Transcriptome analysis unravels RNAi pathways genes and putative expansion of CYP450 gene family in cotton leafhopper Amrasca biguttula biguttula (Ishida)

Amrasca biguttula biguttula is an important pest of cotton and okra in the Indian subcontinent. Presently limited genomic/ transcriptomic information is available for this insect in any open source databases. To initiate molecular studies in this insect, we report first assembled and annotated de novo transcriptome of cotton leafhopper. Out of 75,551 transcripts, 39613 CDS (Coding Sequence) were predicted with 35282 showing positive blast hits with NCBI nr database . From the Gene ontology (GO) analysis, 7431 CDS were annotated. KEGG pathway analysis categorized CDS into 22 different functional categories. The majority of CDS were annotated in signal transduction and transport catabolism pathways. The sequence data was screened for RNAi pathway genes and presence of 37 transcripts associated with this process confirmed the existence of robust RNAi machinery in this insect. The role of core RNAi machinery genes ( Dicer-2 , Ago-2 , Piwi and Staufen ) has been validated through dsRNA feeding studies. The data resource has also been used to identify potential RNAi targets and genes associated with insecticide detoxification specifically CYP 450 family.


Introduction
Numerous hemipteran insects such as cotton leafhoppers, bugs (mealybug, red cotton bug and dusky cotton bug) and aphids are economically important pests and major threat to agricultural crops. Cotton leafhopper or jassid, Amrasca biguttula biguttula (Ishida) (Hemiptera: Cicadellidae), is among the most damaging sucking pests of cotton and okra. Other major hosts for this insect include groundnut, jute, soybean, niger, sunflower, aubergine, potato, mung bean and cowpea. Its severe infestation in the crops causes bronzing or brick red coloring of leaves, which is known as "hopper burn" (Iqbal et al., 2008). Apart from drying of leaves, the leafhopper damage induces shortening of internodes and downward curling of leaves which significantly reduces plant vigour, growth and yield (Somnuck, 2001). This insect also infests many malvaceous and solanaceous crops, however in cotton alone its damage accounts up to 25-45% loss in yield (Dhawan & Sidhu, 1986). Since the mainstay of leafhopper management mainly relies on synthetic insecticides (Gallun et al., 1975), their excessive use has led to the resistance development against major insecticidal groups in this insect (Saeed et al., 2017). However, the molecular basis of resistance needs complete information on the various genes associated with the xenobiotic detoxification. Reportedly, there are three foremost xenobiotic detoxifying enzyme families which play role in metabolic resistance to insecticides are the carboxylesterases (COEs), glutathione transferases (GSTs), and cytochrome P450s (CYP450s) (Ranson et al., 2002). The proteins of these families are associated with the synthesis and breakdown of a large amount of endogenous metabolic moieties, protection against oxidative stress, detoxification of insecticides and the movement of molecules through cells (Le Goff et al., 2003). In Depth knowledge of molecular and biochemical basis of insecticide resistance development would help to devise strategies for its future management which is quite challenging as the various subfamilies of these enzymes are associated with numerous metabolic pathways. To proceed with this research, we have used the present transcriptome to identify potential GSTs, CYP450s, COEs, of A.biguttula biguttula using BLASTx for the first time, which can be used in future to interpret molecular basis of insecticide resistance. Bioassays based few studies have been conducted for continuing insecticide resistance but lack of sequences data resources have restricted the validation of results through molecular tools. Thus, to decipher evolved insecticide resistance at molecular level as well as to develop novel eco-friendly strategies for pest management, genome or transcriptome information is a pre-requisite (Sagar & Balikai, 2014).
Apart from a few studies on mitochondrial-COI based genetic diversity, a recent study on reference genes validation and the demonstration of feeding RNAi in this insect, no genomic or transcriptomic resource is available for A. biguttula biguttula to best of our knowledge (Kranthi et al., 2018;Singh et al., 2018). Since RNAi is a potential present-day tool for exploring functional genomics, holds wide scope for developing new generation insect-pest management strategies. In this endogenous cellular process, the double stranded RNA (dsRNA) when binds to complementary mRNA, directs its cleavage, and eventually blocks the expression of targeted gene which may lead to phenotypic and physiological changes, and insect mortality in some cases (Singh et al. , 2019b;Rodrigues et al., 2018;Burand & Hunter, 2013;Perrimon, Ni, & Perkins, 2010). There are a series of pathway genes that govern this Ai mechanism in an organism starting from uptake of dsRNA by the cell till knockdown of targeted mRNA and disruption of protein synthesis. Therefore, sequence information availability is the prerequisite for initiating RNAi studies in any organism. We have tried to mine the A.biguttula biguttula transcriptome for identification of RNAi pathway genes and validated the same through dsRNA feeding assays. Additionally, we have also identified vital and unique targets amenable for RNAi based on earlier identified targets from other insects, which can be exploited in future to develop management tools in the form of dsRNA based sprays or insect resistant transgenic crops (Artymovich, 2009). The de novo transcriptome of A. biguttula biguttula is the first ever assembled and annotated sequence data resource submitted in NCBI repository. To compare our results, we assembled and annotated the available A. biguttula biguttula transcriptomes (Adult male: SRR9705737, SRR9705738; Adult female: SRR9705739, SRR9705740; Late Nymph: SRR9705741, SRR9705742 and Early Nymph: SRR9705743, SRR9705744). Therefore, this study intends to unveil detailed information about the Cytochrome P450 (CYP450) gene family and RNAi pathway genes, which may be useful for the groups interested to take up molecular studies in this insect.

Sample collection and RNA isolation:
The cotton leafhopper was collected from the insect culture maintained on cotton plants in walk-in environmental chambers of Punjab Agricultural University, Regional Research Station, Faridkot, Punjab, India. Samples for de novo transcriptome included 15-20 individuals of A. biguttula biguttula collected from these cultures. The male and female individuals ((males and females equal in number) were pooled in 0.2 ml TRI-Reagent (Sigma, St. Louis, USA), stored at −80 °C. Total RNA extractions were performed using TRI-reagent (Sigma, St. Louis, USA) as per manufacturer's protocol followed by quality evaluation on a Qubit Fluorometer for the generation of RNA-seq libraries (Supplementary File S1: Figure S1 and Figure S2).

cDNA synthesis and Sequencing
The total RNA extracted in the previous step was subjected to the construction of PE (Paired End) libraries using TruSeq stranded mRNA Library Prep Kit (Illumina) as per manufacturer's instructions. To proceed, the mRNA was converted into First Strand cDNA using poly-T attached magnetic beads and enzymatic fragmentation followed by First double stranded cDNA synthesis using second strand mix and Act-D mix. This double stranded cDNA was purified using Ampure XP beads followed by A-tailing, adapter ligation and enrichment by 10 PCR cycles (10 cycles). These PCR libraries were further evaluated in a 4200 TapeStation system (Agilent Technologies) with High sensitivity D1000 Screen tape as per manufacturer instructions. Afterwards, the PE Illumina library was loaded onto NextSeq 500 for clustering and sequencing which allows it to be read in both forward and reverse directions.
The reads were mapped and aligned back to their reference assembled transcripts using BWA v0.7.12 (Burrows-Wheeler Aligner) for validation of transcriptome (Li & Durbin, 2009). The ORFs were predicted from these transcripts using TransDecoder (Haas et al., 2013) and further annotated using various annotation tools.

Functional Annotation
The sequences (ORFs) obtained were annotated using BLASTx against NCBI nr databases.
The BLASTx resulted accession IDs were used to retrieve gene names or symbols, which were then searched in the species-specific entries of the gene-product tables of GO database.
BLASTx analysis of the resulting proteins was performed with the UniProt, Swiss-Prot, PSD, TrEMBL, RefSeq, GenPept and PDB to have access to UniProt IDs. Accession IDs were searched directly in the dbxref table of GO database. Gene Ontology (GO) of the predicted CDS were determined by the Blast2GO program (Conesa et al., 2005). Afterwards, GO assignments were used to classify the functions of the predicted CDS. GO mapping was carried out in order to retrieve GO terms for all the BLASTx functionally annotated CDS. The completeness and contiguity of the assembled transcriptome was further validated using BUSCO v1.1b1 (Benchmarking Universal Single-Copy Orthologs) (Waterhouse et al., 2017). This analysis was performed using insect (1658) and arthropoda (1066) (Khan et al., 2017;Rodrigues et al., 2017) were identified in this insect from the annotated sequences.

Expression of core RNAi genes across different stages of leafhopper:
To validate the identified RNAi machinery genes from A. biguttula biguttula transcriptome, 1µg total RNA isolated from 15 adults, 20 younger nymphs (1 st , 2 nd and 3 rd instars) and 15 late nymphs ( 4 th and 5 th instars) in three biological replicates as per earlier described protocols was used to synthesize cDNA with First Strand cDNA Synthesis Kit (ThermoFisher Scientific) as per manufacturer's instructions. The expression of core RNAi genes was evaluated across different stages of leafhopper using RT-qPCR and qPCR forward and reverse primers designed using Primer3 software (Untergasser et al., 2007) (Supplementary File S1: Table S1). Quantitative expression studies were carried out using 5µl of 5x SYBR Premix Ex Taq (Tli RNase H Plus) (Clontech takara), 0.2 µl of 10pmol primers and 1µl of 1/10 cDNA template in a 10µl reaction in Lightcyler (Roche).

Functional validation of core RNAi genes: SNF7 functions as a constituent of ESCRT
(Endosomal Sorting Complex Required for Transport) pathway, which has role in cellular functions such as internalization, transport, sorting and lysosomal degradation of transmembrane proteins (Huseth et al., 2016). To validate the functioning of core RNAi genes, Dicer-2, Ago-2, Piwi, and Staufen were knocked-down and their impact on RNAi efficiency of SNF7 was compared with the dsGFP fed control insects. Gene-specific primers with T7 promoter sequence at 5′ ends of both forward and reverse primers were custom synthesized (Eurofin Genomics, Bangalore India) to amplify the template fragments for dsRNA synthesis of Dicer-2, Ago-2, Piwi, Staufen and SNF7 genes (Supplementary File S1: Table S1). The respective gene specific PCR product was amplified using leafhopper cDNA followed by purification using Nucleospin Gel and PCR Cleanup kit (Macherey-Nagel) as per instruction manual. The eluted PCR product was used to synthesize respective gene specific dsRNA using MEGAscript™ RNAi Kit (Thermo Fisher Scientific). Biological samples in triplicate each comprising 15 individuals (5/ tube) of 5 th instar nymphs were released in 1.5 ml tube (lower 1/4th part of the tube was cut and covered with muslin cloth) (Brar et al., 2018). The dsSNF7 (500 ng/μl) was fed along with a diet stretched between two layers of parafilm as per previously described methodology (Brar et al., 2018). The dsRNAs against Dicer-2, Ago-2, Piwi and Staufen genes were evaluated to examine their respective knockdown impact on RNAi of SNF7 gene. For this, 500 ng/μl each of dsDicer-2, dsAgo-2, dsPiwi and dsStaufen was administered in 100 μl of diet  separately for each experimental setup. After 24 hours, the above dsRNAs against respective RNAi gene was replaced by dsSNF7 in the diet of cotton leafhoppers as per methodology described (Supplementary File S1: Figure S3). Live cotton leafhoppers were collected post 48 h from respective feeding chambers, and total RNA was extracted and subjected to expression studies as described in the earlier section. The relative expression of the SNF7 under the influence of each core RNAi pathway gene was calculated using ∆∆CT method in comparison with dsGFP fed control insects after normalization with RPL9 as housekeeping gene .

Annotation, identification and classification of CYP450 gene family
The predicted protein data resource from sequenced transcriptome was used for identification of transcripts associated with CYP450 (cytochrome P450) gene family in A. biguttula biguttula using Hidden Markov Model (HMM) based HMMER v3 (Eddy & Wheeler, 2015 Additionally, the other genes belonging to carboxylesterases (COEs), monooxygenases, and glutathione transferases (GSTs) associated with xenobiotic detoxification were also identified and annotated from this transcriptome using BLASTx search against NCBI nr database.

Expression level of CYP450 genes of A. biguttula biguttula using RT-qPCR.
F5 th instar nymphs (15 individuals per replicate) of two Faridkot (Punjab-India) populations (AbbFdk1 and AbbFdk2) were collected from the cotton fields using aspirators in triplicates.
The two locations were selected based on differential insecticide usage patterns against cotton leafhopper. The Fdk 1 (30.4032° N, 74.4456° E) was a low insecticide usage location while Fdk2 (30.4906° N, 74.6386° E) was a comparatively higher insecticide using area. The collected live individuals were brought to the laboratory and processed for RNA isolation and cDNA synthesis using earlier described methodologies. Primers specific to each selected CYP gene sequence were designed and custom synthesized (Eurofin Genomics, Bangalore India) (Supplementary File S1: Table S1). Randomly selected 9-10 genes representing each clan were evaluated for their expression across these two populations of A. biguttula biguttula. Fifteen genes (3-4 per clan), which showed comparable expression were selected for differential expression analysis between two field populations of leafhopper. The comparative mRNA expression of these genes among two populations was evaluated using Real-time PCR (Lightcycler, Roche) as described in the earlier section.

Transcriptome assembly and annotation
The libraries sequenced on NexSeq 500 (Illumina) using 2 x150 bp PE (paired end) library generated ~9.8 GB raw data (SRR6308174) after eradicating adapter sequences and ambiguous reads. After removing uncertain sequences from the raw data, 28,400,988 (2 x 150 bp) high quality PE reads (QV>20) were obtained, which were used for de novo assembly (Table 1).The reads were assembled using high throughput Illumina NexSeq500 platform and de novo transcriptome assembly generated with the help of Velvet (Zerbino & Birney, 2008) and Oases (Schulz et al., 2012) Fig. S5). Gene ontology of predicted CDS sequences has been represented with the help of WEGO plot, which shows the percentage participation of these genes in different pathways (Supplementary File S1, Fig. S5). The transcripts were functionally annotated using KEGG analysis and KO IDs were assigned to four biological processes, i.e., Metabolism, Cellular, Genetic and Environmental information processing (Supplementary File S1, Fig. S6). The majority of transcripts were grouped under metabolism (33.92 %) followed by cellular processes (20.61%). Individually among these major processes, the majority of transcripts were associated with carbohydrate metabolism ( were "complete" compared to insect and arthropod lineages, respectively. The remaining 4.9-9.0% and 4.6-8.0% were "fragmented", while 2.1-8.8% and 1.2-6.8% were missing compared to insect and arthropod lineages, respectively ( Table 2). The quality of our assembled transcripts was comparable to the earlier assemblies listed in Simao et al (Simão et al., 2015)

Identification of RNAi targets and RNAi machinery genes in leafhopper
The assembled transcriptome can be used as a future resource to identify genes associated with various physiological, biochemicals, hormone biosynthesis and many other processes, which are important from the pest-management perspective in this insect. In the current study, we explored RNAi pathway genes, potential RNAi targets as well genes associated with insecticide resistance in cotton leafhopper. The transcriptome screening suggests the existence of robust RNAi machinery in this insect with 175 identified transcripts associated with 27 RNAi processing genes. After, multiple sequence alignment and removal of duplicates, only 89 transcripts were found to be related with 27 RNAi machinery genes. The details of different transcripts associated with each gene have been summarized in Supplementary File S1: Table   S2. The prime siRNA machinery genes identified in this insect includes SID1, Argonaute-1, Argonaute-2, Argonaute-3, Dicer-1, Dicer-2, Piwi, Aubergine, RNAseIII, helicases and dsRNA binding proteins. The expression of the core RNAi genes showed variation across different insect stages (Fig. 1), however it reconfirmed the presence of these genes in cotton leafhopper.
The expression of SID1, Dicer-2, Ago-1, Piwi, PCDG6, Ago-2 and RNAseIII was significantly higher in adult compared to early and late nymphal instars. However, the expression of few genes such as Dicer-1, Asp and Dwil in late nymph was significantly higher in late nymphs as compared to early nymph and adult. The expression of all the RNAi genes identified was either significantly or numerically lower in early nymph compared to other two stages. The BLASTx analysis for these genes revealed their notable similarity with homologs from other insects groups. Furthermore, the phylogenetic analysis using neighbour joining method of MEGAX (500 bootstrap value) confirmed that the depicted genes were involved in core RNAi pathways through matching metrics with their orthologs from other insect species (Fig. 2).
The sequence specific knockdown of core RNAi pathway genes such as Dicer-2, Piwi,  (Fig. 3). Consecutive knockdown of Staufen followed by SNF7 resulted in 46.7% hampering of RNAi efficiency compared to only dsSNF7 fed insects .
The transcriptome analysis also revealed the existence of novel and potential RNAi targets in A. biguttula biguttula such as chickadee (responsible for female fertility, nourishment of oocytes (Shields et al., 2014) and regulation of germline stem cells (Kiger et al., 2001) (Issa, 2014), T. castaneum (Zhu et al., 2013) and Erthesina fullo (Y. Liu et al., 2015). In the case of cotton leafhopper, the CYP genes belonging to respective clans are clustered as one clade with maximum number of transcripts (79) in Clan 3 (orange). The remaining transcripts grouped into Clan 2 (red) (30), Clan 4 (yellow) (13) and mitochondrial Clan (violet) (11) [(Fig. 5) (Supplementary File 1: Table S6). The remaining uncolored sequences were not grouped into any clan as were too divergent. In our studies we also identified some CSMs such as C-helix, I-helix, K-helix, Heme binding region and PERF motifs by MEME and SALAD ( Supplementary File 1: Fig.S5-S8).
The predicted CSMs from different CYP clans will be helpful in understanding structure and function of CYP family proteins in A. biguttula biguttula. In addition, the transcriptional analysis of 15 representative CYP450 genes of different clans revealed the differential expression between two different insecticide exposed leafhopper populations (Fig. 6). However, this needs to be studied in detail considering more CYP genes and resistant and baseline susceptible populations of leafhopper.
The gene ontology with Blast2GO was conducted to assign GO terms to CYP450s. Out of 182 CYP450s transcripts 116 were mapped and annotated to GO terms, of which 90 belonged to biological process, 140 to molecular function and 84 to cellular component. Among these categories, catalytic activity, binding, membrane and metabolic process were the largest subcategories (Fig. 7). A subsequent analysis of these transcripts with KEGG revealed that four clans contained multiple CYP450 families such as CYP4, CYP6, CYP15, CYP18, CYP49, CYP302, CYP306, CYP307, CYP314, and CYP15. However, CYP4 and CYP6 were found to be largest CYP450 families in this CYPome (Supplementary File S1: Table S5). About 124 out of 235 CYP putative transcripts were annotated in KEGG database and were assigned to six pathways. These pathways include lipid metabolism (Fatty acid biosynthesis), metabolism of terpenoids and polyketides (Insect hormone biosynthesis), environmental information processing (AMPK signalling pathway), Endocrine system (Insulin signalling pathway), metabolism (Cytochrome P450), signalling and cellular processes (Exosome) (Fig. 7). Further, "metabolism of terpenoids and polyketides" pathways included seven CYP450s from Mitochondrial as well as Clan 2. Similarly, four CYP genes from 'fatty acid biosynthesis', two from 'Insulin signaling pathway' and two from 'AMPK signaling pathway' belonged to Clan 2 as well as Clan 3. About 107 CYP genes were found to be involved in 'metabolism by Cytochrome P450' pathway.

Discussion
The de novo transcriptome of A. biguttula biguttula generates the first assembled and annotated The transcriptome was studied to curate the potential RNAi genes that exist in cotton leafhopper transcriptome. The expression studies revealed variability in the core RNAi genes across developmental stages, however the variation in the expression of RNAi genes among different developmental stages has also been reported in earlier studies with T. castaneum (Perkin & Oppert, 2019), T. tabaci (Singh et al., 2019) and P. solenopsis (Singh et al., 2019b).
In depth studies are required to understand the reasons for differential expression of RNAi pathway genes across developmental stages of a particular insect species. The genes from A.
biguttula biguttula such as SID1 and Clathrin heavy chain responsible for dsRNA uptake and commencement of RNAi pathway showed similarity with Z. nevadensis (Rocha et al., 2011;Shih & Hunter, 2011). Two different paralogs of dicers i.e. Dicer-1 and Dicer-2 were identified in the transcriptome which showed high resemblance to hemipteran insects N. lugens and Graminella nigrifrons, respectively. Three paralogs of Argonautes identified from this insect were closely related to Cerapachys biroi, N. lugens and G. nigrifrons. Current study also focuses on functional validation of core RNAi genes through dsRNA mediated knockdown of Dicer-2, Piwi, Argonaute-2 and Staufen. Their knockdown decreased the RNAi efficiency of the target gene SNF7. Many studies have reported that Dicers produce small RNAs that includes siRNA as well as miRNA, these 20-23 nt RNA fragments are important component of target mRNA degrading RISC (RNA-induced silencing complex) (Lee et al., 2004). Similar studies in L. decemlineata demonstrated the role of Dicer-2 in RNAi pathway (Yoon et al., 2016). Since Piwi knockdown led to more efficient RNAi hinderance showed its significant role in RNAi mechanism. PAZ and Piwi are the two domains present in the middle and Cterminal region of argonaute proteins, respectively. Piwi sub family has been characterized from different insect species and consists of 300 aa similar in structure to catalytic domain of RNaseH, which performs a crucial role in slicer (Zhou et al., 2007). The knockdown of Argonaute-2 led to 70.6% blockage of RNAi, which primarily mediate target recognition and form silencing complex (Gu & Knipple, 2013). Previously, it has been reported that in the cell line of L. decemlineata (coleopterans), the knockdown of Dicer-2 and Argonaute partially blocked RNAi in the cells (Yoon et al., 2016). Staufen is a dsRNA-binding protein initially discovered in Drosophila (St Johnston, Beuchle, & Nüsslein-Volhard, 1991) and its role in RNAi machinery has been demonstrated in C. elegans (LeGendre et al., 2013). Stuafen has not been much studied in insects in context to RNAi. However, a recent report in L. decemlineata reveals specific Staufen (StauC) in coleopterans, which predominantly contributes to the RNAi machinery and enhances knockdown efficiency (Yoon et al., 2018). The Staufen bearing five dsRBD (dsRNA Binding Domain) has also been predicted from A. biguttula biguttula transcriptome using sequence analysis software SMART (Letunic & Bork, 2018) (Supplementary File S1: Figure S4). Staufen may be contributing to RNAi in insect's species other than coleopterans, which lack specific StauC or its homologue as its knockdown has hampered the RNAi mechanism in our studies. Earlier studies with Thrips tabaci (Singh et al. , 2019a) and P. solenopsis (Singh et al., 2019b) also reveal the role of Staufen protein in RNAi mechanism.
The variation in dsRNA mediated knockdown efficiency of different genes within the same organism may be attributed to many known and unknown factors such as length of dsRNA, delivery method, target gene etc. (Burand & Hunter, 2013). Our earlier studies also demonstrated the functional feeding RNAi in A. biguttula biguttula through the differential level of knockdown of SNF7, VATPase, AQP and IAP genes .
Insecticide resistance is the major issue in insects of agricultural importance due to continuous and sometimes indiscriminate use of insecticides for their management. The information is lacking on contribution of CYP 450, voltage gated Na channels, GABA receptors and many other genes towards insecticide resistance in cotton leafhopper. Our first sequence data resource provides an opportunity for better understanding of insecticide resistance mechanism at system biology level for this economically important non-model organism.
Resistance at molecular level has been differentially associated with various gene families such as point mutations in ion channel of GABA receptor (cyclodiene insecticides) and vicinity of the acetylcholinesterase active site (organophosphorus and carbamate insecticide resistance), mutations linked to a sodium channel gene (DDT and pyrethroid insecticides), amplification of esterase genes (organophosphorus and carbamate insecticides) as well as uncharacterized mutations leading to up-regulation of detoxification enzymes, such as cytochrome P450 and glutathione S-transferases (many classes of insecticides) (Feyereisen 1995). These gene families are known to be related to insecticide resistance mechanism in insects (Li, Schuler, & Berenbaum, 2006). Thus taking into account the current information generated for various gene families, the detailed studies with resistant and susceptible populations of A. biguttulla biguttulla will help to elucidate and validate the indepth role of various genes in insecticide resistance. Our findings suggest that there is vast diversity in the CYP genes of the cotton leafhopper with different stages and change of fields. Our major focus was on identification of Cytochrome P450 transcripts, which is the vast gene superfamily consisting of numerous families and subfamilies. These genes are majorly associated with detoxification of synthetic chemicals consumed by the pests thus playing a key role in imparting resistance against insecticides due to evolving nature of the CYPome (Li, Schuler, & Berenbaum, 2006). The insect CYPs have been classified into four clans with majority of genes falling in Clan 3 in the present studies as well as in Aedes aegypti (Issa, 2014), T. castaneum (Zhu et al., 2013) and Erthesina fullo (Y. Liu et al., 2015). The CSMs (Conserved Sequence Motifs) present in CYPs are involved in opening and closing of the substrate access/egress channel by modulating the flexible/plastic region of the protein and overall protein stability that affects enzyme function (Oezguen & Kumar, 2011). To date only five motifs i.e. C-helix, I-helix, K-helix, Heme binding region and PERF motifs are known in insect CYP genes (Feyereisen, 2005). The distinct motif located in the C-helix is WxxxR, and the Arg (R residue) is known to form a charge pair with the propionate of the Heme. The CSM present in the middle of the long helix I is GxE/DTT/S, which surrounds a conserved threonine (T-residues) that is responsible for oxygen activation. The PERF motif PxxFxPxRF contributes towards stabilization of the overall structure of CYP protein through a set of salt bridge interactions (E-R-R) with the CSM ExLR of K-helix. The fifth conserved motif is Heme binding region i.e PFxxGxRxCxG/A which carries the cysteine (C residues) to bind with Heme iron on the opposite side of helix I (Sezutsu, le Goff, & Feyereisen, 2013). CYP450s CSMs are important for biocatalyst designing and specifically in context to insects, they have an important role in insecticide discovery (Chandor-Proust et al., 2013). On the whole the information about CYP450 genes, their domains and CSMs will also be useful in unravelling the gene structural changes occurring due to development of insecticide resistance in this insect. In an hemipteran insect E. fullo (yellow spotted stink bug) the CYP genes from Clan 2 and mitochondrial Clan were also involved in Insect hormone biosynthesis and those from Clan3 as well as mitochondrial clan participated in fatty acid biosynthesis (Y. Liu et al., 2015). This indicated that genes from different CYP clans are associated with one or multiple biological pathways. The P450 enzymes in insects are associated with diverse functions ranging from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of xenobiotics (Y. Liu et al., 2015;Feyereisen, 2005). The enzyme family plays a key role in adaptation of insects to toxic compounds in their host plants as well as metabolism of commonly used insecticides (Hodgson 1985). In some insects the detoxification is so active that the insecticide is metabolized before reaching the target site thus rendering organisms resistant to these molecules (N. Liu et al., 2015). The role of various CYP450 genes in insecticide resistance has been reported in many insect species such as Culex pipiens pallens (Chang et al., 2017), Culex quinquefasciatus (Liu et al., 2011), Myzus persicae (Puinean et al., 2010), Tribolium castaneum (Zhu et al., 2010), Drosophila (Le Goff et al., 2003), Anopheles gambiae (Nikou, Ranson, & Hemingway, 2003). It has been evident in many studies that the cotton leafhopper has developed many fold resistance to various classes of insecticides used for its management (Sagar & Balikai, 2014). Thus, identification of P450 genes in this insect will help to understand the molecular mechanisms underlying the insecticide resistance.
in the soybean pod borer (Lepidoptera: Olethreutidae   Sequences in violet colour belong to mitochondrial CYPs, red colored to CYP2 clan, yellow coloured are from CYP4 clan and remaining orange colored transcripts fall under CYP3 clan. Fig. 9 Differential expression of mRNA transcripts of selected CYP450s genes in two differentially insecticide exposed late nymphal populations of Amrasca biguttula biguttula using Real Time-PCR. The error bars represent the standard deveiation (n=15) with 3 replicates each. Fig. 10 Gene Ontology and Pathway analysis of CYP450 genes of Amrasca biguttula biguttula using BLAST2Go (A) and KEGG(B.)