelihood method with 100 bootstrapping replications, utilizing the MEGA-11 software (https://www.megasoftware.net/)²⁶. The resultant tree, along with associated coding genes, was subsequently crafted using the Interactive Tree of Life (iTOL) platform (https://itol.embl.de/)²⁷. Furthermore, the identified cytochrome-coding genes from T. ni, B. mori and S. frugiperda were systematically classified into 3 distinct groups (CYP3, CYP4 and CYP6). This categorization was based on their genetic and evolutionary distribution, juxtaposed against the reference model of B. mori and the presence of particular CYP sub family denoted as CYP3, CYP4 and CYP6. These sequences were renamed according to Dr. Neslson CYP450 nomeclature (https://drnelson.uthsc.edu/).

Conserved domain motif analysis

The motif distribution architecture within the PF00067 domain was systematically analyzed using the MEME database (Multiple Expectation Maximization for Motif Elicitation, https://meme-suite.org/meme/tools/meme). In order to optimize the accuracy of motif identification, a threshold value of 20 was established, and the motif length was constrained within the range of 6 to 50. Subsequently, the conclusive motif structure was derived and visualized through the application of TB-tools software (https://bio.tools/tbtools)²⁸.

Subcellular location, gene structure and chromosomal mapping

The subcellular localization of CYPTni genes was investigated in this study. The WoLF PSORT online tool (https://wolfpsort.hgc.jp/)²⁹ was employed for this purpose, utilizing the peptide sequences of T. ni. The outcomes of this investigation were graphically depicted through the generation of a heat map. The construction of the heat map was conducted with precision using TB-tools software. The structural organization of genes, specifically with respect to intron-exon configurations, was visually depicted using the Gene Structure Display Server (GSDS), an online bioinformatics tool (https://gsds.gao-lab.org/)³⁰. The genomic coordinates of CYPTni genes, encompassing both their start and end positions, were extracted from the Trichoplusia ni genome database. The development of the gene distribution map was facilitated through the utilization of the computational tool TB-tool (https://bio.tools/tbtools)³¹.

Mature MiRNA and synteny analysis

In order to demonstrate the regulatory mechanisms governing CYP450 genes, mature microRNA (miRNA) sequences were procured from the InsectBase 2.0 database (http://v2.insect-genome.com/Organism/774)³² and subsequently aligned with the coding sequence (CDS). The identification and characterization of mature miRNA were employed to unveil their potential influence on the transcriptional regulation of cytochrome genes in the cabbage looper³³. Moreover, in order to find out the prospective genetic framework and paralogous behavior of associated genes, a synteny analysis was performed³⁴.

Transcriptomic analysis of CYP450 genes in response to HDAC inhibitors

We reanalyzed publicly available RNA-seq data from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO), accession number GSE234351 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE234351), generated by Somers et al.³⁵. Raw reads were downloaded and aligned to the T. ni reference genome, summarized to gene-level counts, and assessed for differential expression with standard normalization and multiple-testing correction. Gene expression differences between control and HDAC inhibitor-treated groups (SFN and TSA) were analyzed using an unpaired two-tailed Student’s t-test. Expression values were based on normalized FPKM data, and differences were considered statistically significant at p < 0.05. The primary objective of this study is to scrutinize the impact on gene expression induced by HDAC inhibitors, namely SFN and TSA, administered through a modified artificial diet for T. ni, in comparison to the standard diet.

Results

Identification and characterization the physiochemical properties of CYPTni genes

In this investigation, a total of 19 CYP450 genes were meticulously identified and expressed using a BLAST query sequence that was retrived from NCBI. The BLAST results were compared with identified sequences of CYP450 in B. mori and renamed according to Dr. Nelson’s CYP450 nomenclature. The sequences were also confirmed using InterPro (https://www.ebi.ac.uk/interpro/) and NCBI. Comprehensive physiochemical analyses were conducted, encompassing molecular weight (Mw), isoelectric points (pI), gene localization on distinct chromosomes, coding sequence (CDS), grand average of hydropathy (GRAVY), as well as aliphatic (AI) and instability (II) index, all derived from various databases. The CYPTni genes identified in our study displayed notable diversity in peptide length, with CYP4g15-like exhibiting the lengthiest sequence comprising 566 amino acids, whereas CYP4C1-like-3 featured the shortest sequence with 442 amino acids. Similarly, coding sequence lengths exhibited variability, with CYP6B2-like-2possessing the longest sequence at 1721 nucleotides. The genomic distribution of these genes across nine chromosomes (2, 5, 10, 11, 13, 16, 17, and 21) revealed nearly equal occurrences on both positive (+ tive) and negative (-tive) strands. Molecular weight disparities were observed among the genes, with CYP4g15-likeexhibiting the highest at 64576.66Daltons (Da) and CYP4C1-like-3displaying the lowest at 50260.21Da. Assessment of protein stability through the instability index (II) revealed that genes such as CYP9e2-like, CYP6B6-like-1, CYP6B6-like-2, CYP6B1-like, CYP4C1-1, CYP4d2-like-1, CYP6B5-like, CYP4C1-like-2, CYP4d2-like-2, CYP6B6-like-3, CYP4C1-like-3, and CYP6B2-like-2were deemed stable, as their II values fell below the threshold of 40. The GRAVY scores, indicative of protein polarity, uniformly reflected negative values for all identified genes, signifying their non-polar nature. Furthermore, the pI values, representing the pH at which the net charge of a protein is neutral, ranged from 6.94 in CYP4C1-2 to 8.99 in CYP4C1-1. Additionally, the aliphatic index (AI) spanned from 81.73 in CYP6B2-like-1 to 103.13 in CYP4d2-like-1, as detailed in Table 1.

Table 1.

Show the physiochemical properties of CYP450 genes with gene length (start and end position), gene location, pI, GRAVY, II and mw.

Source accession	Gene ids	Chr. no.	Gene position			Peptide	CDS	Mw	pI	II	AI	GRAVY
			Gene Start	Gene End	Strand
Tni02G02740	CYP6B5-like	2	5,407,949	5,413,611	+	511	1536	58,577	8.84	37.4	89.3	− 0.223
Tni05G04550	CYP4C1-like-1	5	9,065,185	9,070,484	+	500	1503	57,888.17	7.64	40.65	91.62	− 0.207
Tni05G04560	CYP4C1-1	5	9,071,316	9,075,723	+	511	1536	59,590.57	8.99	36.18	96.54	− 0.182
Tni05G04570	CYP4C1-2	5	9,076,674	9,080,852	+	501	1506	57,523.91	6.94	47.86	96.11	− 0.161
Tni10G04110	CYP18a1	10	7,588,257	7,592,371	-	544	1635	61,692.76	6.99	47.21	95.51	− 0.097
Tni11G02860	CYP15A1-like	11	5,406,491	5,416,405	+	506	1521	57,623.53	8.38	47.78	97.04	− 0.163
Tni13G01030	CYP4d2-like-1	13	3,147,200	3,152,595	-	533	1602	60,916.93	8.83	36.21	103.13	− 0.093
Tni13G01040	CYP4d2-like-2	13	3,169,321	3,177,821	-	472	1419	54,363.13	8.97	37.85	99.68	− 0.191
Tni13G02970	CYP4g15	13	7,257,524	7,262,159	+	561	1686	64,386.24	8.79	41.25	94.15	− 0.227
Tni13G02990	CYP4g15-like	13	7,274,884	7,281,584	+	566	1701	64,576.66	8.66	42.4	94.01	− 0.19
Tni16G04090	CYP6B2-like-1	16	7,627,670	7,629,846	+	521	1566	60,370.92	8.17	40.16	81.73	− 0.256
Tni16G04370	CYP6B1-like	16	8,215,694	8,257,235	-	524	1575	60,934.54	8.78	35.8	81.98	− 0.26
Tni16G04380	CYP6B2-like-2	16	8,257,726	8,300,450	-	525	1721	60,930.58	8.36	38.68	84.84	− 0.244
Tni17G05560	CYP9e2-like	17	11,719,375	11,724,374	-	530	1593	61,339.48	8.76	30.17	89.89	− 0.102
Tni21G02450	CYP6B6-like-1	21	6,718,134	6,721,575	-	505	1518	57,536.88	8.6	33.69	93.94	− 0.125
Tni21G02460	CYP6B6-like-2	21	6,725,984	6,727,646	-	505	1518	57,631.98	8.72	34.86	90.89	− 0.136
Tni21G02490	CYP6B6-like-3	21	6,738,743	6,741,564	+	500	1503	57,449.7	7.09	37.86	93.54	− 0.177
Tni21G03620	CYP4C1-like-2	21	9,841,001	9,873,333	+	493	1482	56,693.15	8.43	37.63	97.91	− 0.144
Tni22G05980	CYP4C1-like-3	22	12,758,797	12,763,737	-	442	1329	50,260.21	7	38.12	85.77	− 0.241

Mw: Molecular weight, pI: Isoelectric point, GRAVY: Grand average of hydropathy, II: Instability Index, AI: Aliphatic index.

Comparative phylogenetic analysis of CYP450 genes

In this study, we conducted a systematic curation of genetic sequences corresponding to 19 CYP450 genes from T. ni (cabbage looper), 82 from B. mori (silkworm)and 33 from S. frugiperda (fall armyworm)³⁶. A total of 134 CYP450 gene sequences were utilized to construct a comprehensive phylogenetic tree employing the maximum likelihood (ML) method. The identified genes were systematically categorized into 3 distinct groups, denoted as CYP3, CYP4 and CYP6 based on their presence of subfamilies in CYP450. Group CYP3 comprises 32 CYP450 genes, CYP4 encompasses 48 CYP450 geneswhile CYP6 comprises 54 CYP450 genes (Fig. 1). The bootstrap values were mentioned on each clade emerging from their parent and classified each clade on their respective sequence and values. These genes were classified based on their presence of subfamily domains of CYP3, CYP4, and CYP6.

Fig. 1.

Phylogenetic relationships among 134 CYP450 genes from T. ni, B. mori (Bm), and S. frugiperda (Sf). The tree was constructed using the Maximum Likelihood (ML) method in MEGA v11 with 100 bootstrap replications. Genes are grouped into CYP3, CYP4, and CYP6 families based on sequence similarity and conserved domain architecture.

Conserved domain motif, subcellular and gene localization of CYPTni genes

This study’s motif analysis identifies conserved motifs and associated domains within each CYPTni gene. Figure 2 demonstrates the conservation patterns of motifs 1, 2, 3, 5, 6, 8, 10throughout the majority of CYP450 genes, all of which include essential conserved domains associated with CYP4 and CYP6. Particularly, genes CYP6B6-like-1, CYP6B6-like-2, CYP6B6-like-3, CYP6B5-like, CYP6B2-like-1, CYP6B1-like and CYP6B2-like-2 have more than 90% of conserved motifs. A CYP15A1-like domain is also preserved in one of the discovered genes. Notably, the number of motifs in the CYP450genes in investigation varies, with CYP4C1-like-1having the most and CYP15A1-likehaving the fewest. In accordance with the findings of this research (Fig. 3), the predicted subcellular localization of CYP450 genes includes the extracellular region, mitochondria, cytoplasm, chloroplast, nucleus, Golgi apparatus, endoplasmic reticulum (E.R.), and plastids. Interestingly, the majority of the genes investigated were predicted to show subcellular localization, mostly inside the cytoplasm, plastids, and E.R. The analysis identified the genomic positioning of CYP450 genes across diverse chromosomes in the exploration of chromosomal mapping. The majority of CYP450 gene aggregation was found on chromosomes 13 and 21, which comprise all four genes on each. Furthermore, chromosomes 5 and 16 have an equal number of CYP450 genes, with each having three. As seen in Fig. 4, the remaining chromosomes (2, 10, 11, 17, and 22) have unique CYP450 gene occurrences.

Fig. 2.

The graphical representation depicts the pattern of distribution of the entire group of 20 motifs in CYP450. This distribution strongly depicts conserved domains, which are distinguished as CYP4 and CYP6-like.

Fig. 3.

The graphical representation of CYP450 genes shows the predicted subcellular localization pattern within the cytoplasm, plastids, and E.R.

Fig. 4.

The presence of 19 CYP450 genes in the cabbage looper was shown by chromosomal mapping studies, suggesting an interaction of selection pressure and genomic rearrangement methods.

Mature mirnas, exon-mapping and syntenic analysis

A comprehensive investigation of mature micro-RNAs demonstrates an obvious association with the targeting of 19 identified CYP450 genes, with an emphasis on 33 particular micro-RNAs. Notably, the predicted microRNAs, namely Tni-bantam-2-3p and Tni-miR-2393-1-3p, have a noticeable regulatory impact on the bulk of the CYP450 gene set. Further study reveals that Tni-miR-2393-1-3p interacts with CYP6B1-like, CYP6B2-like-2, CYP9e2-like, CYP6B6-like-1, CYP6B6-like-2, CYP6B6-like-3, CYP4C1-like-2, CYP4C1-like-3, CYP4C1-like-1, CYP4C1-1, CYP4C1-2, CYP18a1, CYP15A1-like, CYP4d2-like-1, and CYP4d2-like-2 genes, effectively influencing cleavage and translation processes. A comprehensive tabulation of detailed information pertaining to these micro-RNAs with targeted genes is provided in Table 1S for reference. The genomic structure and intron-exon distribution across diverse gene loci are illustrated in Fig. 5, based on the findings presented in this investigation. Notably, within the identified CYPTni genes, five exhibit two exon structures devoid of introns, while another five genes contain nine exons. The remaining genes display a range of exon counts, ranging from a minimum of six (CYP18a1) to a maximum of thirteen (CYP6B2-like-2). Regarding the paralogous behavior of CYP450 genes, noteworthy paralogous associations have been discovered on T. ni chromosomes 5, 13, 16, and 21. Tandem duplication events are visible on chromosomes 2, 10, 11, 17, and 22, each of which contains a single gene, as shown in Fig. 6.

Fig. 5.

The intron-exon structure of CYP450 genes is illustrated, and five genes have two exons, while others have six to thirteen exons.

Fig. 6.

The paralogous behavior of all identified CYP450 genes with their gene locations on different chromosomes is depicted in this figure.

Transcriptomic expression of CYPTni in response to HDAC inhibitors

Expression profiling in response to SFN treatment

Sulforaphane (SFN) functions as an inhibitor of nuclear histone deacetylases (HDACs), inducing rapid alterations in enzyme activities, DNA-histone binding, and gene expression. This compound is naturally produced by plants as a defensive mechanism against insect attacks^{35, 37–40}. In order to examine the impact of SFN on insect activities, transcriptomic data sourced from the NCBI GEO was analyzed to assess the significance of associated genes in response to SFN treatment. Notably, the application of SFN resulted in a significant up-regulation of the CYP4C1-1 gene in T. ni (p < 0.05), as determined by Student’s t-test, indicating a pronounced biological response to SFN dosage (Fig. 7).

Fig. 7.

The research contains an extensive study of gene expression profiling of CYP450 genes in response to sulforaphane (SFN) treatment. The regulatory mechanisms that regulate the discovered genes are described and demonstrated in the figure thatdisplaying the gene expression. A vertical bar graph with time intervals on the x-axis and expression levels on the y-axis depicts the expression of the CYP450 genes. Each color represents a specific gene as shown in legends, the error bar is represented by a black colored line (plus cap). Expression values are normalized FPKM means ± SE, and statistical significance was assessed using an unpaired two-tailed Student’s t-test (p < 0.05).

Expression profiling in response to TSA treatment

Trichostatin A (TSA) is a pharmaceutical histone deacetylase (HDAC) inhibitor commonly used as a laboratory positive-control compound. While TSA and other HDAC inhibitors have been investigated experimentally in insects⁴¹, they are not approved or practical as agricultural insecticides. In Trichoplusia ni, TSA is largely ineffective due to strong detoxification mechanisms .Transcriptomic data were utilized to analyze the expression patterns of relevant genes following TSA treatment in order to understand the effect of TSA on the cabbage looper (Fig. 8). The results demonstrate that the CYP6B5-like and CYP4C1-2 genes are significantly up-regulated in response to TSA exposure (p < 0.05), as determined by Student’s t-test. Based on the research outcomes, the identified genes were found to significantly contribute to the detoxification process of the administered therapeutic intervention in T. ni.

Fig. 8.

The study includes a thorough examination of gene expression profile of CYP450 genes in response to Trichostatin A (TSA) treatment. The figure describes and illustrates the regulatory systems that regulate the newly identified genes. Graph displaying the gene expression. A vertical bar graph with time intervals on the x-axis and expression levels on the y-axis depicts the expression of the CYP450 genes. Each color represents a specific gene as shown in legends, the error bar is represented by a black colored line (plus cap). Expression values are normalized FPKM means ± SE, and statistical significance was assessed using an unpaired two-tailed Student’s t-test (p < 0.05).

Discussion

HDACs (Histone Deacetylases) are enzymes that modify histones, which are proteins that assist in binding and arranging DNA within the cell nucleus⁴². Histone acetylation and deacetylation are part of the dynamic process known as epigenetics, which controls gene expression without changing the underlying DNA sequence⁴³. HDAC inhibitors such as SFN and TSA act as insecticides, controlling insect populations⁴⁴. These inhibitors cause fast changes in enzyme activity, DNA-histone binding, and gene expression^{45, 46}. A comprehensive investigation was conducted to determine the regulatory mechanism against the HDAC inhibitors SFN and TSA. This study conducted an extensive genome-wide evaluation of the CYP450 supergene family in T. ni, with a particular focus on the impact of HDAC inhibitors. The primary objective was to understand the complex detoxification mechanisms triggered by these genes.

The findings of the investigation indicate a complex interaction of molecular pathways that contribute considerably to T. ni resistance. To accomplish this, a comprehensive examination of multiple databases, including the Trichoplusia ni genome database, was conducted with the aim of identifying and characterizing the distribution of CYP450 gene clusters throughout T. ni chromosomes. Phylogenetic analysis has been carried out to comprehend the evolutionary relationships and duplication occurrences within the CYP450 supergene family across several insect species. The discovered CYP450 genes were grouped into 3 groups (CYP3, CYP4 and CYP6). Gene categorization correlates to the Dr. Nelson’s nomenclature and classification mechanism⁴⁷. Among all groups, CYP6 has the most CYP450 genes as well as the most CYP450 genes. Furthermore, a closer look at conserved structures within these genes revealed important information about their functional variety and the regulatory complexities that regulate their expression. The investigation of the gene structures additionally identified unusual patterns. Some genes have two or nine exons, whereas others have multi-exonic structures with six to thirteen exons. This finding shows that several regulatory mechanisms influence the expression of these genes^48–50.

The discerned patterns, encompassing paralogous behaviors and segmental duplication events, serve to underscore the dynamic nature of gene evolution and expansion within the T. ni genome^{51, 52}. Furthermore, microRNA (miRNA) analysis unveiled specific regulatory networks associated with distinct CYP450 genes, such as Tni-bantam-2-3p and Tni-miR-2393-1-3p. These findings provide invaluable insights into the post-transcriptional regulation of these genes and their potential roles in modulating defense and chemical detoxification mechanisms^{51, 53, 54}.

Utilizing gene expression profiling data obtained from the NCBI GEO database, we conducted an investigation into the temporal dynamics of T. ni response to resistance against histone deacetylase inhibitors^55–57. Our analysis revealed distinct differential expression patterns of CYP450 genes during SFN treatment, specifically up-regulation of CYP4C1-1, and during TSA treatment, with up-regulation of CYP4C1-like-1and CYP4C1-2. These observations underscore the pivotal roles played by these CYP450 genes in the defense mechanism against HDAC inhibitors. The gene structure and expression of CYP450 genes in T. ni were discovered in this described study. A similar study was reported in fall armyworm in which the 33 CYP450 genes were characterized against the insecticide resistance mechanism. The key gene role was evaluated against four well-known insecticides, such as emamectin benzoate, tetrazolium, cyantraniliprole, and spinetoram, and five genes (Cyp306a1-like, Cyp9e2-like, Cyp6l1-like, Cyp12b1, and Cyp6B2-like) were found to have pivotal roles in the resistance mechanism³⁶. The study gives information on the structure and activities of identified genes, as well as resistance to HDAC inhibitors^58–62. Further study is needed to examine the resistance and detoxification mechanisms triggered by the discovered genes in T. ni⁶³.

Conclusion

A genome-wide investigation of the CYP450 superfamily in T. ni resulted in the identification of 19 distinct genes classified into 3 groups. B. mori, T. ni, and S. frugiperda sequences were utilized in evolutionary and comparative genomics studies. Intron and chromosomal mapping revealed a repetitive gene structure spread across many chromosomes, with significant numbers on chromosomes 13 and 21. Predictive studies of subcellular location were carried out on the discovered genes, which were mostly found in the endoplasmic reticulum (ER). Thirty-three mature microRNAs were investigated in relation to the identified CYP450 genes. Expression patterns of CYPTni genes revealed significant up-regulation results for CYP4C1-1against the treatment of sulforaphane and CYP4C1-like-1and CYP4C1-2genes against the treatment of Trichostatin A. Our study provides valuable insights into the CYP450 superfamily in T. ni through comprehensive genome-wide and transcriptomic analyses. However, these computational findings require further validation to identify each gene’s targeted site and demonstrate the associated resistance pathways involved in detoxification mechanisms. Understanding their specific roles in relation to the targeted sites of HDAC inhibitors under diverse conditions advances this line of research and opens avenues for the manipulation or knockout of resistance-related genes. These results provide a fundamental understanding and provide a strong foundation for future functional and applied studies.

Author contributions

AA and AS carried out research work and wrote the initial draft of the manuscript. AS and MZH conducted the data analysis. MS, QA, MAJ, and SA planned and supervised the study and edited the final version of the manuscript. SA, MAJ, SH, DA, and QA technically reviewed and finalized the draft. All authors reviewed the final version of the manuscript and approved it for publication.

Funding

The authors would like to extend their sincere appreciation to the Ongoing Research Funding Program (ORF-2025-165), King Saud University, Riyadh, Saudi Arabia.

Data availability

The datasets used and/or analyzed during the current study has been available in the manuscript.

Data transparency: Rest assured, I have ensured that all data, materials, software applications, and custom code supporting the claims made in this article are in full compliance with field standards. It is important to note that I have taken into account the possibility of individual journal policies regarding research data sharing, considering the norms and expectations of our discipline.

Declarations

Competing interests

The authors declare no competing interests.