Efficacy of Certain Pesticides Against Larvae of Tomato Leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

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Recently the tomato leafminer, Tuta absoluta become one of the most devastating pests of tomato in Egypt.Chemical control has been the main method of controlling it.To select the best insecticide for control this insect, susceptibility tests were made under laboratory conditions.Nine insecticides were tested against field T. absoluta 2 nd instar larvae.Imidacloprid and Thiocyclam-H.O. were the most powerful insecticides and the insect had no resistance to them.Lufenuron, Dinotefuran, Fenoxycarb, Diflubenzuron and Phenthoate gave moderate toxic effect and resistance level except Phenthoate and Fenoxycarb.The two bio-insecticides had the lowest effect on larvae with low level of resistance, but Bacillus thuringiensis kurstaki is more toxic than Nuclear Polyhedrosis Virus (NPV).Treatment of T absoluta 2 nd instar larvae with LC 50 of tested insecticides decrease the total protein content in the whole body homogenate of treated larvae compared with that of control insects.A significant depletion of protein concentration presented in Phenthoate (-46.8%),Imidacloprid (-43.0%) and Dinotefuran (-39.1%)treatments.LC 50 treatment of tested insecticides elevated the activity of Cytochrom P450 (PCMANdemethylase monooxygenase) and Superoxide dismutase (SOD) enzymes in tissues of treated larvae than control.The highest significant increase in PCMAN-demethylase monooxygenase activity (157.1%) was detected in tissues of Phenthoate larvae.The more pronounce increasing in (SOD) activity was observed in Imidacloprid larvae.Fractionation of the total protein contents in whole body homogenate of T. absoluta larvae on SDS-PAGE revealed that the depletion of protein concentration correlated to disappearance of several proteins and reduction of bands intensity in treated larvae compared with control.The highest difference in protein pattern (37%) presence between control and treated larvae with Phenthoate , Imidacloprid and Dinotefuran.The aforementioned results revealed that the all tested insecticides had toxic effect on the tomato leafminer and caused biochemical disturbance in their bodies.So that, we can be use small amount from the effective chemical insecticides in rotation with IGRs and bio-insecticides in integrated pest management (IPM) program of this pest.
The insect deposits eggs usually on the underside of leaves, stems and to a lesser extent on fruits.After hatching, young larvae penetrate into tomato fruits and leaves on which they feed and develop creating mines and galleries.Tomato plants may be attack at any developmental stage, from seedlings to mature stage (OEPP, 2005).
Chemical control has been the main method of control used against T. absoluta and the growers normally choose the insecticide in a diversity of options officially registered and recommended (Braham andHajji, 2011: Hanafy andEl-Sayed, 2013).The farmers apply insecticide from 8 to 25 times in a season (Temerak, 2011).The indiscriminate use of synthetic chemical pesticides to control this pest resulted in the rapid development of resistance (Dittrich et al., 1990) and several risks as harmful pesticide residues in fruits (Malhat et al.,2012), undesirable effects on humans and natural environments, eliminated natural enemies from crop ecosystems and estimates proved that only a small portion of pesticides applied to the crop reaches the target pest while the major portion reaches to the nontarget organisms (Erayya et al., 2013).
Applying new types of insecticides, originated from natural agents or products that disrupt the physiological processes of the target pest, could be useful alternatives in the integrated management approach (Parsaeyan et al., 2013).
Microbial biopesticides are environmentally safe, self perpetuating in nature, specific to target pests etc.Among the microbial biopesticides, bacterial, fungal and virus products occupy a special space in managing several pests (Bidyarani-Devi et al., 2016).Bacterial insecticides were the earliest developed and the most widely used microbial pesticides in the world.One of the most common bacterial insecticides contains Bacillus thuringiensis (Bt), which specific to larvae of lepidopteran pests (Peng, 1992).The application of B. thuringiensis in Egypt started at 1960 against young larvae of the cotton leaf worm, Spodoptera littoralis (El-Husseini, 1981).Each of the three subspecies of B. thuringiensis attacks larvae of a specific order, i.e., B. thuringiensis kurstaki for Lepidoptera, B. thuringiensis israelensis for Diptera and B. thuringiensis tenebionis for Coleoptera (El-Husseini, 2006).Viruses are sub microscopic, intracellular and obligate pathogenic entities with nucleic acid and protein.These viruses are often genus or species specific and highly virulent to their hosts.Several groups of viruses having a potential in controlling many pests.Among these groups of viruses, Nuclear Polyhedrosis Virus (NPV) which belongs to the family baculoviridae was exploited widely as a microbial control (Herniou et al., 2012).Entomopathogenic viruses are currently used as alternatives to traditional insecticides.Its use should not be generalized because each pest has its own case.In specific cases, viruses proved very effective in managing populations of certain pests as Lepidoptera and Hymenoptera (El-Husseini, 2006).The virus enters the nucleus of infected cells, and reproduces until the cell is assimilated by the virus and produces crystals in the fluids of the host.These crystals will transfer the virus from one host to another (Chiu et al., 2012).NPV forming polyhedra like occlusion bodies kills most important crop pests such as Helicoverpa armigera and S. litura (Bidyarani-Devi et al., 2016).
The exposure to insecticide can lead to physiological and behavioral changes in the organism (Hyne and Maher, 2003).These changes in pest leading to insecticide avoidance, altered penetration, sequestration, and target site alteration or bio-degradation.Metabolic changes occur through increased biodegradation of the insecticide, usually through overproduction of detoxification enzymes (Hemingway and Ranson, 2000).Cytochrom P450s are the primary enzyme family associated with resistance to most insecticides.Elevated levels of P450 activity are frequently observed in resistant insects (Hemingway and Ranson, 2000;Brooke et al., 2001).Also, pesticides and allelochemicals cause oxidative stress, characterized by exposure to in vivo excessive reactive oxygen species (ROS) is involved in organisms (Amin and Hashem, 2012).Reactive oxygen species cause oxidative stress leading to the damage of biomolecules such as proteins, lipids, and nucleic acids, resulting in disturbance of homeostasis and cellular death if not eliminated (Hermes-Lima and Zenteno-Savin, 2002).Insect's posses an antioxidant defense system that consists of both enzymatic and non-enzymatic components.
Superoxide dismutase enzyme (SOD) is one from the enzymatic components of antioxidant defense system (Krishnan and Kodrík, 2006;Krishnan et al., 2009).
This work aims to evaluate toxic and biochemical effects of the chemical and bio-insecticides on second instar larvae of the tomato leaf miner, T.absoluta.

Bioassay:
Leaf-dip bioassay method used to detect the response of second instar larvae of T. absoluta F1 progeny to tested insecticides.
Ten serial aqueous concentrations of each pesticide were prepared.Fresh tomato leaflets were individually dipped in each prepared concentration of the tested insecticides for 10 seconds with genital agitation, control leaflets were dipped in water only (Five replicates for each insecticide concentration and control), then the leaflets were left to dry.The dried leaflets were placed on a slightly moistened filter paper covering the bottom of clean glass cages (15D× 15H) to keep the leaf material turgi1d throughout the bioassay period.Ten 2 nd instar larvae were carefully placed using a fine soft brush in each cage and kept under lab conditions (IRAC, 2010).Mortality was recorded after 72 hr. of treatment and corrected by Abbott's formula (Abbott, 1925).
Sub lethal concentrations, LC 50 and LC 95 of treated larvae were calculated by using SAS probit program (SAS, 1997).
To assess the resistance of a given population, the resistance coefficient (Wegorek et al., 2011) was calculated as follows: Resistance Coefficient (RC) = LC 95 of field insects/ recommended field concentration.

Biochemical assay:
After toxicological experiments, the survivor larvae of T.absoluta from untreated (control) and LC 50 treatments were removed and frozen for subsequent biochemical analysis.The total protein content, fractionated proteins and activity of Cytochrom P450 (PCMA Ndemethylase monooxygenase) and Superoxide dismutase (SOD) enzymes were determined in the whole body tissues of control and treated larvae.

Protein and enzymes extract:
Samples of 500 mg from control and treated larvae were homogenized in 1 ml Sodium Phosphate buffer (0.1M pH7) using Teflon glass homogenizer and centrifuged at 10.000rpm for 15 min at 4C (five replicates of each sample).The supernatant was used as a source of protein and SOD enzyme tests.

Total content of proteins:
Total protein content was determined based on Biuret test (Henry, 1964), using Kit purchase from dp international laboratory.A mixture of 1.0 ml of the total protein reagent (0.2N Sodium hydroxide, 18mM/L Sodium Potassium tartrate, 12mM/L Potassium iodide and 6mM/L Cupric sulfate), 20μl of each sample and 20μl of deionized water then incubated for 5 minutes at 25 o C. Read and recorded the absorbance at wave length of 546 nm versus the reagent blank as reference and Standard.The total protein concentration in the whole body homogenate of control and treated larvae was represented as g/mg body weight of insects.

Activity of Superoxide dismutase (SOD) enzyme:
The activity of Superoxide dismutase enzyme (SOD) was measured according to method of Beauchamp and Fridovich (1971) with some modification by Krishnan et al., (2002) tests the ability of SOD to inhibit the reduction of Nitro blue tetrazolium by the superoxide anion generated photo chemically.One milliliter of assay mixture consisted of 50 mM Sod. Phosphate buffer, pH 7.8, 13 mM Methionine, 75 µM Nitro blue tetrazolium, 2 µM riboflavin, 0.1 mM EDTA, and enzyme extract.Riboflavin was added last, the samples were placed inside a light box contains three comptalux bulbs (100 W, Philips Ltd,) and the reaction was allowed to run for 15 min.The reaction was stopped by switching off the light.A non irradiated reaction mixture, which was run in parallel, did not develop color and served as a control.The absorbance was read at 560 nm.One unit of activity is the amount of protein required to inhibit 50% initial reduction of NBT under light.
Total protein content and enzyme activities of all samples are reported as mean ± standard error and statistically analyzed using Excel Microsoft Office and Student's t-test Program.Differences were considered significant at p 0.05 level.

Fractionation of proteins:
The basic principle of proteins fractionation by electrophoresis is the movement of the charged molecules towards an electrode with the opposite charge through a supporting medium.Proteins of tissues were separated on 11% Sodium Dodecyl Sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970).In a solution of SDS and 2-mercaptoethanol, proteins dissociated into subunits with rod like shape, the rod diameter is thought to be constant, while the long axis varies in proportion to the molecular weight.The later value can be determined by comparing the electrophoretic mobility of unknown proteins with the mobility of known standard protein markers.After fractionation process the gel was photographed, scanned and analyzed with Gene Tools program.Similarity index, commonality band ratio and polymorphism in samples protein were calculated according to Nei and Li (1979).

Bioassay:
Toxic effect of the nine tested insecticides against 2 nd larval instar of field T. absoluta was shown in Table 1 2).

Activity of Cytochrom P450 (PCMANdemethylase monooxygenase) enzyme:
The activity of Cytochrom P450 (PCMAN-demethylase monooxygenase) was highly increased in the whole body homogenate of T. absoluta larvae post treatment with LC 50 of the tested insecticides than control ones (Table2).This activity reached to 2.8±1.24n moles of p-chloroaniline/mg -1 protein/ min -1 in tissues of control insects.The highest significant increase in enzyme activity (157.1 %) was recorded in tissues of treated larvae with Phenthoate.Also, a very high elevation of enzyme activity (125.0,67.9, 64.

Activity of Superoxide dismutase (SOD) enzyme:
The activity of Superoxide dismutase (SOD) enzyme increased in T. absoluta 2 nd instar larvae after exposure to LC 50 of the tested insecticides (Table 2).The highest increase in enzyme activity was detected in Imidacloprid (93.3%) treated larvae.Avery high significant increase (83.2, 70.1, 66.3, and 49.7%) presented in Fenoxycarb, Thiocyclam-H.O., Phenthoate and B. thuringiensis treated larvae, resp.Dinotefuran treatment produced significant elevation (39.1%) of enzyme activity but Lufenuron, Diflubenzuron and NPV caused a slight activation (13.9, 9.2 and 4.3%, resp.) of larval enzyme.Mean of total protein concentration and enzyme activity values in the same column followed by different letters are significantly different (P < 0.05).Change%= mean of treated larvae-mean of control larvae / mean of control larvae x 100

Fractionation of proteins:
The total protein contents in control and LC 50 treated 2 nd instar larvae of T. absoluta were fractionated on SDS polyacrylamide gel electrophoresis (Fig. 1 and Table 3).Nineteen bands appeared in control and treated larval tissues according to their molecular weights and relative mobility on the gel.The total numbers of protein bands were 19, 14, 13, 15, 13, 12, 12, 14, 14 and 12 appeared in the whole body homogenate of control (C), B. thuringiensis (Bt), NPV(V), Diflubenzuron (D), Lufenuron (L), Phenthoate (P), Imidacloprid (I), Thiocyclam-H.O.(T), Fenoxycarb (F) and Dinotefuran (Df) treated larvae.There were eleven common bands; 1,2,4,6,7,10,11,15,16,17and 18 with Molecular Weights of 201.42,194.56,170.89,130.78,115.23,65.36,57.55,26.65,21.37,17.52 and 14.13 (KDa) and Relative Mobility values of 0.053, 0.097, 0.171,0.248,0.283,0.494,o.536,0.772,0.808,0.834 and 0.863 (Rm),respectively, appeared in control and treated insects.Band no.9 (83.57KDa and 0.412 Rm) was common in all larval samples except Df.Band no.5 ( 159   (+) Present (-) Absent DISCUSSIONS Data revealed that the chemical pesticides proved high toxic effect on field T. absoluta 2 nd instar larvae than IGRs and bio-insecticides.Results showed that Imidacloprid and Thiocyclam-H.O. are the highly effective insecticides and the insect had no resistance to them.Lufenuron, Dinotefuran, Fenoxycarb, Diflubenzuron and Phenthoate gave moderate toxic effect and resistance level except Phenthoate and Fenoxycarb.The two bio-insecticides had the lowest effect on larvae with low level of resistance.These results are in conformity with the findings of several researchers such as Santos et al., (2011) who mentioned that the chemical pesticides continue to be an important component of insect pest management even with the development of other control methods.Susceptibility of field populations of T.absoluta to insecticides was positively correlated with the number of chemical sprays in the field.The reduction of susceptibility observed, is more probably due to crossresistance with other insecticides more widely applied in the past (i.e.organophosphates and pyrethroids).The resistant populations have been collected in locations where the presence of the pest has lead to the use of a high number of insecticide sprays during the crop season (Reyes et al., 2012).The reducedrisk insecticides, Imidacloprid providing rapid knockdown and mortality followed by residual antifeedant activity on rose chafer beetle adults (Isaacs et al., 2002).Indoxacarb, Spinosad, Imidacloprid, Deltamethrin and B. thuringiensis var.kurstaki have successfully been used to control of T. absoluta larval infestations in Spain (Russell, 2009).Moussa et al., (2013) Nazarpour et al., (2016) indicated that Bt, Azadirachtin and mixture of them significantly suppressed the larval density and caused 100% reduction in fruit and foliage damage compared to the untreated plants.Microbial biopesticides acts as a solution as they are environmentally safe, self perpetuating in nature, specific to target pests etc.Among the microbial biopesticides, viruses after bacterial and fungal products occupy a special space in managing several pests (Bidyarani-Devi et al., 2016).
Insect Growth Regulators (IGRs) have a much slower mode of action than synthetic chemical insecticides but they cause malformations of treated insects.IGRs include juvenile hormone mimics (Fenoxycarb) and chitin synthesis inhibitors (Lufenuron) inhibit the production of chitin, a major component of the insect exoskeleton.The treated insects became unable to synthesize a new cuticle, and therefore unable to successfully moult into the next stage (El-Aswad, 2007).Treatment of S. littoralis last instar larvae with Novaluron (IGR) resulted in some features of impaired morphogenesis and remarkably accelerated the ovarian maturation because Novaluron prohibited this vital process (Hamadah et al., 2015).
Proteins are important for individual level fitness associated traits such as body size, growth rate, and fecundity, and at higher levels of organization they have been linked to population dynamics, life histories and even biological diversification (Fagan et al., 2002).Treatment of T. absoluta larvae with the LC 50 of tested insecticides produced high significant reduction in larval tissue proteins, especially Phenthoate and Imidacloprid treatments.The same observations were detected by Abdel-Hafez et al., (1988) who mentioned that the IGR; Diflubenzuron and Triflumuron treatments reduced the level of proteins and free amino acids in laboratory and resistant strains of S. littoralis.Etebari et al., (2005) showed that many insecticides treatments decrease feeding efficiency and protein amount of insects.The decreasing of the total soluble protein contents in supernatant of the homogenated Musca domestica larvae post-LC30 treatment with B. thuringiensis β exotoxin as compared to control (Abuldahab et al., 2011).Piri et al., (2014)  Biochemical mechanisms including the insensivity of target sites to insecticides and enhanced detoxification rate by several detoxifying mechanisms.MFO family (Cytochrom P450s) acts as effectively reducing the efficacy of insecticides on pests (Wang et al., 2009).High increase of Cytochrom P450 (PCMAN-demethylase monooxygenase) activity was detected in T. absoluta larvae treated with LC 50 of the tested insecticides.Phenthoate and Imidacloprid treatments had the highly significant increasing effect on insect enzyme.The evaluating mechanisms would be involved in insecticide resistance of T. absoluta insect, presenting an increased MFO activity in populations (Reyes et al., 2012).The enhanced oxidative metabolism mediated by cytochrom P450 monooxygenase was a major mechanism for insecticide resistance in the western flower thrips (Chen et al., 2011).By pinpointing the key enzymes associated with insecticide resistance we can begin to develop new tools to aid the implementation of control interventions and reduce their environmental impact on Earth.Recent technological advances are helping us to build a functional profile of the P450 determinants of insecticide metabolic resistance in mosquito insects (David et al., 2013).
Cells have interdependent antioxidant defense mechanisms that protect against damage from oxidative stress.Insect's posses an antioxidant defense system that consists of both enzymatic and non-enzymatic components.The enzymatic components are superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase ( (Krishnan and Kodrík, 2006;Krishnan et al., 2009).The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of prooxidant endobiotics and xenobiotics (Kolawole et al., 2014.).The reactive oxygen species, such as superoxide radical (O 2 -), hydrogen peroxide H 2 O 2 , and hydroxyl radical (OH -) are generated in aerobic organisms under normal metabolism and when exposed to various abiotic and biotic factors during life spans (Halliwell and Gutteridge, 2001).LC 50 treatment of the tested insecticides caused elevation of SOD activity in tissues of treated larvae and Imidacloprid had the superior effect.Al-Barty (2014) studied the effects of Methylamine avermactine on the oxidative stress indicator, and antioxidant enzyme superoxide dismutase activity in Sitophilus oryzae tissues.There were significant increases in SOD activities in the LC 50 treated insect compared to the control.The insecticide treatment causes an increase in oxidative stress which induces antioxidant defense mechanisms.SOD was stimulated by scavenging superoxide radical to protect the insect from insecticide stress.
Fractionation of the total protein contents in the whole body homogenate of T. absoluta larvae on SDS-PAGE revealed that the depletion of protein concentration correlated to disappearance of several proteins and reduction of bands intensity in treated larvae compared with control ones.The highest difference in protein pattern (37%) presence between control and Phenthoate, Imidacloprid and Dinotefuran treated larvae.These findings are in accordance with results obtained by Zidan et al., (2002) who mentioned that treatment of the spiny bollworm, Earias Insulana larvae with LC 50 of Abamectin and Esfenvalerate caused disappearance and appearance of numerous protein bands with different intensity.Treatment of the med fly, Ceratitis capitata with LC 50 from Vertimec, sumi-gold and Tracer caused disappearance, appearance and degrees of difference (22, 29 and 38% resp.) in fractionated proteins of insect tissues compared with control (Radwan and El-Malla, 2008).

CONCLUSION
The tested insecticides are effective against the tomato leaf miner, T. absoluta 2 nd instar larvae.Chemical insecticides proved high toxic effect on insect than IGRs and bio-insecticides.Imidacloprid and Thiocyclam-H.O were more potent toxicant and the insect had no resistance to them.Lufenuron, Dinotefuran, Fenoxycarb, Diflubenzuron and Phenthoate gave moderate toxic effect and resistance level except Phenthoate and Fenoxycarb.The two bio-insecticides had the lowest effect on larvae with low level of resistance.Treatment of tomato leaf miner larvae with the LC 50 of tested insecticides produced a high significant reduction in larval tissue proteins.This depletion in protein concentration was correlated to disappearance of several proteins and reducing of bands intensity in treated larvae.LC 50 treatment of tested insecticides cause elevation of detoxification enzyme, Cytochrom P450 (PCMAN-demethylase monooxygenase) and antioxidant enzyme, superoxide dismutase.Finally, all tested insecticides had toxic effect on the tomato leafminer and caused biochemical disturbance in their bodies.So that, we can be use small amount from the effective chemical insecticides in rotation with IGRs and bio-insecticides in integrated pest management (IPM) program of this pest.
.22 KDa and 0.202 Rm) shared in five samples C, D, T, Fand Df.Also, band no.13 (38.68 KDa and 0.667 Rm) appeared in five samples C, Bt, V, D and L. Band no.8 (94.49KDa and 0.376 Rm) shared in four samples C, D, T and F. Band no.14 (32.16 KDa and 0.710 Rm) presented in C & Bt and disappeared in other samples.Bands no. 3, 12, 19 (M.W. 185.38, 45.95&8.56KDa and 0.125, 0.623&0.930Rm) are specific proteins to control larvae and disappeared in tissues of treated ones.The denistometeric scanning of protein pattern revealed that the concentration of bands in treated larvae was very low compared with those of control.The highest commonality band ratio and similarity index (1) with no difference in SDS protein pattern presented between V&L, P&I and T&F treated larvae.A highest similarity and commonality band ratio (0.96 and 0.90) and the lowest polymorphism (10%) in protein bands were presented between Bt & V larvae.Also, a high similarity and commonality band ratio (0.92 and 0.84) with low polymorphism (16%) in protein bands were presented between Df & P & I.A moderate value of similarity, commonality and polymorphism in protein (0.88 and 0.79 and 21% , rep.) was presented between Control & D and (0.85, 0.74 and 26%, rep.) in Control & Bt, T and F larvae, resp.Low value of similarity and commonality (0.81 and 0.68) and high polymorphism (32%) were detected between Control &V & L larvae,.The highest difference (37%) and very low similarity and commonality protein band ratio (0.77 and 0.63) were presented between Control & P, I and Df larvae.

Fig. 1 :
Fig.1: SDS Polyacrylamide gel of denatured protein patterns in the whole body homogenates of Tuta absoluta 2 nd instar larvae treated with LC 50 of tested insecticides .Lane: M for the protein molecular weights marker.Lane :C for control larvae, Lanes; Bt, V, D, L, P, I, T, F and Df for treated larvae with LC 50 of B.thuringiensis, NPV, Diflubenzuron, Lufenuron, Phenthoate, Imidacloprid, Thiocyclam H.O., Fenoxycarb and Dinotefuran respectively.Protein band numbers are indicated on the right side of the gel.Molecular weights of marker bands are indicated on the left side of the gel.