Monitoring Resistance in the Whitefly Bemisia tabaci (Homoptera: Aleyrodidae) to the Efficiency of Three Insecticides in Relation to Some Detoxification Enzymes

The journal of Toxicology and pest control is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the interaction between insects and their environment. The goal of the journal is to advance the scientific understanding of mechanisms of toxicity. Emphasis will be placed on toxic effects observed at relevant exposures, which have direct impact on safety evaluation and risk assessment. The journal therefore welcomes papers on biology ranging from molecular and cell biology, biochemistry and physiology to ecology and environment, also systematics, microbiology, toxicology, hydrobiology, radiobiology and biotechnology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Since 1930s the whitefly B. tabaci became the first whitefly species to be implicated as a vector of the cotton leaf curl disease in Sudan and Nigeria.With the exception of cotton and few other crops, agriculturalists are much more concerned about the whitefly as a vector than a pest.
Many workers have pointed out the increase prevalence as well as expanded distribution of whitefly born viruses, during the last decade and the devastating impact yield losses range from 20-100% depending on the crop, season, vector prevalence and other factors (Sastry and Singh 1973;Horowitz et al.,1984;Brown and Bird 1992).
On the other hand, some authors recorded that B. tabaci damage a wide range of crops not as a vector but as a feeder.Mound (1965) experimentally demonstrated that general weakening of the plants due to whitefly infestation could cause serious reduction of cotton yield due to in part to a decreased number of bolls and in part to a decline in weight of seed and lint per boll.Heavy colonization of B. tabaci can lead to serious direct and indirect damage Basu1995 and Lemos et al.,(2003).
Neonicotinoid have established themselves world-wide, as key components in insect control programs, because of their unique chemical and biological properties, such as, broadspectrum insecticidal activity, low application rates, excellent uptake and translocation in plants, new mode of action and favorable safety profile.As first neonicotinoid, confidor and the second generation is actara Nauen et al., (2003); Mainfisch et al., (2001 andWakita et al., (2003).
Insects contain numerous enzymes with different substrate spectra.It is a fact that development of more active hydrolytic detoxification systems by an insect species is the most probable explanation of self resistance (Casida, 1958;Oppenoorth and Asperen,1960).Also, Loxdale and Lushai,1988;Al-Beltagy et al., 1993 andZhang et al., 2015 reported that the resistance in insects to several insecticides was conferred by high carboxyl esterase activity.

Insect populations Field populations:
The whitefly adults of Bemisia tabaci used were collected from cabbage fields of Sharkia, Beni-suif and Giza Governorates in October which following the insecticidal applications by different types of insecticides.
Adults SPWF (sweet potato whitefly) were collected from field using custom made battery operated suction sampler (Dittrichetal., 1990).Adults were randomly collected across a representative collage growing area from two or more fields.Samples collected were pooled in wide mouth glass jars and kept in cool box during the transport from the field to the laboratory.

Laboratory strain
The laboratory reference strain of B. tabaci (Genn), originated from field collection over vegetables and ornamentals.This strain has been reared in laboratory culture for 30 generations under standard conditions at 26 ±1c 0 and 70 ±5 R.H, and a photoperiod of 16 :8 (light :dark) as described by Coudriet et al., (1985).

Insecticides used:
All the tested insecticides were from neonicotinoids group as follows:
The mixture was incubated for exactly 15 min.at 27 o C, then 1 ml of diazoblue color reagent (prepared by mixing 2 parts of 1% diazoblue B and 5 parts of 5% sodium lauryl sulphate) was added.The developed color was reading at 600 or 555 nm for α-and β-naphthol produced from hydrolysis of the substrate, respectively.α-and β-naphthol standard curves were prepared by dissolving 20mg αor β-naphthol in 100 ml phosphate buffer, pH7 (stock solution).Ten milliliters of stock solution were diluted up to 100 ml by the buffer.Aliquots of 0.1, 0.2, 0.4, 0.8, and 1.6 ml of diluted solution (equal to 2.4, 8.16 and 32 µgnaphthol) were pippeted into test tubes and completed to 5 ml by phosphate buffer.One milliliter of diazoblue reagent s was added and the developed color was measured as mentioned before.

Glutothion-S-Transferase assay
GST activity was determined based on the technique of Habig et al. (1974) using 1-chloro-2,4-dinitrobenzene (2,4-CDNB) as a substrate.The reaction mixture comprised of 10 μL reduced glutathione (GSH) (10 mM) in sodium phosphate buffer (100 mM, pH 6.5) and 10 μL of the enzyme solution.The reaction was initiated by adding 10 μL of 2,4-CDNB (6 mM in methanol) resulting in a final volume of 30 μL.The plates were immediately transferred to absorbance microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).The reactions were allowed to continue for 5 min and absorbance readings were taken at 340 nm automatically once per min against blanks (wells containing all reaction components except the enzyme solution).The increase in absorbance was linear throughout the 5 min reading interval.An extinction coefficient of 9.6 mM -1 cm -1 was used to calculate the amount of 2,4-CDNB conjugated.

Mixed Function Oxidases (MFOs) assay
MOs activity were detected through the transformation of pnitroanisole to p-nitrophenol through O- demethylation via the enzyme pnitroanisole-O-demethylase based on the methods of Hansen and Hodgson (1971) with slight modifications.The standard incubation mixture contained 1 mL sodium phosphate buffer (0.1 M, pH 7.6), 1.5mL enzyme solution, 0.2 mL NADPH (final concentration 1 mM), 0.2 mL glucose-6-phosphate (final concentration 1 mM) and 50 μg glucose-6-phosphate dehydrogenase.The reaction was initiated by the addition of p-nitroanisole in 10 μL acetone to give a final concentration of 0.8 mM and was incubated for 30 min at 37°C.The incubation period was terminated by the addition of 1 mL HCl (1N), and pnitrophenol was extracted with CHCl3 and NaOH (0.5 N).The absorbance of NaOH solution was measured at 405 An extinction coefficient of 14.28 mM - 1 cm -1 was used to calculate the concentration of 4-nitrophenol.

Total protein:
Total protein content was determined according to Bradford (1976).

Statistical analysis:
The percentage mortality of treated larvae was corrected against that of the control using Abbott's formula (Abbott, 1925).Then, the corrected mortality was subjected to Probit analysis (Finney, 1971).Data of the biochemical assays were analyzed using one-way analysis of variance (ANOVA).When the ANOVA statistics were significant (p< 0.05), the means were compared by Duncan's multiple range test.All the analyses were computed by IBM ® SPSS ® Statistics 21.0 (IBM Corp., Armonk, NY, USA.

RESULTS AND DISCUSSION
Baseline data from susceptible strains are a prerequisite for understanding the development of resistance in the field insecticides in populations to the tested insecticides.Because resistance is a genetically based shift in population response, resistance monitoring is added by the initial quantification of responses to toxic substances by susceptible strain (Robertson et al., 2007).
Data presented in Table ( 1) showed that Sharkia field population was the most resistance one to the novel compounds, followed by Giza field population.While Beni-suif field population was the most susceptible population with LC50 0.12, 0.03 and 0.25 for actara, confidor and pymetrozin, respectively.Confidor was the most potent one among the tested compounds, this means that the confidor has a great effect.Afzal and Basit (2002) reported that confidor was the most effective insecticide moong four insecticides.Also, actara exhibits exceptional systemic characteristic and provides excellent control of a broad range of commercially important pests, such as aphids, jassids and whiteflies (Mainfisch et al., 2001).Although neonicotinoids proved high efficacy, it is important to bear in mind that the probability of build up a resistance to them (Olson et al., 1996;Zhou et al., 2004;Mota-Sanchez et al., 2006).Qi-AiMing et al., 2004 reported that confidor was the most effective insecticide for both whitefly and black thrips, also Dewar et al., 2004 reported that the confidor gave good control of aphids and whitefly.
In general Sharkia population exhibited no wide range of resistance ratio to tested insecticides Table (1).In other words, chess insecticide indicated increase in resistance to be ranged from 1.9, 12.6 and 28.6 folds for Beni-suif, Giza and Sharkia populations, respectively while RR values were doubled or tripled for actara with values 2.4, 7.2 and 14.8 folds for Beni-suif, Giza and Sharkia respectively.For confidor, 1.5, 17.5 and 24.5 folds for Beni-suif, Giza and Sharkia, respectively.
Table 1: LC50, LC90 values and Resistance ratios for Bemisia tabaci strains collected from three Egyptian Governorates to three neonicotinoid compounds compared to the susceptible laboratory strain.

Detoxifying enzymes activity: α-esterase activity:
Treatment of B. tabaci with the LC50 neonicotinoides revealed a significant decrease (p< 0.05) in αesterase activity of the samples collected from Giza, Beni-suif and Sharkia governorates compared to the susceptible laboratory strain (Table2).

GST activity:
B. tabaci collected from Giza, Beni-suif and Sharkia Governorates showed a significant increase (P˂ 0.05) in GST activity compared to the laboratory strain.

MFOs activity:
MOs activity compared to the laboratory strain by contrast, showed that MFOs activity of the strains collected from Giza, Beni-suif and Sharkia Governorates was significantly increased (p< 0.05) (Table 2).
Typically, enhanced metabolic detoxification among insects may contribute to insecticide resistance (Low et al. 2007), the metabolic detoxification system in insects consists of three major groups of enzymes.The phase I detoxification enzymes, acting on a broad range of substrates directly to reduce their toxicity, are represented by cytochrome P450.The phase II enzymes, including GST, UDP-glucuronosyltransferases (UGTs), and α -esterase, facilitate the excretion of hydrophobic toxic compounds by improving their hydrophilicity.Several studies indicated that increased levels of detoxification gene expression are known to result in increased levels of detoxifying enzymes that are responsible for insecticide resistance (Karunker et al. 2008;Liu et al. 2011;Schuler, 2011;Gong et al. 2013,;Zhang et al. 2015).