Toxic and biochemical effects of different insecticides on the tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Toxic effect of dinotefuran, imidacloprid, fenoxycarb, phenthoate and thiocyclam H.O insecticides on greenhouses population of the tomato leafminer, Tuta absoluta (Meyrick) was evaluated in laboratory. Data revealed that the five tested insecticides had high contact toxic effect on moths and 3 instar larvae of the insect. Moths were more susceptible to the effect of tested insecticides than larvae. Imidacloprid was the superior toxicant against moths and larvae, so it had a very low resistance coefficient (RC= 0.01 and 0.13). Phenthoate and thiocyclam-H.O. had high toxic effect on two stages. Dinotefuran seemed to have low effect on moths and the same trend was observed with fenoxycarb on larvae. The activity of acetycholinesterase (AChE), glutathione-S-transferase (GST) and monooxygenase (PCMAN-demethylase) was higher in whole body homogenate of untreated moths (1.5, 1.2 and 1.58 times, respectively), than that of 3 instar larvae. The exposure of moths and 3 instar larvae to LC30, LC50 and LC80 of tested insecticides caused significant reduction (51.11and 25.00%) or increasing (41.78 and 28.77%) in activity of AChE, respectively. Phenthoate treatments reduced AChE activity, but imidacloprid and dinotefuran induced this activity in LC80 treated insects .Low insecticide treatments produced slight induction (1.41-11.90%) of GST, other two treatments produced reduction or increasing enzyme activity of treated insects. LC80 of fenoxycarb had moderate induction (24.7927.90%) of moths and larvae GST. A positive correlation between the insecticide concentration and the activity of monooxygenase PCMAN-demethylase was observed in treated insects with five insecticides. Elevation of enzyme activity ranged from 1.59 to 52.12% in moths and 5.8359.17% in larvae. Phenthoate and imidacloprid produced the higher induction effect of treated insect enzyme than the other three insecticides.


INTRODUCTION Tomato, Lycopersicon esculentum
Mill is a vegetable crop of large importance throughout the world.It is the first horticultural crop in Egypt.Tomatoes are grown both under plastic covered greenhouses and in open field.The tomato leafminer, Tuta absoluta Meyrick, (Lepidoptera: Gelechiidae) is a serious pest of both outdoor and greenhouse tomatoes.It was originated from South America (Giordano and Silva, 1999) and was recently introduced in Europe and subsequently spread throughout the Mediterranean Basin and Europe (EPPO, 2011).The serious outbreak was reported in many countries, Belgrade (Toševski1 et al., 2011), Greece (Roditakis et al., 2010), Brazil (Siqueira, et al., 2000a, b), and Egypt (Mohammed, 2010).Since the time of its initial detection, the pest has caused serious damages to tomato in invaded areas and it is currently considered a key agricultural threat to European and North African tomato production (Desneux et al., 2010, Garcia andVercher, 2010).
Chemical control is the main method for controlling the tomato leafminer, T.absoluta (Siqueira et al., 2001;Galdino et al., 2011).In Argentina organophosphates were initially used for T. absoluta control then were gradually replaced by pyrethroids during the 1970s.During the early 1980s, cartap which alternates with pyrethroids and thiocyclam were sprayed showing the good effectiveness of the former.During the 1990s, insecticides with novel mode of actions were introduced such as abamectin, acylurea, insect growth regulators, tenbufenozide and chlorfenapyr (Lietti et al., 2005).Spinosad and Indoxacarb are effective against larvae of T.absoluta in Spain (SEWG 2008).Chemical pesticides continue to be an important component of insect pest management even with the development of other control methods (mass-trapping, plant resistance...).The use of insecticides based on different chemistries and with varying modes of action is an important component of an integrated pest management strategy.Hence, insecticides will continue to be an integral component of pest management programs due mainly to their effectiveness and simple use (Braham and Hajji, 2011).
Sublethal insecticide exposure can lead to physiological and behavioral changes in the organism (Hyne and Maher 2003).These responses can be measured using specific biomarkers that provide a measure of sublethal effects, e.g., "fitness" of the survivors (McCarthy and Shugart 1990).Three such biomarkers are acetycholinesterase (AChE), glutathione-S-transferase (GST) and mixed function oxidase (MFO).Acetycholinesterase is the target enzyme for organophosphate and carbamate pesticides, which act by inhibiting its activity.GST is involved in the detoxifcation of a wide range of xenobiotic chemicals.In insects, GST plays an important role in biotransformation of various insecticides (Motoyama 1980, Lamoureux andRusness 1987); including the degradation of some organophosphorus compounds (Yang 1976).MFO was effectively reducing the efficacy of insecticides on pests (Wang et al., 2009).This work aims to evaluate the efficiency of some insecticides on adult moths and third instar larvae of tomato leaf miner, T. absoluta.Effect of the insecticide treatments on activity of acetycholinesterase, glutathione-Stransferases and PCMA N-demethylase monooxygenase in treated insects was also, determined.

1-Insect
Samples of differant instars T. absoluta larvae were collected in March 2011 from commercial tomato greenhouses and kept under laboratory conditions (25 ± 2° C, 65 ± 5% R.H. and a photoperiod of 16 L:8 D) on untreated tomato plants until emergence of the moths from F1generation in Central Agriculture Pesticides Laboratory.

2.1-Adulticidal test
Vial method (Plapp et al., 1987;Snodgrass1996) was used to evaluate the toxicity of five formulated insecticides against adult moths (oneday old) of T.absoluta.Five mL of each concentration of tested insecticides was pipette into 100mL (9Dx15H) glass scintillation vial (five replicates for each treatment).The vials were placed on a hot dog roller (heating elements removed) which was operated until all acetone was evaporated leaving behind insecticidal residues inside the vials.Ten adults were added to each vial containing insecticide concentration and control (treated with acetone only).Vials were then closed with clean muslin squares secured with rubber bands.Vials were kept under lab conditions.The effect of insecticides was determined after 12h of application and expressed as percent mortality of moths at each concentration.

2.2-Larvicidal test
Filter papers impregnated with insecticide molecules method (Salazar andAraya, 1997& 2001;Siqueira et al., 2000a, b) was used to evaluate the contact action of the tested insecticides against larvae of T.absoluta.Filter paper (Whitman no. 1 cellulose filter paper 9cm) was putted in glass Petri dish with the same diameter then 1 ml of each insecticide concentration was pipette on it, control treatments were applied with acetone only.Five replicates for each treatment and control were used.Ten 3 rd instar larvae were added to each replicate after the filter paper was dried and kept in lab conditions for 12h, then the mortality counted.

3-Enzyme activity
After experiments of toxicology, untreated control and survivor treated (LC 30 , LC 50 and LC 80 ) moths and 3 rd instar larvae of T.absoluta were removed and frozen for subsequent enzyme analysis.Acetycholinesterase (AChE), glutathione-S-transferase (GST) and monooxygenase (PCMAN-demethylase) activities were measured in all frozen samples.

3.1-Enzyme extract
For AChE and GST activities ,500 mg of control and each of the treated insects were homogenized in 1 ml sodium phosphate buffer(0.1M,pH7)using Teflon glass homogenizer and centrifuged at 10.000g for 15 min at 4C ( five replicates of each sample).The supernatant was used as a source of enzyme.

3.5-Data analysis:
Enzyme activities were expressed as mean ± standard error (S.E.) and statistically analyzed by using SPSS program V.13.Differences were considered significant at p 0.05 level.
The third instar larvae were lowest susceptible (LC 50 = 3.30 -6.88 ppm) to effect of tested insecticides than moths.The adult and neonate of insects were more susceptible to effect of the insecticides than old larva and pupa (Campanhola and Plapp, 1989;Abdel-Rahman et al., 2002).Comparison the LC 95s with the recommended concentration of insecticides revaled that the larvae had low resistance coefficient values (0.24-1.79) to thiocyclam-H.O., phenethoate and Dinotefuran .This value was increased to 5.24 with fenoxycarb treatments, which pointed to high resistance of T. absoluta larvae to this insecticide by contact application.
Susceptibility of field populations of T.absoluta to insecticides was positively correlated with the number of chemical sprays in the field (Reyes et al., 2012).

Enzyme activities
Biochemical evaluations revealed that the enzymatic systems involved in the susceptibility of T absoluta to insecticides.The activities of Acetycholinesterase (AChE), Glutathione-S-transferase (GST) and monooxygenase (PCMAN-demethylase) were higher in whole body homogenate of untreated moths (1.5, 1.2 and 1.58 fold, resp.), than that of 3 rd instar larvae.These enzymes were decreased or increased in body homogenate of treated moths and larvae than the untreated ones (Tables 3& 5).
Treatment of T. absoluta moths and 3 rd instar larvae with LC 30 , LC 50 and LC 80 of tested insecticides produced a significant decrease in AChE activity of insect body homogenates except imidacloprid and dinotefuran (Table 3).There is a negative correlation between the insecticide concentration and the enzyme activity in moth tissues.Phenthoate caused high significant reduction (33.31, 46.40 and 51.11 %)   nmoles of acetylthiocholine hydrolyzed/mg protein - 1 /min -1 ).A medium increase (28.77%) in this activity was detected in LC 80 treatment of dinotifuran (Table 3).If AChE activity is reduced by  50%, it is associated with mortality and knockdown (Edwards and Fisher, 1991).Mortality of aphid which exposed to dimethoate in laboratory was negatively correlated with cholinesterase activity (Booth et al., 2007).
The PCMAN-demethylase activity in moths and 3 rd instar larvae (3.78±1.33 and 2.40±0.82nmoles of pchloroaniline/mg protein -1 /min -1 , resp.), was increased with treatments; there is a positive correlation between the concentration of insecticide and the activity of enzyme in moths and larval homogenates.The high significant increase in enzyme activity (52.18 and 59.17%) was recorded with LC 80 phenthoate in moth and larval homogenates.Dinotefuran caused the lowest induction (18.25 and 27.50%) to moths and larvae enzyme.The evaluating mechanisms would be involved in insecticide resistance of populations of T. absoluta, presenting an increased MFO activity in populations (Reyes et al., 2012).It appears that enhanced oxidative metabolism mediated by cytochrom P450 monooxygenase was a major mechanism for insecticide resistance in the western flower thrips (Chen et al., 2011).

In conclusion
The five tested insecticides had high contact toxic effect on moths and 3 rd instar larvae of T. absoluta, so that we can use them in control of this insect in greenhouses and open fields.Imidacloprid was the superior toxicant against to moths and larvae of this insect.Phenthoate and thiocyclam H.O. had high toxic effect on two stages.Dinotefuran seemed to have moderate effect on moths and the same trend was observed with fenoxycarb on larvae.The activity of acetycholinesterase (AChE), glutathione-S-transferase (GST) and monooxy-genase (PCMANdemethylase) was higher in whole body homogenate of untreated moths (1.5, 1.2 and 1.58 times, resp.), than that of 3 rd instar larvae.The exposure of moths and 3 rd instar larvae to LC 30 , LC 50 and LC 80 of tested insecticides caused significant reduction or slight increasing in activity of AChE and GST.
Elevation of 97) 0.30 CL = confidence limits.RC = resistance coefficient Toxic and biochemical effects of different insecticides on the tomato leafminer 5 Table 2: Susceptibility levels of tomato leafminer, Tuta absoluta 3rd instar larvae to tested insecticides CL = confidence limits.RC = resistance coefficient.

Table 1 :
Susceptibility levels of tomato leafminer, Tuta absoluta adult moths to tested insecticides

Table 3 :
Activity of Acetycholinesterase enzyme in whole body homogenates of treated Tuta absloluta with different concentrations of tested insecticides (nmoles of acetylthiocholine hydrolyzed/ mg protein -1 /min -1 ) Change%= mean activity of treated-mean activity of control/ mean activity of control x100 Mean activity values in the same column followed by different letters are significantly different (P< 0.05).

Table 4 :
Activity of Glutathione-S-transferase enzyme in whole body homogenates of treated Tuta absoluta with different concentrations of tested insecticides (nmoles of CDNB conjugated /mg protein -1 /min -1 ).
S.E =standard error.Change%= mean activity of treated-mean activity of control/ mean activity of control x 100 Mean activity values in the same column followed by different letters are significantly different (P < 0.05).