Introduction
Tomato (Solanum lycopersicum) is one of the most important vegetable crops cultivated throughout the world. It is also known as protective food due to its special nutritive value (Chaudhary et al., Reference Chaudhary, Sharma, Singh and Nagpal2018). In India, tomato is the third most prominent vegetable after potato and onion. India ranks second in the area as well as in the production of tomato. However, tomato cultivation is seriously affected by pests and diseases both in field and polyhouse conditions. Some common tomato insect pests are stink worm, cutworm, tomato hornworm, aphids, whiteflies, flea beetles, spider mites, slugs, nematodes, moths, and leaf miners including south American tomato pinworm (Gatahi, Reference Gatahi2020). Among all these pests, the South American tomato moth, Phthorimaea absoluta (Meyrick) (formerly, Tuta absoluta) (Lepidoptera: Gelechiidae) is one of the key devastating insect pests of tomato throughout the world (Montella et al., Reference Montella, Schama and Valle2012). It originated in South America, spread first to Spain in 2006 and then to rest of the Europe (Desneux et al., Reference Desneux, Wajnberg, Wyckhuys, Burgio Arpaia, Narvaez-Vasquez, González-Cabrera, Catalán, Tabone, Frandon, Pizzol, Poncet and Urbaneja2010). In India, the first report was in Karnataka during 2014 (Sridhar et al., Reference Sridhar, Chakravarthy, Asokan, Vinesh, Rebijith and Vennila2014). The larvae can cause 80–100% damage by feeding on mesophyll tissues by leaving the epidermis intact, forming irregular leaf mines, burrowing the stalks, apical buds, green, and ripen fruits (Garzia et al., Reference Garzia, Siscaro, Biondi and Zappala2012).
In India, an ad hoc management was recommended to manage P. absoluta but farmers started spraying many broad-spectrum insecticides such as indoxacarb, flubendiamide, cyantraniliprole, and spinosad without proper implementation of integrated pest management practices. Due to indiscriminate and continuous usage of the same insecticides, outbreaks of the pest in many prominent tomato-growing belts of South India were noticed during 2019 and 2020. Reduced susceptibility of P. absoluta to frequently used insecticides like flubendiamide, cyantraniliprole, and indoxacarb was observed in most commonly tomato-growing states in south India (Prasannakumar et al., Reference Prasannakumar, Jyothi, Saroja and Ramkumar2021). In many parts of the world, resistance development in P. absoluta was reported due to undesirable changes in the gene pool that govern resistance owing to repeated use of the same class of insecticides (Lietti et al., Reference Lietti, Bottom and Alzogaray2005; Grant et al., Reference Grant, Jacobson and Ilias2019; Inak et al., Reference Inak, Ozdemir, Abdullah, Zelyü, Demir, Roditakis and Vontas2021).
One of the important mechanisms of resistance development in insects is enhanced rates of detoxification. Detoxification of insecticides usually happens through an increase in enzymatic activity, either one or combination of more. Out of possibly 100 detoxifying enzymes produced by an insect, the oxidative enzymes such as cytochrome P450-dependent mono-oxygenases and carboxylesterases are most commonly involved in the detoxification of insecticides (Konus, Reference Konus2014). Biochemical estimation of these enzymes by biochemical assays helps in understating the metabolic resistance development mechanisms (Kerns and Gaylor, Reference Kerns and Gaylor1992). Similarly, the genes which are involved in the development of resistance play a crucial role. In many lepidopteron insects, cytochrome P450 (CYP450) genes involvement in insecticide resistance development was reported but such a report lacks in P. absoluta. Information along with biochemical and molecular bioassays, life history parameters such as egg laying capacity, development time, and egg fertility in resistance population further helps in understanding insecticide resistance and to device effective management of the pest under field conditions. The present study was thus carried out to know the effect of insecticide resistance on biochemical enzymes and their gene expression, and life history traits over the generation to commonly used insecticides viz., flubendiamide, cyantraniliprole, and indoxacarb from field population of P. absoluta viz., Bangalore, Kolar, Madurai, Salem, and Anantapur districts under laboratory conditions. The information generated in the present study helps in understanding insecticide resistance development in P. absoluta and development of insecticide resistance management strategy to manage the resistant populations in future.
Materials and methods
Maintenance of P. absoluta populations and healthy tomato plants
Phthorimaea absoluta populations were collected from different states of south India during 2019–2020 from infested greenhouse and open field tomato crops (table 1). The leaves that were infested with P. absoluta larvae were collected and brought to lab and immediately transferred on to fresh healthy leaves to avoid starvation. The box containing the infested leaves was maintained separately (location wise) in the lab at the Division of Crop Protection, ICAR-Indian Institute of Horticultural Research (IIHR), Bangalore till adult emergence. After emergence, the adults were transferred into insect-proof rearing cages containing 2–3 healthy tomato plants. The rearing cages were placed at 26 ± 1°C, 65% RH, and 16:8 h light:dark photoperiod.
Survey details of Phthorimaea absoluta collected on different tomato fields
In the present study, the Bangalore population was used as a susceptible population as the pest was first reported at IIHR in 2014 (Sridhar et al., Reference Sridhar, Chakravarthy, Asokan, Vinesh, Rebijith and Vennila2014). The susceptible culture was maintained in a separate room by not exposing to any of the insecticides for 12 generations.
Healthy tomato plants were maintained in large insect-proof cages inside net house without spraying any insecticides. The plants were screened for the presence of pests every day; if any infestations found were removed manually by cutting the infested leaves or by destroying the whole plant. The healthy plants were used for maintaining the insect culture for all bioassay studies.
Insecticides and bioassays
The insecticides used in the present study include commercial formulations of the diamide group – cyantraniliprole 10.25 SC (DuPont, France) and flubendiamide 39.35 SC (Bayer crop Science AG, Germany), and indoxacarb 14.5 SC (Syngenta, Basel, Switzerland) which were classified under various categories based on mode of actions by Insecticide Resistance Action Committee (IRAC), and majorly being recommended for P. absoluta management in India (table 2). Leaf dip bioassay of the above insecticides on P. absoluta (II instar larvae) was carried at every generation (G1–G20); for each generation, the LC50 values calculated through probit analysis and the resistance ratios (RRs) were estimated as per IRAC (2013) and Prasannakumar et al. (Reference Prasannakumar, Jyothi, Saroja and Ramkumar2021).
Insecticides used in resistance bioassay studies under laboratory condition
Esterase and cytochrome P450 assays
Preparation of cytosols from P. absoluta larvae for CYP450 and esterase enzymes was done according to Lowry et al. (Reference Lowry, Rosebrough, Farr and Randall1951) and Prasannakumar et al. (Reference Prasannakumar, Jyothi, Saroja and Ramkumar2021). Peroxidation of 3, 3`, 5, 5`-tetramethyl-benzidine activity by CYP450s in P. absoluta was determined following Brogdon (Reference Brogdon1989). The absorbance was measured in UV-VIS carry 60 spectrophotometer (Agilent Technologies Pvt. Ltd USA) at 630 nm for 10 min. The total enzyme activity was quantified as nmol per mg protein per min using cytochrome C as a standard curve. Esterase-α-NA enzyme activity was determined by using α-naphthyl formation (Kranthi, Reference Kranthi2005). The estimation was carried out according to Prasannakumar et al. (Reference Prasannakumar, Jyothi, Saroja and Ramkumar2021).
Life history trait studies: fecundity, development time, and egg fertility
Life cycle of P. absoluta was conducted in the laboratory at 26 ± 2°C temperature, 65 ± 5 relative humidity (RH) (%), and 16:8 h light:dark photoperiod. Fecundity was observed by keeping five pairs of pupae in a cage (45 × 45 × 45 cm, 96 × 26 meshes) with healthy tomato plant with honey (10%) syrup as a substrate food. Once the adult emerged (after 48 h), the plants were carefully removed from the cage and new plants supplied for oviposition. The removed plants were closely observed under light, and the eggs were collected and maintained on new plants for further studies. The newly hatched first instar neonates were individually released on each leaf and kept on glass petri-plates (9 cm diameter), containing wet filter paper and the larval developmental period was recorded. Daily, a new leaf was provided for the larvae to avoid the stress. After pupation, the pupae were left in the petri-plates to get adults, and once adults emerged, they were transferred to 250 ml transparent bottle covered with black muslin cloth provided with 10% honey solution. Data on formation of pupa, adult emergence, and its longevity were recorded. For all resistance and susceptible culture, the experiment was conducted in three replicates.
Identification of genes involved in resistance development
Primers, total RNA extraction, and cDNA synthesis
As there was no meaningful expression of esterases in the insecticide-resistant P. absoluta population, only the expression of the CYP genes was selected for the study. For primer design, the CYP genes of the lepidopteron family were chosen based on their previous known history in association with insecticide resistance through Blastn analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with 90% query coverage (Supplementary table 1). The conserved region was selected by using Clustal W multiple sequence alignment and degenerate primers were manually synthesized using PrimerQuest software. The efficiency of the primer was checked by Oligo Analyzer Tool (https://www.idtdna.com/calc/analyzer).
The field population of P. absoluta larvae collected from different locations as mentioned above were exposed to insecticides through leaf dip bioassay. The survived larvae from the insecticide bioassays were selected for extraction of total RNA. The total RNA was isolated with 20 mg insect samples in the presence of liquid nitrogen, and ground to fine powder with mortar and pestle using ISOLATE II RNA MINI KIT (Bioline, USA) as per manufacturer's instructions. The quality and quantity of RNA were determined by denatured agarose gel electrophoresis and measured using spectrophotometer, SpectraMax® Model No. M2 (Molecular Devices, CA, USA).
The first strand cDNA synthesis was carried out using 1 μg of total RNA by using OneScript® cDNA Synthesis Kit (abm, Canada) according to the manufacturer's instructions and the cDNA was diluted to 1:10 ratio. The polymerase chain reaction (PCR) (Effendorf Roche Applied Science, Basel, Switzerland) was carried out in 25 μl of PCR mixture that contains 2.5 μl of 10× PCR buffer, 4 μl of 2.5 mM dNTPs (Deoxynucleoside triphosphates), 0.5 μl of 10 pM (forward primer), 0.5 μl of 10 pM (reverse primer), 0.20 μl (5 U) of LA Taq DNA Polymerase (Takara Bio, USA), and 5 μl of diluted cDNA as template. Thermo cycling conditions for PCR include an initial denaturation at 95°C for 3 min; 15 cycles of 98°C for 10 s, 62°C for 30 s, 68°C for 1 min 30 s, another 20 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 1 min 30 s; followed by a final extension of 72°C for 10 min. The post-PCR products were separated by electrophoresis in a TAE (Tris-acetate-EDTA) buffer containing 1.2% agarose gel (40 mM Tris-acetate [pH 8.0], 1 mM EDTA [Ethylenediamine tetraacetic acid]) at 100 V for 1 h. The gel was visualized in ultraviolet light.
The amplified products were eluted using gel extraction kit (Nucleospin® Extract II, Macherey Nagel, Germany) as per the instructions. The eluted products were ligated into pTZ57R/T (T/A cloning vector) (Thermo Scientific, USA), according to the manufacturer's protocol. The ligated products were cloned into E. coli (DH5 α cells), by heat shock method, then the transformed cells were spread on LB agar plates containing X-gal, IPTG (Isopropyl ß-D-1-thiogalactopyranoside), and ampicillin (100 μg ml−1) as antibiotic. The plates were then incubated at 37°C for overnight. The plasmids were isolated using alkaline lysis method and the clones confirmed by plasmid mobility check compared with control plasmids (PTZ57R/T without insert). Sequencing was performed for selected clones in triplicate in an automated sequencer (ABI prism® 3730 XL DNA Analyzer; Medauxin, Bengaluru). A homology search was done using NCBI-BLASTn (http://blast.ncbi.nlm.nih.gov/) and sequence alignment was performed using BioEdit version 7.0.9.0. The gene sequences were aligned using the Clustal W program in BioEdit.7.0.
Evaluation of CYP gene expression through RT-qPCR
Expression of the identified CYP genes was performed using RT-qPCR (Real-Time Quantitative Reverse Transcription PCR) with their gene-specific primers (Supplementary table 2) with the internal reference gene, RPS-13 (Yang et al., Reference Yang, Wang, Huang, Lv, Liu, Bi, Wan, Wu and Zhang2021). The cDNA from G1 and G20 population was prepared and diluted at 10 ng μl−1 before setting the reaction. The amplifications of the genes were carried out with 20 μl reaction mixture that consists of 10 μM of each of the primers,10 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus), and 30 ng of cDNA template. The RT-qPCR reactions were carried out in a Light Cycler® 480 Real-Time PCR System (Roche Applied Science), one cycle at 95°C for 1 min, followed by 40 cycles of 95°C for 10 s and 59°C for 30 s, followed by melting curve analysis of 95°C for 5 s, 59°C for 1 min, and 95°C for each cycle (acquisition mode: continuous, acquisitions: 5 per °C). The relative quantification of genes was done using △△Ct (delta-delta cycle threshold) method followed by one-way analysis of variance for testing the level of significance (P = 0.05) (Livaka and Schmittgen, Reference Livaka and Schmittgen2001).
Data analysis
Mortality was recorded after 24, 48, 72, and 96 h of insecticidal exposure. The LC50 (Lethal Concentration 50) values were calculated by Probit analysis using IBM SPSS 21 software. For each generation, the RRs were calculated by dividing the LC50 value of each resistance strain by the LC50 value of the susceptible strain of P. absoluta (Konus, Reference Konus2014). The change in the enzymes activity was carried by measuring difference between measured esterase and monooxygenase (MFO)-CYP450 activities with Student's t-test.
Results
Resistance to flubendiamide
The population from Bangalore, Madurai, Salem, and Anantapur showed a significant increase in RRs to flubendiamide under laboratory conditions. The RR of Bangalore population ranged from 1.34 (G1) to 10.96-fold (G21). The LC50 for G1 and G21 generation was found to be 9.40 and 76.45 mg l−1, respectively (table 3). Similarly, the Madurai P. absoluta population showed RR that ranged from 0.87 (G1) to 7.4-fold (G20) with the LC50 6.09 for G1 and LC50 49.56 mg l−1 for G20 (table 3).The RR of Salem population ranged from 1.05 (G1) to 9.3-fold (G20). The LC50 for G1 was 9.14 and LC50 for G20 was found to be 64.83 mg l−1. Likewise, the RR of Anantapur population ranged from 4.64 (G1) to 9.5-fold (G18), with the LC50 32.34 and 66.85 mg l−1, respectively (table 3).
Insecticide resistance ratio (RR) of different P. absoluta population over the generations to flubendiamide
LC50, lethal concentration which kills 50% of the exposed population; χ2 chi-square (observed); n, sample size; LCL (95%), lower confidence limit; UCL (95%), upper confidence limit; RR, resistance ratio, determined by dividing the LC50 of field population by LC50 of susceptible population.
a Susceptible population from Bangalore without exposed to insecticide.
Increased esterase activity was observed in the flubendiamide-resistant population (G15 lab) compared to the susceptible population. The highest activity of the enzyme (5.6-fold) was recorded in the Bangalore population followed by Anantapur (5.31-fold), Salem (5.09-fold), Madurai (5.05-fold), and Kolar (4.2-fold). A similar pattern of increased esterase enzyme activity was observed across the resistant population compared to their respective field population (G1) (table 6). The activity of MFO-CYP450 was also increased in the flubendiamide-resistant (G15 lab) population than susceptible one (lab susceptible). The highest activity was recorded in the Anantapur population (7.82-fold), followed by Bangalore (7.75-fold), Madurai (6.49-fold), and Salem (5.93-fold). When compared to their respective field population (G1), the activity of MFO-CYP450 was increased in the lab-resistant population (table 7).
Resistance to cyantraniliprole
The populations from Kolar, Madurai, Salem, and Anantapur showed variable RRs under laboratory conditions. The RR of Kolar population ranged 2.24 (G1) to 10.98-fold (G20). The LC50 for G1 generation was 20.22 and 99.12 mg l−1 for G20 generation (table 4). Madurai P. absoluta population showed RR that ranged from 1.21 (G1) to 9.20-fold (G20) with the LC50 of 10.96 and 83.12 mg l−1 for G20 (table 4). The RR of Salem population ranged from 1.45 (G1) to 9.27-fold (G20). The LC50 for G1 of Salem population was 13.11 and 83.56 mg l−1 for G20. The RR of Anantapur population ranged from 3.267 (G1) to 10.36-fold (G18). The LC50 for G1 generation was 29.49, whereas for G18, 93.56 mg l−1 (table 4).
Insecticide resistance ratio (RR) of different P. absoluta population over the generations to cyantraniliprole
LC50, lethal concentration which kills 50% of the exposed population; χ2(observed) chi-square; n, sample size; LCL (95%), lower confidence limit; UCL (95%), upper confidence limit; RR, resistance ratio, determined by dividing the LC50 of field population by LC50 of susceptible population.
a Susceptible population from Bangalore without exposed to insecticide.
The highest esterase activity in cyantraniliprole-resistant population (G15 lab) was in Anantapur population (4.96-fold) followed by Kolar (4.61-fold), Salem (4.35-fold), and Madurai (4.01-fold), compared to the susceptible population. Similarly, esterase activity was increased in lab-resistant population compared to their respective field population (G1) (table 6). The MFO-CYP450 activity also rose in cyantraniliprole-resistant population (lab) than the susceptible population (lab). The maximum activity was in Anantapur (7.18-fold) followed by Madurai (6.55-fold), Kolar (5.87-fold), and Salem (5.63-fold). A similar trend of increased activity of MFO-CYP450 was noticed when compared to field population (G1) (table 7).
Resistance to indoxacarb
The populations from Kolar, Madurai, Salem, and Anantapur showed variable RRs under laboratory conditions to indoxacarb. The RR of Kolar population ranged from 2.07 (G1) to 9.35-fold (G20). The LC50 for G1 Kolar population was 22.85 mg l−1 and G20 generation 103.25 mg l−1 (table 5). Similarly, Madurai population showed the RR range of 0.98 (G1) to 8.35 mg l−1 (G20) with the LC50 of 10.88 mg l−1 and for G20, the LC50 was 92.12 mg l−1. No significant indoxacarb RR was observed for the Bangalore population (table 5). The RR of Salem population ranged from 1.44 (G1) to 8.87-fold (G20). The LC50 for G1 was 12.77 and for G20, 97.956 mg l−1. The Anantapur population RR ranged from 3.00 (G1) to 9.27-fold (G18) with LC50 of 33.21 and 102.23 mg l−1, respectively (table 5).
Insecticide resistance ratio (RR) of different P. absoluta population over the generations to indoxacarb
LC50, lethal concentration which kills 50% of the exposed population; χ2 (observed) chi-square; n, sample size; LCL (95%), lower confidence limit; UCL (95%), upper confidence limit; RR, resistance ratio, determined by dividing the LC50 of field population by LC50 of susceptible population.
a Susceptible population from Bangalore without exposed to insecticide.
The highest esterase activity was recorded in Kolar (5.67-fold), followed by Salem (4.54-fold), Anantapur (4.58-fold), and Madurai (3.90-fold) compared to susceptible population (lab). Likewise, esterase activity was also significantly increased in resistant population compared to their respective field populations (G1) (table 6). The activity of MFO-CYP450 was highest in Anantapur (6.74-fold) followed by Madurai (6.69-fold), Kolar (5.96-fold), and Salem (5.94-fold) compared to the susceptible population. A similar trend of increased activity of MFO-CYP450 was observed compared to their respective field population (G1) (table 7).
Activity of esterase enzyme in different field population of P. absoluta
N, sample size taken for analysis.
a Esterase activity value of lab resistance population by esterase activity value of susceptible population.
b Esterase activity value of lab resistance population by esterase activity value of field population.
c No significant resistance (P < 0.05).
Activity of MFO-CYP450 enzyme in different field population of P. absoluta
N, sample size taken for analysis.
a MFO-CYP450 activity value of lab resistance population by MFO-CYP450 activity value of susceptible population.
b MFO-CYP450 activity value of lab resistance population by MFO-CYP450 activity value of field population.
c No significant resistance detected (P < 0.05).
Life history trait studies: fecundity and development time
Mean duration of various development stages in different populations is given in table 8. The eggs were hatched in 4 days in both susceptible and resistant population under laboratory conditions. The larval period of the susceptible population ranged from 8 to 11 days, while the flubendiamide-, indoxacarb-, and cyantraniliprole-resistant populations took 12–16, 11–16, and 13–16 days to complete the larval period, respectively. A variable mean larval development period was observed in different insecticidal resistant populations. The mean larval period was found to be 9.00 days for the susceptible population, 14.66 days for the flubendiamide-resistant population, 14.00 days for the indoxacarb-resistant population, and 14.4 days for the cyantraniliprole-resistant population. The average time of development was 1.8, 2.6, 2.6, and 2.0 days to complete first, second, third, and fourth instars, respectively, for susceptible population. The flubendiamide-resistant population took 3.6 days to complete the first instar, 4.1 days for the second instar, 2.4 days for the third instar, and 4.75 days for the 4th instar. In case of indoxacarb-resistant population, the average days to complete first instar was 3.6 days, second instar 3.6 days, third instar 2.8 days, and fourth instar 4.6 days. Likewise, cyantraniliprole-resistant population took 3.6, 3.8, 2.6, and 4.4 days to complete first, second, third, and fourth instars, respectively. The average pupal period was found to be 5.25 days in the susceptible population, 7.6 days in the flubendiamide-resistant population, 7.33 in the indoxacarb-resistant population, and 8.0 days in the cyantraniliprole-resistant population. Adult longevity ranged from 9 to 11 days for susceptible population, whereas 12–15 days in flubendiamide, 12–14 days for indoxacarb, and 11–16 days for cyantraniliprole-resistant population.
Biology of P. absoluta insecticide resistant population
Identification of genes involved in resistance development and its expression
Resistant populations of P. absoluta from different places were analyzed to identify the CYP genes that were associated with insecticide resistance. The prominent resistant population was selected from the bioassay experiment to identify the CYP450 genes. The flubendiamide-resistant population from Bangalore (G15), cyantraniliprole from Kolar (G14) and Salem (G14), and indoxacarb from Kolar (G14) were used in this experiment. The four novel CYP genes were identified and deposited in NCBI gene bank. The genes CYP248f (flubendiamide) (Acc No MZ720787), CYP272c, CYP724c (cyantraniliprole), (Acc no. MZ720788 and MZ723792), and CYP648i (indoxacarb) (Acc No. MZ720788) were probably involved in the development of resistance in P. absoluta. The sequencing results were subjected to NCBI-BLASTn analysis.
Relative expression analysis was performed for four putative CYP genes that were identified through PCR studies. The relative expression of CYP248f gene in the flubendiamide-resistant population was 0.79-fold (G20 generation), in cyantraniliprole-resistant population, the relative expression of CYP272c and CYP724c genes was 0.53-fold (G20 generation) and 4.37-fold (G20 generation), respectively. Similarly, the relative expression of CYP648i gene in indoxacarb-resistant population was 10.6-fold (fig. 1).
Relative expression of CYP genes in P. absoluta. (a) CYP248f gene in flubendiamide-resistant population, (b, c) CYP272c and CYP724c genes in chlorantraniliprole-resistant population, (d) CYP648i gene in indoxacarb-resistant population.
Discussion
The present study investigated the development of insecticide resistance in P. absoluta for flubendiamide, cyantraniliprole, and indoxacarb under laboratory conditions over the generations. The activity of the enzymes involved and the expression of the probable genes were also studied along with the important biological traits. In 2019, the pest outbreaks of P. absoluta were reported in major tomato-growing regions of Karnataka (Kolar) and Andhra Pradesh (Anantapur) (Prasannakumar et al., Reference Prasannakumar, Keshava Rao, Saroja and Jyothi Yadav2020). The pest populations were collected from these outbreak areas and preliminary studies were carried out to know the resistant levels. The variable RRs across the locations in G1 generation were observed due to different patterns of insecticide usage, indiscriminate and continuous spraying of same insecticides, and ignorance or a lack of concern in dealing with usages of insecticides by the farmers (Prasannakumar et al., Reference Prasannakumar, Jyothi, Saroja and Ramkumar2021). Further, an increase in the RRs across the generation was noticed owing to an increase in the activity of MFO-CYP450 and esterase (ESTα-NA) enzymes. The different patterns for MFO-CYP450 and esterase (ESTα-NA) activities in the resistance populations of P. absoluta from Bangalore, Kolar, Madurai, Salem, and Anantapur resulted in the development of resistance. The elevated production of these enzymes (P450s and esterases) in the insect gut degrades and detoxifies the insecticides (Hemingway and Ranson, Reference Hemingway and Ranson2000; Montella et al., Reference Montella, Schama and Valle2012; Karaagac and Sakine, Reference Karaagac and Sakine2015). The enzymes from esterase family hydrolyse ester bond in a wide range of insecticides; therefore, these might have played a key role in insecticidal resistance (Bornscheuer, Reference Bornscheuer2002). Further, the CYP-P450 (super family is the largest family of plant metabolic enzymes) are highly divergent and have important functions in detoxification of various drugs, pesticides, plant toxins, chemical carcinogens, and mutagens. They also metabolize endogenous compounds such as hormones, fatty acids, and steroids (Yang and Liu, Reference Yang and Liu2011). The MFO-CYP450 activity was increased in all three resistance populations of different locations indicating that the enzymes have major role in insecticide resistance development by directly involving insecticide metabolic detoxification (Reyes et al., Reference Reyes, Rocha, Alarcón, Siegwart and Sauphanor2012).
The biological trait studies showed a significant increase in the duration of larval, pupal period, adult longevity, and total developmental time than susceptible population. The increased trend of developmental period in all the stages may be attributed to altered hormonal titers such as juvenile hormone and molting hormones in the insecticide-resistant population (Harrison and Lynn, Reference Harrison and Lynn2008; Cai et al., Reference Cai, Bai, Wei, Lin, Chen, Tian, Gu and Murugan2016; He et al., Reference He, Sun, Tan, Sun, Qin, Ji, Li, Zhang and Jiang2019). In contrast, in aphid, Rhopalosiphum padi, the sub lethal effects of beta-cypermethrin and indoxacarb insecticides significantly affected the female's sexual organs or ovipositing mechanism that resulted in reduced fecundity and longevity. However, in subsequent generations, development of nymphs was prolonged by sub lethal concentrations of the insecticides (Zuo et al., Reference Zuo, Wang, Lin, Li, Peng, Jaime, Piñero and Chen2016). Therefore resistant adults develop more slowly than susceptible ones (Arnaud et al., Reference Arnaud, Brostaux, Assie, Gaspar and Haubruge2002).
A comparative study between G1 and G15 indicated increased RR in flubendiamide, cyantraniliprole, and indoxacarb-resistant populations (table 9). The activity of MFO-CYP450 and esterases was estimated in 15th generation, a significant increase in their activity was noticed according to their RR levels across different resistant populations. In the present study, the 10-fold resistance was recorded for flubendiamide and cyantraniliprole on continuous exposures till 17–19 generations in Bangalore, Kolar, and Anantapur due to an increase in enzymatic activity. The biological trait studies showed a significant increase in the larval developmental period and adult longevity in the resistant population. In general, if the larval duration extended, the crop would likely get more damaged but prolonged availability of host (larval stage of the pest) for natural enemies may result in more predation and parasitization. Since adult longevity was more in resistant population their migration and spread will also be more.
Comparative studies of resistance ratios among P. absoluta population
a RR of G1/RR of G15 of respective population.
CYP450s are the major genes associated with resistance to most of the chemical insecticides (Nahlen et al., Reference Nahlen, Clark and Alnwick2003; Brooke et al., Reference Brooke, Kloke, Hunt, Koekemoer, Temu, Taylor, Small, Hemingway and Coetzee2008; Zhang et al., Reference Zhang, Yao, Wang, Liu, Lanting, Wang and Xu2019). We attempted for the first time to study the CYP450 genes in P. absoluta and identified four novel genes viz., CYP248f (flubendiamide), CYP272c, CYP724c (cyantraniliprole), and CYP648i. The relative expression of the identified genes compared to their respective G1 (generation one) population further indicate their involvement in resistant development. The expression of CYP248f gene in flubendiamide-resistant population changed from 0.38-fold (G1 generation) to 0.79-fold (G20 generation). Similarly, in cyantraniliprole-resistant population, the expression of CYP272c was increased from 0.121-fold (G1 generation) to 0.53-fold (G20 generation) and CYP724c changed from 1.67-fold (G1 generation) to 4.37-fold (G20 generation). In the indoxacarb-resistant population, the expression of CYP648i gene changed from 8.1-fold (G1 generation) to 10.6-fold (G20 generation); the increase in the activity of these directly depends on resistant development because expression levels of the genes increased with increased RR over the generation (Daborna et al., Reference Daborna, Lumb, Boey, Wong, Constant and Batterham2007). Many studies have reported the development of insecticide resistances in P. absoluta from many regions where the pest was previously reported on tomato (Siqueira et al., Reference Siqueira, Guedes and Picanco2000; Marcela et al., Reference Marcela, Eduardo and Raul2005; Astor and Scals, Reference Astor and Scals2009; Reyes et al., Reference Reyes, Rocha, Alarcón, Siegwart and Sauphanor2012; Roditakis et al., Reference Roditakis, Skarmoutso, Staurakaki, Marıadel, Vidal, Bielza, Haddi, Rapisarda, Rison, Bassie and Teixeiraf2013; Prasannakumar et al., Reference Prasannakumar, Keshava Rao, Saroja and Jyothi Yadav2020). Extensive use of high potent diamide group in Greek on the tomato led to the development of 14-fold resistance for cyantraniliprole and 11-fold for flubendiamide resistance in P. absoluta (Roditakis et al., Reference Roditakis, Vasakis, Grispou, Stavrakaki, Nauen, Gravouil and Bassi2015). Likewise, variable resistance levels to abamectin, cartap, and permethrin were also reported from five different regions of Brazil due to long periods with a high frequency of usage of the chemicals against P. absoluta (Siqueira et al., Reference Siqueira, Guedes and Picanco2000). The resistance of P. absoluta to flubendiamide (750-fold) and cyantraniliprole (860-fold) in Kuwait was due to differential selection pressures in glasshouse conditions and application of different amount of insecticide in the field (Jallow et al., Reference Jallow, Dahab, Albaho, Devi, Dawood and Binson2019). In European/Asian regions, low to moderate resistance to emamectin benzoate (15-fold), spinosad (RR: 33-fold), indoxacarb (RR: 13–91-fold), and chlorantraniliprole (RR: 64-fold) was due to target site mutation at glutamate-gated chloride channel, the gamma amino butyric acid gene fragments (Roditakis et al., Reference Roditakis, Vasakis, García-Vidal, Aguirre, Rison, Lutun, Nauen, Tsagkarakou and Bielza2018).
Conclusions
The present study showed that P. absoluta under laboratory selection was 10-fold more resistant to insecticides. Cytochrome P450 was an important metabolic detoxifying enzyme that was found in resistance populations. The relative expressions of cytochrome P450 genes in resistant population indicate their involvement in resistance development. Educating the farmers about proper use of chemical insecticides would help in slow downing the resistant development in future.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485323000081
Acknowledgements
The authors are grateful to the Director, ICAR-IIHR and Head, Division of Crop Protection, ICAR-IIHR, for supporting and giving valuable suggestions throughout the study. The farmers of the visited places are greatly acknowledged for providing the inputs and co-operating throughout the study. We are also thankful to DST-SERB, New Delhi for financial funding through ECRA project (ECR/2017/000715).
Author contributions
N. R. P. and N. J. planned, designed, executed, and prepared the draft. G. R., S. S., and V. S. were involved in survey and culture maintenance. K. P. and R. A. worked on molecular aspects and final checking of the draft.
Conflict of interest
None.
