Comparative Toxicity and Synergism in Two-Spotted Spider Mite (Tetranychus urticae) Under Disruptive Selection

The two-spotted spider mite Tetranychus urticae Koch is the major pest of a wide range of outdoor and protected crops worldwide (Gupta and Gupta, 1985; Kumar et al., 2003). Its control has been mainly based on the use of acaricides. The sustained use of acaricides beside its high reproductive potential and short life cycle led to the rapid development of  resistance against  most of the commonly used acaricides for which, its control has become problematic in many areas (Nauen et al., 2003; Pottelberge et al., 2009). Organophosphates (OP) are among the first chemical groups used to control T., urticae and to which the species developed resistance. The first reported cases of failure of OP to control T., urticae go back to the late 1940s. In many cases, especially in greenhouses, resistance appeared quickly after the first contact of T., urticae with the chemical (Pottelberge et al., 2009). Resistance of T., urticae to METI (mitochondrial electron transport inhibitor) acaricides has been also found in many countries (Stumpf, and Nauen, 2001). Several field populations of T., urticae have already developed high levels of fenpyroximate resistance despite its short-term used (Cho et al., 1995). The present article reports on the resistance of malathion and fenpyroximate in two-spotted spider mite under disruptive selection for 20 generations. Diagnostic concentrations were determined and several acaricides compounds were tested in inbred mite lines and evaluate comparative toxicity for each acaricide, also the role of synergists as enzymes inhibitors was discussed.

Rearing of two-spotted spider mite and acaricides bioassay
 
The experimental work was undertaken using two-spotted spider mite (T., urticae Koch) females of the strains of our breeding homozygous for susceptibility to the toxic effects of selective acaricides. The mites were reared continuously on bean plants, Canavalia ensiformis L., under laboratory conditions at 25 ± 1°C, 70 ± 5% RH and a 16h photoperiod. Acaricides bioassay was conducted using the dipping method and mortality was calculated 24 h after treatment. Individual mite survival was determined by touching each mite with a fine brush.
 
Selection for resistance and susceptibility
 
Two lines of gravid females were selected for resistant against malathion and fenpyroximate and disruptive selection was conducted using the diagnostic concentrations of each acaricide (LC₉₅ × 2 for the mites of a susceptible strain). In each line, the mites were bred in families from single females after an inbred cross. Survivors of 2-3 families which showed less susceptibility of resistance line and 2-3 families which showed high susceptibility of  sensitive line after 24 h were used to initiate the next generation. In each generation, ten females were taken from each of the two or three, families were used for the acaricide bioassay and the remaining were kept to produce the next generation.
 
Comparative toxicity of acaricides
 
Comparative toxicity relationships between malathion/ fenpyroximate and five other acaricides were evaluated on selected resistant (R) and susceptible (S) strains of T., urticae of each line. The acaricides used were malathion (50%EC), fenpyroximate (Ortus 5% EC), dimethoate (B-58 40 g/L EC), bifenthrin (Talstar 10 g/L EC), bromopropylate (Neoron 50% EC), abamectin (Vertimec 18% EC) and pyridaben (Sanmite 20% WP). Mortality was assessed 24h after treatment in the same manner as described earlier.
 
The role of the synergists (PBO and butyphos) as enzymes inhibitors
 
Acaricides with synergists Tributyl phosphate (butyphos) and piperonyl butoxide (PBO) were tested on T., urticae strains in the ratio (1:1) and (1:0.01) (acaricides: synergist). These ratios are selected based on preliminary studies. Synergistic PBO (C9H30O5) was used as an inhibitor for monooxygenases, while synergistic butyphos (C₁₂H₂₇OS₃P) was used as an inhibitor for carboxylesterases.
 
Statistical analysis
 
The LC95 values were calculated by the prophet’s method according to Litchfield and Wilcoxon (Belenkiy, 1959). The sample rate of the arithmetic average of the mite lethality percentage error in mite generations under disruptive selection was calculated by the following formula: 

Sp= average of lethality percentage error, p= lethality percentage, n= the total number of mites.
 
while the coefficient of relative dispersion of the variable was calculated by (Urbah, 1964) formula:     

 
       

 
υ = Coefficient variation, x= The aggregate average lethality rate (%).

Expression of resistance to malathion and fenpyroximate in 20 generations
 
Breeding two-spotted spider mites for resistance or susceptibility to malathion and fenpyroximate has revealed similar patterns of variability in the expression of resistance in the generations under disruptive selection (Tables 1 and 2). Females families which showed absolute resistance to acaricides produced new families with a different number of individuals surviving after exposure to the diagnostic concentrations of these toxic agents. The aggregate statistical average percentage of resistant mites, showing no significant changes in the resistant lines between 20 generations of each acaricide, where the aggregate average lethality rate (%) of the first and second ten generation resistance of malathion strains was x=30.57±3.9 and x=28.95±3.3, respectively, with coefficient variation (%) n=12.7±0.24 and n=11.4±0.18, respectively, while the aggregate average lethality rate (%) of the first and second ten generation resistance of fenpyroximate strains was x=32.3±3.8 and x=37.5±3.9, respectively, with coefficient variation (%), n=11.7±0.2 and n=10.4±0.19, respectively.
 

 

        
Disruptive selection of 20 generation females of T. urticae, according to the criteria of the presence or absence trait resistance to malathion or to fenpyroximate revealed the features of manifestations, related to the biology of haplo-diploid breeding. Virgin females of the mite produce only haploid males and fertilized females produce diploid daughter females and haploid males. Heterozygous female based on resistance to acaricide produces males of two genotypes with allele determining sign of resistance to acaricide and without such alleles are susceptible to acaricide males. Inbred reproduction of T. urticae is not limited to the number of generations and does not cause any signs of inbred depression, being in this type of normal biological process. In such inbred families, 60% of males smoked with already fertilized females (Oku, 2010; Oku and Beuken, 2017). The number of females without resistance under the action of diagnostic concentrations of acaricides in isolated inbred families obtained from one resistant parental female, it was on average 30% (Table 1 and 2).
        
In inbred families of T. urticae, obtained from sisters of females 100% - but mortality in the selection of acaricides, remained alive after treatment with diagnostic concentrations of malathion and fenpyroximate 5-15% females (Table 1 and 2). It was assumed that the alternation of acaricides OP and METI groups should be one of the preferred variants of the rotation due to significant differences in the mechanisms of their action on the nervous system of arthropods (OP) and the complex energy and respiratory processes occurring on the mitochondrial membrane (Dekeyser, 2005). The acaricides  resistance gene of individual chemical classes initiates restoration of physiological functions are disturbed by toxicant regulation of ion channels, conformation of receptors, etc. normalizing flow of biochemical processes through these functions is regulated. Enzymatic “detoxification” of pesticides discussed in literary sources (Leeuwen et al., 2010) by analogy with the processes taking place in the circulating fluids of warm-blooded animals in arthropods with built-in cellular structures, toxicants cannot occur.
 
Efficacy of certain acaricides on T. urticae strains
 
The toxicity bioassay data determined on adult females of T. urticae by the dipping application. Data revealed that, all the tested acaricides significantly affected of T. urticae strains. Bromopropylate was the most effective compared to the tested acaricides of malathion and  fenpyroximate strains,where the average lethality rate (%) of resistance and susceptibility malathion strains was, x=74.15±4.3 and x=92.5±2.4, respectively, with coefficient variation(%), n=5.8±0.35 and n=2.6±0.13, respectively (Table 3), while the average lethality rate(%) of resistance and susceptibility fenpyroximate strains was x=92.4±2.5 and x=100, respectively,  with coefficient variation (%), n=2.7±0.14 and n=0, respectively (Table 4). Abamectin was more active in fenpyroximate resistant strains than malathion resistant mites, (Table 3 and 4). Information primary molecular disorders caused by brompropylate missing, according to the results of these experiments, it can be concluded that these violations prevent the manifestation of the regulatory action of carboxylesterase isozymes on the permeability of the cell plasma membrane.
 

 

               
Both genes of resistance to the toxicants group OP and ÌΕÒÈ, stimulate the same complex biochemical processes, to ensure the viability of the cells. Results show that, similar values of mortality rate when cross-testing toxicants these chemical groups (Table 3 and 4). Toxicological experiments with the genotypes of T. urticae, showing signs of resistance to acaricides of these chemical classes. The immediate cause of death of arthropods by poisoning them with acaricides, acute toxic effects are a critical violation of the homeostasis of base cations Na+, K+, Ca2+ and Mg2+ in cellular and intracellular fluids (Sundukov, 2012). Pyrethroids compounds are reported to be upsetting the operation mechanism of sodium ion channels (Tsagkarakou et al., 2009). Avermectins are inhibitor receptors of neurohormonal mediators L-glutamate, histamine, serotonin, gamma-aminobutyric acid (Zhao and Salgado, 2010). Rai and Singh (2008) reported that the effectiveness of dicofol was reducing mite (T. urticae) population on okra.
 
Effect of synergistic Tributyl phosphate (butyphos) and piperonyl butoxide (PBO)  on lethality rate % of resistance strain T. urticae by malathion and fenpyroximate acaricides
 
Tributyl phosphate (butyphos) killed T. urticae females, with a lethality rate% (x=100, 34.9) in the ratio (1:1) and (1:0.01) (malathion: synergist) respectively, while the lethality rate% by diagnostic concentration was (x=15.8) in malathion resistance strain. Synergistic butyphos was more significant effect in fenpyroximate resistance strain than malathion, where the lethality rate% of fenpyroximate acaricide was (x=100, 88.9) in the ratio (1:1) and (1:0.01) (fenpyroximate: synergist) respectively, while by diagnostic concentration it was (x=41.3), (Fig 1).
 

       
PBO also, was killed T. urticae females, but with no significant effect in malathion resistance strain, where the lethality rate% of synergistic PBO with malathion was (x=25.8), while the lethality rate% by diagnostic concentration of malathion acaricide separately, was (x=21.48). In fenpyroximate resistance strain, synergistic PBO with fenpyroximate together was more significant effect on lethality rate% than effect of diagnostic concentration of fenpyroximate acaricide separately, where the lethality rate% of synergistic PBO with fenpyroximate was (x=100), while it was (x=23.33) by diagnostic concentration of fenpyroximate acaricide separately (Fig 2).
 

 
Results of enzymes inhibition assays are given in (Fig 1 and 2). Inhibition of monooxygenases andcarboxyles-terases by synergists PBO and butyphos was found. In the present study malathion and fenpyroximate resistance strains of T. urticae analyzed, showed a different response to synergists; the lethality rate% for each synergist with acaricide analyzed was more significant than effect acaricide separately. The use of synergists is an invaluable tool in acaricides resistance mechanism and detoxification pathway in mites and insects. The most important effects of synergists are inhibition enzyme (Valles et al., 1997), and alteration of cuticular penetration rate of insecticide (Gunning et al., 1995). PBO also inhibited carboxylesterases activity, but it did not inhibit completely, as found other authors (Young et al., 2005).  

The method of the disruptive selection of two-spotted spider mites by the presence or absence of the acaricide resistance feature allows to obtain guaranteed homogeneous genotypes in comparison with population samples. The obtained results of the comparative toxicity of acaricides of the chemical groups OP and METI for mites resistant to malathion and fenpyroximate strain indicate that the genes of resistance to these toxins normalize biochemical processes of the same system. This is a qualitative composition of the cytoplasm, dependent on the regulatory functions of the plasma membrane and the complex biochemical processes at the mitochondrial membrane, dependent on the composition of the cytoplasm. The results shown in this study indicate that, synergists butyphos and PBO were inhibited carboxylesterases and monooxygenases enzymes, although PBO did not inhibit monooxygenases completely.