Molecular Study on Field Evolved Resistance of Red Palm Weevil (Rhynchophorus ferruginous) and its Management through RNAi

The date palm is an important fruit tree but due to severe attack of pests and diseases 30% yield losses were recorded throughout the world (El-Juhany and Loutfy, 2010). R. ferrugineus is regarded as tissue borer that have the ability to destroy 28 species of palm trees throughout the world. The enormous losses induced annually by this invasive pest in the Middle East are around $US 130 million (Faleiro, 2006; El-Sabea et al., 2009) In Pakistan, recently, red palm weevil (R. ferruginous), dusky cotton bug, (Oxycarenus hyalinipennis) and nucleopolyhedrovirus from Armyworm (Spodoptera litura) have been collected, molecularly identified and characterized from Pakistan (Ahmad et al., 2018a, b; Manzoor et al., 2018).

For the past six decades, synthetic pesticides are being implemented as pest control regardless of their negative impacts such as environmental pollution, health hazards, resistance development in insects (Tiwari et al., 2011b) and contamination of foods due to residual effect (Silva et al., 2012). The parasites and predators are also killed due to no target specificity of these insecticides. To handle this burning pest, molecularly constructed insecticides specific to insect species may be helpful.

RNA interference is a reliable molecular technique employed in insect pest management and operational gene expression due to the systemic nature of dsRNA (Sachs-Ericsson et al., 2007; Price and Gatehouse, 2008; Scharf et al., 2008; Huvenne and Smagghe, 2010; Whitten et al., 2016).Plenty of genes have been isolated and targeted for RNAi in various insects such as chitinase in Tribolium castaneum, Ostrinia nubilalis, Nilaparvata lugens, Mythimna separata, Manduca sexta, Drosophila melanogaster, Chilo suppressalis, Bombyx mori, Anopheles gambiae and many other economically serious pests (Shen and Jacobs, 1997; Zhu et al., 2008b; Khajuria et al., 2010; Zhang et al., 2011; Huang et al., 2012; Pan et al., 2012; Tetreau et al., 2015; Xi et al., 2015; Su et al., 2016; Cao et al., 2017).

Cytochrome P450 mono-oxygenase is one of the exceedingly vital groups of enzymes which are important for metabolism in arthropods. Explicit gene silencing has been directed via ingestion or incorporation of dsRNA in Caenorhabditis elegans, tick and flatworms, respectively (Newmark et al., 2003; Soares et al., 2005). In the same manner the gene suppression through microinjection in last instar larvae as well as adult of T. castaneum (Bucher et al., 2002) and insects such as Ostrinia nubilalis larvae for dsRNA delivery (Wang et al., 2011) has been practiced. Catalase and Olfactory Co-Receptor (Rfer Orco) down regulation in Rhynchophorus ferrugineus has been achieved in near past (Al-Ayedh et al., 2016; Soffan et al., 2016).

The purpose of this investigation was to evaluate parathyroid insecticide resistance level in R. ferruginous population in association with expression of P450 gene and to enhance the effectiveness of this pesticide by knocking down the expression of resistance P450 gene. This was achieved via injection of dsRNA to the thorax region of R. ferruginous.

Adults, larvae and pupae of R. ferruginous were collected from four provinces of Pakistan, Punjab (31.1704° N, 72.7097°E) Baluchistan (28.49° N, 65.0958° E), Sindh (25.8943°N, 68.52°E) and KPK, 34.95°N, 72.331°E) from field and various palm orchards of the public and private sectors and rearing was done at Integrated Genomics, Cellular, Developmental and Biotechnology Laboratory (IGCDB), Department of Entomology, University of Agriculture, Faisalabad. Collections were made with the help of pheromone traps as well as handpicking and transferred into insect rearing boxes (15cm × 10cm × 5cm) for rearing and bioassays purposes. Laboratory susceptible (SG7-without pesticides exposure) and resistant (RG6-contiuously exposed to pesticides for 6th generations (G6) population of R. ferruginous parathyroid insecticide were developed and maintained under laboratory controlled conditions (25±5 °C, R.H 60±5%) with 12:12 (L: D) periods. Different concentration (10-500 ppm) of tested insecticide (Parathyroid) was applied against R. ferruginous of laboratory and field collected population along with control and mortality data after 24h were recorded regularly.

Total RNA isolation from R. ferruginous samples collected from fields as well as laboratory susceptible and resistant population was done by using isolation kit (Promega WI., USA). Picodrop-200 was used to measure the quality and quantity of the RNA. The cDNA synthesis was doneusing kit (iScript synthesis kit, CA, Bio-Rad, USA). Expression of the gene P450, beta actin and gfp was determined according to the protocol of (Ahmad et al., 2013a; 2014). The primers for Beta actin, P450 and gfp (control) genes are shown in Table I. Gel electrophoresis on 1.5% gel stained with ethidium bromide was carried out in 1% TAE buffer. There were five replicates consisting of 10 insects including three technical replications.

Double stranded RNA specific for P450 gene (mono-oxygenase) was synthesized according to the manufacturer protocol through RNAi-kit (Austin, TX) following the methodology of Al-Ayedh et al. (2016). Using reverse and forward primers with T7 promoters designed for P450 gene were amplified to get339 bp from the cDNA synthesized (Table I). There were 35 cycles consisting of 72 °C for 60 sec, 55 °C for 45 seconds and 94 °C for 30 seconds with 10 minutes elongation steps at 72 °C for the PCR amplifications.. PCR amplified product was sent for purification and sequencing to Macrogen for verification of the desired sequence. P450 ds RNAwas generated and then suspended in the DEPC water and quantified using Nanodrop (Pico-200). Agarose gel electrophoresis (1.5 %) was performed for the verification of amplified genes. . The concentration of tested genes used in RNAi experiment were made up to (1µg/uL in ddH20) and kept at -20 °C. The same protocols and methods were used for constructing of the double stranded RNA for green fluorescent protein (gfp) employed for control.

P450-dsRNA was micro-injected at a concentration of 0.5, 1.5, 2 and 3 ug/insect in the thoracic region of 5th, 10th instar larvae and adult of R. ferruginous and observed for a times period of 4h-48h (Fig. 1). For the purpose of negative and positive control, DEPC water and dsGFP were used for the microinjection. These treated insects were then dissected 24-48h after application and forgut, midgut and hindgut were extracted for further study. Three replications were made with ten larvae per replication. Treated and control larval population were placed in the cups and allowed to feed on the artificial diet.

Knockdown effect for P450 gene was studied through PCR. For each test sample, about 25µl reaction mixture was prepared consisting of RNA free water (8.5 µl), cDNA template (2 µl), and primers (1µl reverse primer and 1µl forward primer) including 12.5µl PCR mix. RT-PCR (real time) was done according to Ahmad et al., 2013, 2014.

Table II. The probit analysis showing Resistance Ratios (RR) in Lab R(G6)= laboratory resistant, FP= Field resistant population from Punjab, FK= Field resistant population from KPK, FS= Field resistant population from Sindh and FB= Field resistant population from Baluchistan of RPW through employing 0-, 10-, 50-, 100 plus 500 ppm insecticides concentrations.

PCR condition were used as 95°C for 30 seconds followed by the other cycles at 95 °C for 5 seconds and lastly at 60 °C for 30 seconds. Analysis of the melting curve was done by the final elongation step of 10 and 15 minutes at 95°C and 60°C respectively with three replications. Beta actin expression was used as standard to standardize the P450 expression by following the 2-∆∆Ct procedure of Livak and Schmittgen (2001).

Protein was extracted from dsP450 treated and control R. ferruginous samples according to prescribed procedure (Ahmad et al., 2013b). Transverse electrophoresis was used for the blotting on PVDF (Perkin Elmer) membrane at 4°C at 100V in a glycine buffer (0.192 mM) and Tris base (0.025 mM). At room temperature, immune labeling was implemented. Tween 20 was used for the incubation of PVDF membranes for 1 hour. Membrane washing was done with Tween 20 (0.05%). Membranes were incubated with laboratory prepared polyclonal antibodies (Anti-P450 1/4000) for two hours and then diluted with Tween 20. PBS Tween 20 (0.05%) was used for washing (4x5 min. and 1x15min.). Chemi-luminescent reagent kit (Thermo Fisher) was used to express the labeling in the dark room. Light films (Kodak Biomax) were used to expose the membranes.

R. ferruginous were reared and maintained in cages box and fed on artificial diet for a period of two hours and then transferred to other cages. Efficacies of the dsRNA were assessed in the R. ferruginous applied with different concentrations (0, 0.5, 1, 1.5, 2 and 3µg/insect). The plates were soaked in the insecticides solution for 30 seconds in acetone and then air dried about 1h prior to insect placement according to the methods of Killiny et al. (2014). The cypermethrin at dosage rate of the 0.02 ng Al/ml (acetone) were used. The mortality data was recorded after every 24 hours. For P450-dsRNA, ds-RNA-gfp was implemented as control treatment. There were five replications having five insects from RPW tested population. Each population was applied with four treatments such as RNase free water, cypermethrin treated discs, dsRNA-P450 treated RPW and cypermethirn.

Collected data were analyzed through ANOVA and DMR (Tukey’s Multiple Range Test) used for mean’s comparison at 5% significance level by SAS18. Means were expressed as standard error (SE). Insecticide resistance and activity of the P450 was examined through Probit analysis. Student-T significant test was used to find the comparisons among the treatments and genes expression at 5% significance level.

Comparative resistance development was studied in Laboratory susceptible, laboratory resistant and four field populations (FP, FK, FS and FB) collected from four provinces {Punjab (P), Sindh (S), Baluchistan (B) and KPK (K) the Pakistan (Table II). The observed resistance ratio (RR) for laboratory resistant population of generation (G6) was 178.5945 fold as compared to laboratory susceptible generation (G7). The field populations of Sindh (FS) and Punjab (FP) depicted 15.5 and 7.32 fold more resistance whereas resistance ratio of field population of KPK (FK) and Baluchistan (FB) population was 4.3 and 1.98 fold more as compared to laboratory susceptible ones.

Through RT-PCR, Cytochrome P450 gene expression was studied in larval and adult population collected from different places before the application of dsRNA-P450. Adult male (M), female (F) and Last stages of larval instars showed maximum expression of P450 gene expression as indicated by band intensity. Bands intensity in gel for various larval stages has the expression of P450 gene with increasing trend from L1 to L10. The expression of P450 was highly enhanced in larval stages L7-L10 as compared to L1-L3 as compared to constant expression of B-actin reference gene (Fig. 2).

Infusion of larvae (10th instar) with dsRNA-P450 at different concentrations exhibited significant knockdown of P450 gene. After 24-48h of dsRNA-P450 injection, the knockdown effect for P450 was enhanced as dose rate increased from 0-3 µg/insect (Fig. 3). The high intensity of amplified bands was observed in the foregut of L10 with H20 and 0µg / insect (Wells 1-2) while the bands intensities was reduced gradually with increased doses showing down regulated expression of P450 gene in R. ferruginous (Wells 3-7). The 40-50 percent larvae was found dead after 48h and 10 percent larvae was found abnormal with passage of time as shown in Fig. 3)

The ds-RNA application in R. ferruginous (male and female) was confirmed by western blot analysis. The controlled palm weevil, R. ferruginous exhibited maximum amplification of P450 expression. When R. ferruginous was treated with different concentrations of dsRNA-P450 (0.5µg, 1µg and 3µg) and constant doses of dsRNA-gfp (control), a decreasing expression of P450 gene was observed. The activity of P450 was decreased tremendously by increasing the concentration of applied dsRNA-P450. The complete cytochrome P450 down-regulation was achieved when 3µg/ insect concentration of dsRNA-P450 was injected to the body of R. ferruginous. Although 0.5 µg/insect concentration of dsRNA-P450 suppresses the P450 gene but no significant results were noted. It was observed that target P450 expression levels and dsRNA treated proteins expressions are directly correlated (Fig. 4).

The expression was also studied with qRT-PCR after the application of dsRNA-P450 and dsRNA-gfp. The treatment with ds-RNA-P450 induced reduced expressions of P450 gene at 1-2 ug/individual. Each treatment sample of ds-RNA-P450 indicated positive correlation as down regulation of P450 expression with increased dose rate (1-2 ug/individual). The target gene P450 expression was investigated in ds-GFP treated insect samples used as control treatments. The relative gene expression of P450 in R. ferruginous exhibited significant down regulation (0. 2-0.5 *P<0.05) compared with dsRNA-GFP control treatments (1 *P<0.05). The dsRNA P450 employed with 2µg/larva displayed maximum down regulation (0.03-0.1 ***P<0.005), compared with as treated with 1µg/adult or larvae (0.2-0.3*P<0.05). But the down regulation of P450 was higher in larva (0.03-0.2) as compared to adults (0.3-0.4). The control gene Beta actin demonstrated constant expression level in all dsP450 and dsGFP treated insect (Fig. 6). Statistically no significant differential gene expression was recorded in reference gene Beta-actin. The results proved that the dsRNA-P450 was found target specific and induced maximum mortality.

The effectiveness of cypermethrin against the different laboratory susceptible, resistant, and Punjab field collected larvae, male and female (SL, SM and SF; FL, FM and FF and RL, RM and RF) population of R. ferruginous were assessed. Its effectiveness was investigated before and after the application of dsRNA-P450 (Fig. 5). R. ferruginous Punjab and laboratory resistant populations exhibited minimum susceptibilities for cypermethrin mixed in artificial diet at LD50 recommended dose rates. RNase-free water treatments were used as control treatments. The pesticide treated SL, SM, SF, RF and RL populations’ showed more significant (P<0.01) relationship and mortality was (Susceptible 84±3, 75±2, 73±6), (Field 63±4, 60±2, 55±5), (Resistant 38±1, 33±1, 32±2) respectively, comparing with H2O treatment pool of RPW (susceptible 11±4, 11±3, 8±2), (field 12±4, 11±2, 13±2), (resistant 6±2, 9±3, 8±1). When these population were treated with dsRNA P450 at least 12h before pesticide treatment, “Susceptible, Field and Resistant population of R. ferruginous showed significant interaction (P<0.005) and mortality was increased (susceptible 98±6, 95±2, 91±4), (field 90±2, 83±6, 81±7), (Rresistant 78±4, 73±4, 72±3), respectively) after the application of dsRNA-P450 (Fig. 7).

Various genes that play an important role in the growth and development of R. ferrugineus are diverse in chemical composition and enzymatic properties. In R. ferrugineus, glycoside hydrolase 18 (GH18), playing an important role in the chitin synthesis of mature eggs of R. ferrugineus (Abdel-Banat et al., 2018).

According to Timmons and Fire (1998) feeding or micro-injection are the key methods associated with the novel RNA-interference expertise and its utilization for arthopodal control in the field conditions (Zhou et al., 2008; Rangasamy and Siegfried, 2012). These techniques have both benefits in addition to limitations. Passionate training is requisite in micro-injection technology comprising injection point, needle size for efficacious dsRNA penetration in test insects and appropriate calibration and optimization is needed for assortment of volume (Yu et al., 2013). The dsRNA-P450 delivered through micro-injection reduces adequacy for the induction of RNAi (Hunter, 1999). Potency of the double stranded RNA is diminished inside the gut of insect body due to harsh environmental conditions (Rajagopal et al., 2002; Surakasi et al., 2011) making it difficult for quantifying the amount of dsRNA. In the present study, R. ferrugineus was injected with dsRNA-P450 by micro-injection via ventral micro-inoculation to suppress the Cytochrome P450 gene in RPW.

The current work was supported by the previous work done to suppress a number of genes in insect pests including Blattodea, Hymenoptera, Neuroptera, Dictyoptera, Orthoptera, Diptera, Lepidoptera, Hemiptera and Coleoptera. These orders have been showing amenability to RNAi mediated down regulations of target genes (Tomoyasu et al., 2008; Bellés, 2010; Mao et al., 2010; Wuriyanghan et al., 2011; Cheng et al., 2012). Moreover, the Cytochrome P450 provides gossypol resistance to insects and is also a pesticide detoxifying gene. Turner et al. (2006) observed that the reduction in the mRNA in E. postvittana is induced by highly concentrated (4000 ppm) dsRNA but it doesn’t cause mortality. Insect disease resistance against D. citri is supported by the metabolic mechanism which is enhanced by over-expression of gene (cytochrome P450) and detoxifying enzymes (Tiwari and Siddiqi, 2012; Tiwari et al., 2011a, 2011b; Killiny et al., 2014), Helicoverpa armigera was subjected to dsRNA down regulation of CYP6AE14 gene and retarded growth was observed (Mao et al., 2007), cytochrome P450 monooygenase genes suppression was also noted in Aphis gossypii (Peng et al., 2016). Catalase and Olfactory Co-Receptor RferOrco down regulation in R. ferrugineus has been done to control this invasive pest (Al-Ayedh et al., 2016; Soffan et al., 2016).

El-Sheshay et al., (2013) proved that gene (P450) in the D. citri can be down regulated by a small amount of dsRNA (1µg/insect). However, in present work 3µg/insect concentration was sufficient to down regulate the Cytochrome P450 gene. The solicitation of dsRNA through micro-injection provided significant results showing shortened life span upon dsRNA in D. citri as compared to control. In some cases it causes 40-50% mortality of O. nubilalis (Wang et al., 2011). Double stranded-RNA target design is likewise pretty supportive in case of non-target arthropods in the fields (Whyard et al., 2009). In light of the fact, dsRNA specificity suggested a helpful invention to control the resistance in the R. ferrugineus as demonstrated in both exposed as well as resistant R. ferrugineus population. Contrarily, no impact of dsRNA-gfp was detected on mortality as well as Cytochrome P450 gene expression in control treatments. One of the most vital limitations of dsRNA is its difficult and costly for large scale. Moreover, different insect species lack the RNA interference machinery and concerned genes with high transcriptional rate so the impact of RNA interference can be evaded by them (Upadhyay et al., 2013). The dsRNA delivery in the H. armigera and D. vergifera introduced through plant mediated RNA interference shows positive correlations (Baum et al., 2007; Mao et al., 2007). The dsRNA could be applied in the absence of interferon regulated innate immunity for maximal effect of RNAi (Clemens and Elia, 1997). According to some researchers, the injection of bacteria with dsRNA provides a potential feasible delivery of dsRNA as well as engaging them to plants in foliar form with appropriate transfection reagent therefore, In future, dsRNA efficacy with any other microbial formulations or mixtures will be evaluated for the purpose of dsRNA application in field conditions against R. ferruginous.

Authors are highly grateful to Punjab Agriculture Research Board (PARB) under grant (PARB-802) to conduct research, Higher Education Commission of Pakistan (HEC) under grant (No.204535/ NRPU/R&D/HEC/14/159) and Framework of Institutional Cooperation Program (FICP-Pak-Norway Grant) (to Dr. Jam Nazeer Ahmad and Dr. Samina Jam Nazeer Ahmad) for providing funds to establish well equipped Laboratory (IGCDB) at University of Agriculture, Faisalabad, Pakistan.

The authors declare no conflict of interest.