Trichlorfon and spinosad resistance survey and preliminary determination of the resistance mechanism in Pakistani field strains of Bactrocera dorsalis

The use of insecticides has been a primary tool to manage Bactrocera dorsalis in Pakistan; however, recent reports of field control failures necessitate mapping out the insecticide resistance problem. Therefore, eight field strains from Pakistan, were evaluated for their resistance against trichlorfon and spinosad. Compared with a reference strain, six field strains showed high levels of resistance to trichlorfon, while two field strains expressed intermediate resistance. In case of spinosad, five field strains fell in the susceptible range, whereas, the rest of the strains represented minor resistance. Correlation analysis between LD50 values of trichlorfon and spinosad of all the field strains revealed non-significant association, suggesting the possibility of lack of cross-resistance between both insecticides. Synergism bioassays implementing S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) revealed that the LD50 values of trichlorfon in the presence of either DEF or PBO in seven field strains were significantly reduced. However, DEF and PBO had a non-significant effect on synergizing spinosad toxicity. The results revealed resistance to trichlorfon in field strains of B. dorsalis, which might be metabolic-based. Absence or minor resistance to spinosad and lack of cross-resistance to trichlorfon, suggest that spinosad could be a potential candidate for managing B. dorsalis.

Assessment of insecticide resistance is very important to devise an effective management strategy against insect pests 11 . Detection of insecticide resistance in B. dorsalis can help to adopt alternate measures to slow the spread of resistance. Therefore, keeping in view the economic importance of B. dorsalis and field control failures, a study was planned to assess the level of resistance to one of the most commonly used insecticide (trichlorfon) and the newly introduced insecticide (spinosad) for the management of B. dorsalis in Punjab, Pakistan. The data could be helpful to devise an effective management strategy for B. dorsalis.

Materials and Methods
Insects. Eight field strains of B. dorsalis collected from different localities and hosts (Table 1) were maintained under the laboratory conditions 25 ± 2 °C and 12 L: 12D photoperiod, to get the homogenous and sufficient number of insects in bioassays. The adults were maintained in mesh cages (40 × 30 × 30 cm) provided with maizebased artificial diet 4 and fresh fruits for egg laying. A laboratory reference strain (Ref.) was maintained in 2013 from a strain of insects inhabiting untreated guava trees at the University of the Punjab, Lahore. The Ref. strain was maintained in the laboratory as described above.
Bioassays. Assays were done following the methodology described by Hsu, et al. 1 . Working insecticide solutions, with a range of doses having >0% and <100% mortality, were prepared in acetone. One-microliter of the appropriate solution was applied onto the thoracic tergum of adult flies (3-5-day-old) using a micropipette (0.1-2 µL, Acura ® manual 825, Socorex, Switzerland). Flies in the control group received acetone only. The range of doses tested varied for different strains being tested. For spinosad, the range of doses was 0 to 32 ng/fly for the Ref. strain, and 0 to 96 ng/fly for field strains. For trichlorfon, doses used to test ranged from 0 to 48 ng/fly (Ref. strain) and 0 to 2460 ng/fly (field strains). Treated flies were shifted to perforated plastic jars (250 ml) containing a cotton swab (2 cm) moistened with a liquid food [sugar(4 parts):yeast(1 part):water(5 parts)] and kept under said laboratory conditions. For the synergism bioassays, PBO and DEF were applied onto the dorsal thorax of all the strains at the rate of 1 µg per fly for two hours before the insecticidal bioassays. Mortality data were recorded 48 h after the insecticide treatment.
Data analyses. Mortality scores were analyzed by Probit analysis using SPSS 16, to calculate median lethal dose (LD 50 ) values. Resistance ratios (RRs) were calculated by dividing LD 50 value of a field strain by the corresponding LD 50 value of the Ref. strain. Jin, et al. 12 criterion was used to classify resistance levels: susceptible (<3-fold RR), minor resistance (3-5-fold RR), low resistance (5-10-fold RR), intermediate resistance (10-40-fold RR), high resistance (40-160-fold RR), and extremely high resistance (RR >160-fold). Significant differences between any two (laboratory Ref. vs field strains) LD 50 values were determined by calculating the 95% fiducial limit (FL) of the RR values (SR) at the LD 50 level. If the 95% FL includes 1, the RR values are not significantly different. Synergism ratios were calculated by dividing the LD 50 value without synergist by the LD 50 value with synergist (PBO or DEF), and the significance of synergistic effects were assessed by calculating the 95% FL of the synergism ratio (SR) at the LD 50 level 13 , as stated above.
Ethical statement. The article deals with B. dorsalis which is an invertebrate. The study was conducted according to the standard guidelines and regulations. The study/bioassay protocol was approved by the research projects evaluation committee of the Institute of Agricultural Sciences, University of the Punjab, Lahore.  (Table 2). These values were used as baselines for evaluating resistance in different field strains.  Table 2). Spinosad showed the highest toxicity to the JG, BR, LR, RN and SA strains (all were at par based on overlapping 95% FLs) followed by the MN and KR strains, while the FD strain showed the least susceptibility to spinosad. Correlation analysis between LD 50 values of trichlorfon and spinosad of all the field strains revealed non-significant association (r = −0.43; p = 0.28), suggesting the possibility of lack of cross-resistance between both insecticides. Synergism experiment. The results of synergism bioassays implementing DEF and PBO are shown in the Table 3. The results revealed that the LD 50 values of trichlorfon in the presence of either DEF or PBO in all the field strains, except the SA strain, were significantly reduced (based on 95% FLs of SRs didn't include 1) when compared with the LD 50 values of their respective field strains without any synergist (Table 3). However, DEF and PBO had a non-significant effect on synergizing spinosad toxicity in all the strains of B. dorsalis (data not shown here).

Discussion
Synthetic insecticides have been excessively used for the management of B. dorsalis in Punjab, Pakistan; however, there is no report on the development of insecticide resistance from studied localities. In the present study, eight field strains of B. dorsalis were evaluated for their resistance against tichlorofon and spinosad. The results have confirmed the development of intermediate to high levels of resistance to trichlorfon, but fortunately the field strains were susceptible to spinosad since all of them exhibited less that 10-fold RRs which cannot warrant their status of being resistant 11,14 .
This study provides first information regarding resistance to trichlorfon and susceptibility status of spinosad in B. dorsalis from Pakistan. The most probable reason for the high level of resistance to trichlorfon could be due to the fact that the strains were collected from areas with intensive use of trichlorfon for the last many years, since this chemical has been recommended as an important tool for the management of tephritid flies in Punjab, Pakistan 2 . Trichlorfon has also been recommended for the management of tephritid flies in other countries with the reports of resistance development as a consequence of intensive exposures. For instance, a low level of trichlorfon resistance (RR = 10) has been observed in a field strain 15    field strains of B. dorsalis from south China have been reported with moderate levels of resistance to trichlorfon 6,7 . Some field strains of B. cucurbitae from Hainan Island, China, has also been reported to have minor resistance against trichlorfon 12 . Similarly, moderate to high levels of resistance to tricholrfon in B. zonata has been reported from some parts of the Punjab province, Pakistan 2,9 .
In the present study, five of the field strains of B. dorsalis were found susceptible to spinosad while three strains had minor resistance to spinosad. This might be due to the fact that spinosad has recently been included in management programs for tephritid flies. Previously, field strains of B. zonata from some parts of Punjab have also been reported susceptible to spinosad 2 . However, spinosad resistance in Bactrocera spp. has been reported from other countries, and it was linked with a wide spread application of spinosad in baits or cover sprays. For example, B. dorsalis from Taiwan 5 , B. cucurbitae from Hawaii & Taiwan 16 and China 12 , and B. oleae from the United States 17 , have been reported with varying levels of resistance to spinosad.
Synergism bioassays in the presence of enzyme inhibitors (PBO, DEF) were conducted in all the strains of B. dorsalis since such bioassays could help to assess the preliminary mechanisms of insecticide resistance 25 . DEF helps to identify if the mechanism of resistance is oxidase-and/or esterase-based, because it inhibits the activities of oxidases and esterases. Similarly, PBO also helps to identify the oxidase-based mechanism of resistance, since it is an inhibitor of mixed function oxidases 26 . The results of the present study revealed that resistance to trichlorfon in all the field strains, except the SA strain, could be due to the activities of oxidases and/or esterase since both enzyme inhibitors synergized the toxicity of trichlorfon. However, there was no effect of either PBO or DEF in synergizing the toxicity of trichlorfon in the susceptible reference strain. Similarly, there was no synergistic effect of both enzyme inhibitors on the toxicity of spinosad in all the strains of B. dorsalis, suggesting the possibility of resistance mechanisms other than metabolic-based. Previously, Hsu, et al. 1 has also reported the synergistic effect of DEF on the toxicity of trichlorfon in B. dorsalis. There was generally no synergistic effect of enzyme inhibitors on the toxicity of spinosad 20,[27][28][29] in other insect pests. However, there is a further need to assess the exact mechanism of resistance in the present strains of B. dorsalis by doing biochemical analyses and molecular techniques.
Ironically, intermediate to high levels of resistance to trichlorfon were observed in Pakistani strains of B. dorsalis. This resistance might be the result of over-reliance on trichlorfon in the chemical management plans for B. dorsalis in Punjab, Pakistan, and is most probably mediated by enhanced activities of detoxifying enzymes. Simultaneously, no or minor resistance to spinosad was observed in the present study with no cross-resistance to trichlorfon. This is encouraging and provides a window to use both insecticides in rotation along with other integrated management practices for B. dorsalis.  Table 3. Effect of enzyme inhibitors on toxicity of trichlorfon in laboratory and field strains of Bactrocera dorsalis. ns = non-significant. *Synergism ratio calculated by dividing the LD 50 of trichlorfon of a locality by the LD 50 of trichlorfon with synergist of the respective locality.