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Research in Resistance Management

RAPD-PCR Analysis to Monitor Imidacloprid Resistance in Cotton Whitefly

Smriti Sharma, V. K. Dilawari and V. K. Gupta
Department of Entomology, Punjab Agricultural University, Ludhiana-141004, India.

ABSTRACT RAPD-PCR analysis of imidacloprid treated whiteflies produced amplicons (amplified fragments) which were categorized into four groups namely fragments amplified i) in the control (untreated population) but disappeared or reappeared in subsequent generations due to insecticidal pressure, ii) in the imidacloprid treated population (both dead and alive) in different generations, iii) in the individuals surviving the effect of imidacloprid pressure and iv) in the imidacloprid treated dead individuals but were absent from the treated live individuals. Analysis of amplicons indicated that imidacloprid treatment produced distinct genetic alterations in surviving whiteflies. The potential markers for imidacloprid resistance in whitefly were identified from F6, F9 and F11 generations.

KEY WORDS: Bemisia tabaci, molecular markers, insecticide, generations

INTRODUCTION Cotton whitefly, Bemisia tabaci (Gennadius) (Hemiptera : Aleyrodidae) is one of the most devastating agricultural pests worldwide as it affects the yield of a broad range of agricultural, fiber, vegetable and ornamental crops (Cahill et al., 1996). Whitefly infestation affects growth, flowering, boll formation, seed and lint development, and thus causes loss of quality and quantity of cotton produce. Worldwide, in many agricultural systems high levels of resistance in whitefly has been reported for many insecticides such as organophosphates, carbamates, synthetic pyrethroids, chlorinated hydrocarbons, several insect growth regulators and neonicotinoids (Prabhaker et al., 1985, Ahmed et al., 1987, Prasad et al., 1993, Mohan and Katiyar 1995, Cahill et al., 1996, Elbert and Nauen 2000).

Random amplified polymorphic DNA-Polymerase chain reaction technique (Williams et al., 1990) has been used previously for population genetic studies of a number of insects including aphids (Black et al., 1992), grasshoppers (Chapco et al., 1992), fruit flies (Haymer and McInns, 1994) and blow fly (Stevens and Wall, 1995). RAPDs are viewed as having several advantages over other molecular markers and DNA fingerprints as the technique randomly samples the genome and hence multiple amplifiable fragments are present for each primer (Lynch and Milligan, 1994). Amplification of genomic DNA by the RAPD-PCR was used to differentiate between deltamethrin resistant and susceptible Culex pipiens pallens (Zhu et al., 1998) to study nucleotide divergence and insecticide resistance in aphids (Komazaki et al., 1998).

In spite of the work done on various insecticides, information on the molecular diagnostic techniques for monitoring imidacloprid resistance in whitefly populations is lacking. In view of the above, attempts have been made to identify such molecular markers by exposing whiteflies to imidacloprid selection pressure for eight generations (F0 and from F5 to F11). After the selection, resistance ratio increased by 11.7 folds and this was correlated with the markers amplified in the survivors.

MATERIALS AND METHODS

Selection procedure

Standard leaf disc dip method of bioassay with slight modifications (Elbert and Nauen 2000) was used to treat whiteflies with imidacloprid (0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, 0.00312% with five replications each). Adults of mixed sex (25-30) collected with mouth aspirators were released in each ventilated Petri dish. Petri dishes were held at 27±2ºC, R.H. 75±10% and photoperiod of 12:12 (light: dark). After 24 hours concentration-mortality regression was worked out using Probit analysis package POLO-PC (Le-Ora software 1987 based on Finney 1971) and living whiteflies of control and treated tests were released separately in rearing cages on untreated young plants. Sampling was done both from dead and live whiteflies of treated and untreated control populations and were stored in 70 per cent ethanol in 1.5 ml Eppendorf tubes in deepfreezer at -35ºC for further analysis.

Living whiteflies (F0) after treatment were released on a fresh plant to obtain a batch of next generation while maintaining discrete generations. Adults that emerged from these plants were considered as F1. Similar procedure was followed for all the succeeding generations. The number of adults subjected to selection in each generation varied depending upon the number and vigor of the adults of the preceding generation. Whiteflies of generation F6-F9 and F11 were exposed to single selective concentration chosen equivalent to LC50 (Bloch and Wool 1994).

DNA extraction and Quantification

For total DNA isolation, 8-14 whiteflies (dead and live) in each generation were thoroughly macerated individually with micro pestle in a 1.5 ml Eppendorf microcentrifuge tube containing 50µl lysis buffer (50 mM KCl, 10 mM Tris-Cl pH 8.4, 60 µg ml-1 proteinase K (Merck, >30 mAnson units/ mg), 0.45% Nonidet NP-40 and 0.45%Tween 20). DNA extraction procedure involved 65ºC for 45 min, 95ºC for 10 min, centrifugation at 13200 rpm for 3 min, two folds dilution with Mili-Q quality autoclaved water, -20ºC for storage. Prior to RAPD characterization DNA quality assessed by agarose gel electrophoresis (0.7% prepared in TAE buffer) was of high molecular weight with DNA band near the wells and no streaking or RNA band. DNA concentration assessed at 260 nm in Biophotometer® was ~15-30 ng/ µl on an average.

PCR and RAPD analysis

Forty Operon primers (Operon Technologies, Almeda, U.S.A.) belonging to OPA, OPB, OPC, OPE, OPF and OPH series were initially screened and out of those, 10 primers showing good amplification with discrete fragments and polymorphism were selected for studying insecticide resistance (Table 1).

The PCR reaction was performed in a 500 µl PCR tube with a reaction volume of 25 µl containing final concentrations as 30-60 ng/ µl DNA, 1.6 nM Primer (Operon Technologies, Almeda, U.S.A.), 1X Reaction Buffer (Biogene U.S.A.), 1.5 mM MgCl2 (Biogene, U.S.A.), 0.2 mM dNTP mix (MBI, Fermentas), 2.5 U Taq polymerase enzyme (MBI, Fermentas). To avoid evaporation one drop of sterilized mineral oil was overlaid and the reaction was set in the Thermal Cycler with following thermal profile using 40ºC as the annealing temperature [found to be optimum from amplified comparative profiles obtained for different polymerization experiments set at different annealing temperatures (37-40ºC) (data not shown)]: 95ºC - 5 min, 94ºC - 1 min, 40ºC - 1.5 min, 72ºC - 2 min (94ºC - 1 min, 40ºC - 1.5 min, 72ºC - 2 min, 40 cycles), followed by 72 ºC for 20 min as final elongation step and 4ºC for storage until use.

The DNA fragments in the PCR amplified products were separated in 1.4% agarose gel (in TAE buffer having 3 µl (of 1 mg ml -1) ethidium bromide/ 100 ml) run in 1X TAE buffer at 5V/cm for 1 hr with a 100 bp molecular weight standard run along the samples. Gels were photographed by Ultralum Gel Documentation system for scoring.

RESULTS

Total and polymorphic fragments amplified by selected primers

The primers amplifying more than 50 per cent polymorphic fragments were used in all subsequent studies on treated whiteflies in each generation. Per cent polymorphic amplicons (amplified fragments) by each primer are listed in Table 1. On an average, each primer amplified 54 fragments in all the generations studied and the number of polymorphic amplicons were 42 with 77.8 per cent polymorphism. Maximum number of polymorphic fragments were amplified using primer OPB 05 (92.3%). Total number of PCR amplified fragments by each primer in all generations ranged from 39 (OPC 04) to 67 (OPB 10).

The number of amplicons per primer and the number of polymorphic fragments in each generation are shown in Table 2. Maximum number of fragments were amplified in the field population (F0) and maximum polymorphism in F5 (83.1%). More than 65 per cent polymorphism in the amplified fragments was obtained in all the generations. Increased polymorphism increases the chances of effective detection of genetic variation that has arisen due to insecticidal pressure.

Identification of PCR based markers

The specific amplicons were divided into the four categories. First category included fragments amplified in the control (untreated) population but disappeared or reappeared in subsequent generations after the insecticidal treatment with imidacloprid. These fragments indicated a genetic change in the whiteflies due to insecticide treatment in the form of alteration in the nucleotide sequences that resulted in disappearance of fragments which were originally present in the untreated population. Second category included the PCR products which appeared in imidacloprid treated population (both dead and alive) in different generations. This category indicated a significant deviation in the genetic organization of whiteflies resulting from the imidacloprid selection pressure and the change was depicted in variation in the banding pattern of treated whiteflies from that of control. Third category was of the fragments amplified in imidacloprid treated dead individuals but were absent from the treated living individuals. These amplicons indicated that the selection pressure created by insecticidal treatment has led to mutation in the genome of whitefly and due to these alterations in the genetic structure of the survivors, the primer does not anneal and hence, leads to disappeared band. Most important category included the PCR amplified products that appeared only in the individuals surviving the effect of imidacloprid selection. The potential molecular (RAPD) markers for imidacloprid resistance in whitefly were identified from F6, F9 and F11 generations as they showed distinct genetic variation among the imidacoprid treated survivors and the susceptible insects which died after the treatment (Table 3).

As evident from the table OPB 05 primer amplified maximum polymorphism (92.3%) along with maximum polymorphic distinguishing fragments that could serve to develop SCAR (Sequence characterized amplified regions) markers for imidacloprid resistance followed by OPB 10, which had lesser polymorphism (80.6%) in the amplified fragments but amplified three potential markers.

DISCUSSION The amplicons that were either amplified or those that disappeared in the individuals surviving the effect of imidacloprid selection pressure can serve as the potential RAPD markers for the identification of resistance at an early stage and could help in the pest management programmes. Early detection of resistance is helpful in the identification of effective insecticides to manage the pest. Present work indicated that RAPD fragments are useful as genetic markers to identify insecticide resistance, but a number of factors can complicate their use and interpretation e.g. DNA quality, primer sensitivity and co-migration of non homologous fragments (Stevens and Wall, 1995).

Significance of the fragments amplified only in the survivors is that these show the development of variants in the resistant whitefly population after the selection pressure and hence can be used as possible markers to identify resistant individuals from a field population. In the generations except F6, F9 and F11, the variability was not stable and inconsistency may be due to the random changes in the genome after selection of whiteflies to the insecticide so were not included to identify markers for resistance. Complete segregation of the survivors from the dead individuals in these generations indicated that a distinct variation occurred in the living individuals, which separated them from dead individuals indicating possibility of development of resistance in the survivors.

For developing a repetitive marker, selection should be carried out till the resistance stabilizes as the first few generations developed resistance to imidacloprid at a slow rate followed by a rapid increase in the succeeding generations (Prabhakar et al., 1997). Gradual increase in the initial stage may be due to the fact that the resistance genes are rare in the population but subsequently by selection, as the expression of these genes increases, resistance also increases (Prabhakar et al., 1997). Earlier research indicated that the inheritance of insecticide resistance, which is known to involve gene amplification, could be unstable in the absence of selection and insecticide resistance is also believed to arise from selection acting at random variation i.e. it is pre-adaptive (Devonshire and Field, 1991). Sethi et al. (2002) suggested that insecticide resistance in whitefly against imidacloprid is controlled by more than one gene. Also, dominance estimates indicated that insecticide resistance was completely recessive. Moreover, whiteflies are arrhenotokous and in haplodiploidy fertilized eggs give rise to females, which are heterozygous and unfertilized eggs produce males, which are haploid. This method of reproduction allows rapid selection at later stages and fixation of resistance genes (Denholm et al., 1998). Thus, these factors along with the random changes in the genome affect the amplification of a repetitive RAPD marker. So, the selection pressure carried out for more number of generations can result in increase in the homogeneity of whitefly response and also in discreet markers.

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