1 College of Agriculture, Liaocheng University, Liaocheng 252059, China
2 College of Plant Protection, Shandong Key Laboratory of Pesticide Toxicology and Application Technique, Shandong Agricultural University, Tai’an 271018, China
3 Institute of Zhenjiang Agricultural Sciences Jiangsu Hill Region, Jurong Jiangsu 212400, China
4 College of life science and technique, Taishan University, Tai’an 271000, China
Using microtitration method, the relationship between Phenoloxidase activity and the resistance of the diamondback moth Plutella xylostella (Linnaeus) to the novel insecticide butane-fipronil was determined in vitro. After selection of the tenth-generation by butane-fipronil, the resistance of the fourth instar larvae was increased 83.80-fold as compared to the susceptible strain. Phenoloxidase activity of the resistant strain (POr) was 1.29-fold higher than the susceptible one (POs). However, the Km and optimum pH values were similar in resistant and susceptible strains, which were 1.11 mM and 6.5, respectively. Both POr and POs have maximum stability at pH values less than 7.0, although POs was less stable at lower pH values than POr. In addition, the thermal stabilities of the two phenoloxidase were very similar. It is suggested that PO may play an important role in the increasing resistance of pests to pesticides.
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The diamondback moth, Plutella xylostella (Linnaeus), is one of the most widely distributed insects in the world and lives in more than 80 countries [1Harcourt, DG Biology of the diamondbackmoth, Plutella maculipennis (Curt) (Lepidoptera: Plutellidae) in eastern Ontario. I Distribution, economic history, synonomy and general description. 37th Report of the Quebec Society for the Protection of Plants, 1956, 155-160.]. The indiscriminate use of insecticides coupled with the ability of P. xylostella L. to produce multiple generations per annum and the high migratory potential of the species has led to its resistance to many insecticides [2Sayyed, AH; Wright, DJ Fipronil resistance in the diamondback moth (Lepidoptera: Plutellidae): inheritance and number of genes involved J. Econ. Entomol, 2004, 97, 2043-2050. [http://dx.doi.org/10.1603/0022-0493-97.6.2043] [PMID: 15666763] , 3Sayyed, AH; Omar, D; Wright, DJ Genetics of spinosad resistance in a multi-resistant field-selected population of Plutella xylostellaPest Manag. Sci, 2004, 60, 827-832. [http://dx.doi.org/10.1002/ps.869] [PMID: 15307676] ]. It has been shown that the resistance occurring included alterations to insecticide target sites and powerful metabolic resistance catalyzed by some enzyme such as mixed function oxidase, esterase or glutathione-s-transferases [4Denholm, I; Devine, G Insecticide resistance In: Encyclopedia of Biodiversity; Levin, SA, Ed.; Academic Press: San Diego, 2001; pp. 465-477. [http://dx.doi.org/10.1016/B0-12-226865-2/00158-9] ]. For example, the chlorfluazuro-resistant P. xylostella has higher glutathione S-transferase-3 (GST-3) gene expression and GST activity, suggesting that GST-3 is involved in chlorfluazuron detoxification in diamondback moths [5Sonoda, S; Tsumuki, H Studies on glutathione S-transferase gene involved in chlorfluazuron resistance of the diamondback moth, Plutella xylostellaL. (Lepidoptera: Yponomeutidae) Pestic. Biochem. Physiol, 2005, 82, 94-101. [http://dx.doi.org/10.1016/j.pestbp.2005.01.003] ]. While no differences in detoxication enzyme activities of permethrin-selected diamondback moths compared to non-selected populations have been found, the observed pyrethroid resistance is most likely due to decreased target site sensitivity [6Yu, SJ; N guyen, SN Insecticide susceptibility and detoxication enzyme activities in permethrin-selected diamondback moths Pestic. Biochem. Physiol, 1996, 56, 69-77. [http://dx.doi.org/10.1006/pest.1996.0060] ].
Fipronil is the first member of the phenylpyrazole group of insecticides, and is highly toxic to both piercing–sucking and chewing insects. In addition, it has shown excellent activity against a broad spectrum of insect orders [7Grant, DB; Chalmers, AE; Wolff, MA; Hoffman, HB; Bushey, DF Fipronil: action at the GABA receptor Rev. Toxicol, 1998, 2, 147-156.], with no obvious cross-resistance to other insecticides with different mechanisms of action [8Colliot, F; Kukorowski, KA; Hakins, DW; Roberts, DA Fipronil: A new soil and foliar broad spectrum insecticide Proc. Brighton Crop Protect. Conf-Pests Dis, 1992, 29, 34.]. Its resistance mechanism was demonstrated as involving low cuticle permeability, monooxygenase metabolism and increased activity of detoxification enzymes in Musca domestica, Plutella oxylostella,and Chilo suppressalis [9Wen, ZM; Scott, JG Genetic and biochemical mechanisms limiting fipronil toxicity in the LPR strain of house fly Musca domestica. Pest. Sci, 1999, 55, 988-992. [http://dx.doi.org/10.1002/(SICI)1096-9063(199910)55:10<988::AID-PS53>3.0.CO;2-E] -2Sayyed, AH; Wright, DJ Fipronil resistance in the diamondback moth (Lepidoptera: Plutellidae): inheritance and number of genes involved J. Econ. Entomol, 2004, 97, 2043-2050. [http://dx.doi.org/10.1603/0022-0493-97.6.2043] [PMID: 15666763] ]. Butene-fipronil is a novel compound obtained via the structural modification of phenylpyrazole. It has similar activity as fipronil in controlling pests on rice and vegetable crops, and has low toxicity to fish, which makes it an excellent pesticide [13Niu, HT; Luo, WC; Jiang, GQ; Zhu, XF Bioactivity of butene-fipronil and its field efficacy against diamondback moth, Plutella xylostella (L) Acta Phytophylacica Sin, 2007, 34, 316-320.].
Phenoloxidase (PO) (EC 188.8.131.52), also known as tyrosinase, is one of the key enzymes of the insect molting process, which is involved in hardening and stabilizing the newly secreted exoskeleton. Phenoloxidase catalyzes the oxidation of various phenolic compounds to the corresponding quinones, and then quinones combine with proteins to form the hard dark-colored layer of the cuticle [14Suderman, RJ; Dittmer, NT; Kanost, MR; Kramer, KJ Model reactions for insect cuticle sclerotization: Cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols Insect Biochem. Mol. Biol, 2006, 36, 353-365. [http://dx.doi.org/10.1016/j.ibmb.2006.01.012] [PMID: 16551549] ]. Phenoloxidases have been proposed to participate in cuticle tanning. Arakane et al. reported that phenoloxidases extracted from Tribolium castaneum are required for larval, pupal, and adult cuticle sclerotization and pigmentation [15Arakane, Y; Muthukrishnan, S; Beeman, RW; Kanost, MR; Kramer, KJ Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning Proc. Natl. Acad. Sci. USA, 2005, 102, 11337-11342.]. Phenoloxidase is also a key enzyme of insect immune response, and can be found in cuticular matrix [16Ashida, M; Brey, PT Role of the integument in insect defense pro-phenol oxidase cascade in the cuticular matrix Proc. Natl. Acad. Sci. USA, 1995, 92, 10698-10702. [http://dx.doi.org/10.1073/pnas.92.23.10698] [PMID: 11607587] [PMCID: PMC40679] ], hemocytes [17Riberio, C; Simòns, N; Brehélin, M Insect immunity: the haemo-cytes of the armyworm Mythimna unipuncta (Lepidoptera: Noctuidae) and their role in defence reactions.In vivo and in vitro studies J. Insect Physiol, 1996, 42, 815-822. [http://dx.doi.org/10.1016/0022-1910(96)00048-0] ], and hemolymph [18Hillyer, JF; Christensen, BM Mosquito phenoloxidase and defensin colocalize in melanization innate immune responses J. Histochem. Cytochem, 2005, 53, 689-698. [http://dx.doi.org/10.1369/jhc.4A6564.2005] [PMID: 15928318] ].
It is known that insecticide resistance is due to behavioral changes, physiological modifications, and metabolic detoxification [19Liu, NN; Zhu, F; Xu, Q; Pridgeon, JW; Gao, XW Behavioral change, physiological modification, and metabolic detoxification: mechanisms of insecticide resistance Acta Entomol. Sin, 2006, 49, 671-679.]. Insect cuticle penetration is involved in physiological modifications, and some insecticides must pass through the insect cuticle in order to reach the site of action before lethal effects can occur. Some reports indicated that decreased penetration confers 2-3 fold resistance by itself [20Plapp, FJ; Hoyer, RF Insecticide resistance in the housefly: decreased rate of absorption as the mechanism of action of a gene that act as an intensifier of resistance J. Econ. Entomol, 1968, 61, 1298-1303. [PMID: 5680754] , 21Sawicki, R Interaction between the factor delaying penetration of insecticides and the demethylation mechanisms of resistance to organophosphorus-resistant houseflies Pestic. Sci, 1970, 1, 84-87. [http://dx.doi.org/10.1002/ps.2780010302] ]. There have been no reports regarding phenoloxidase (PO) activity in insecticide resistance, although PO is involved in hardening and stabilizing the newly secreted exoskeleton. In fact, more attention must be paid to this enzyme when studying insecticide resistance, because PO is one of the key enzymes of the insect molting process. In addition, PO influences the cuticle structure to allow insecticide penetration.
In the present paper, PO activity and the properties of the resistant and the susceptible diamondback moth P. xylostella L. are investigated, the relationship between PO and pest resistance are also discussed. The results of this study may lead to a better understanding of the mechanism of pest resistance.
MATERIALS AND METHODOLOGY
Chemicals and Instruments
L-Dihydroxyphenylalanine (L-DOPA) was purchased from Sigma (USA), and butane-fipronil (97%) was supplied by Dalian Raiser Pesticide Co. LTD (China). All other reagents were local products of analytical grade. The 96-well microtiter plate reader used was a Thermo Mustiskan Mk3 (Thermo electron corporation).
The susceptive diamondback moth strain was reared on Chinese cabbage Brassica parachinesi (Bailey) in a greenhouse at 25±1 °C with a 14:10 h light:dark cycle, and had never been exposed to insecticides [22Wang, SD; Luo, WC; Xu, SJ; Ding, Q Inhibitory effects of 4-dodecylresorcinol on the phenoloxidase of the diamondback moth enzymatic properties of Plutella xylostella (L) (Lepidoptera plutellidae) Pestic. Biochem. Physiol, 2005, 82, 52-58. [http://dx.doi.org/10.1016/j.pestbp.2004.12.005] ]. Half of the susceptive strain population was selected for resistance to butane-fipronil for 10 generations to make the resistant strain, using dipping methods described by Ismail F. [23Ismail, F; Wright, DJ Cross-resistance between acylurea insect grossth regulators in a strain of Plutella xylostella (L)(Lepidoptera: Yponomeutidae) from Malaysia Pestic. Sci, 1991, 33, 359-370. [http://dx.doi.org/10.1002/ps.2780330309] ]. Briefly, butane-fipronil was dissolved in acetone and five serially diluted concentrations were prepared with distilled water containing 0.05% triton X-100. Cabbage leaves (4 cm × 4 cm) were cut and dipped in the serially diluted solution for 10 s. Three-day-old third instar larvae of parental generations were allowed to feed on the butane-fipronil treated leaf for 48 h, followed by a fresh leaf until it pupaed.
Resistant Selection Bioassay
Technical grade butane-fipronil was dissolved in acetone and five serially experimental concentrations were prepared after protest to larvae of diamonback moth. Then fourth-instar larvae (2-3 mg/larva) were treated with 0.3 μL of the dilution on the mesothorax notum by a hand microapplicator to determine the median lethal concentration (LC50). Each test was repeated six times and acetone treatment was used as a control [24FAO. Method for diamond back moth (Plutella xylostella L.) FAO Plant Prot, 1979, 27, 44-46.].
Preparation and Purification of PO
Fourth instar larvae (about 4 g) of the two strains were homogenized using a blender in ice-cold extraction buffer [20 mL, 0.02 M phosphate buffer solution (PBS), pH 6.5]. The homogenates were centrifuged at 6 770 g for 30 min. The supernatant was collected and brought to 40% saturation with solid ammonium sulfate. The precipitate was collected by centrifugation at 6 770 g for 30 min, redissolved in a minimum volume of the same buffer, and dialyzed against 20 mM PBS (pH 6.5) for 40 h. The partially purified enzyme was used in the present investigation.
Total protein concentration was determined by Coomassie blue G-250 dye-binding using bovine serum albumin as the standard [25Bradfold, M.M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding Anal. Biochem, 1976, 72, 248-254. [http://dx.doi.org/10.1016/0003-2697(76)90527-3] ].
Phenoloxidase activity was assayed by determining the initial linear increase in absorbance at 490 nm using a Thermo Mustiskan Mk3 microtiter plate reader at room temperature, using L-dihydroxyphenylalanine (L-DOPA) as the substrate. The reading was taken instantly after enzyme addition.
Assay of Optimal pH and Stability of PO Under Different pH Conditions
The optimum pH and the pH stability were determined according to Xue et al. [26Xue, CB; Luo, WC; Chen, QX; Wang, Q; Ke, LN Enzymatic properties of phenoloxidase from Pieris rapae (Lepidoptera) larvae Insect Sci, 2006, 13, 251-256. [http://dx.doi.org/10.1111/j.1744-7917.2006.00091.x] ]. Briefly, to determine the optimum pH, 140 µL of different buffers (pH range: 4.4-9.0), and 40 µL of 10 mM L-DOPA were added to a microtiter plate well. Next, 20 µL of enzyme (103.55mg/mL, as same below) was added to each reaction mixture, and the plate was read immediately to record the absorbance at 490 nm at room temperature.
To determine the pH stability of PO, 20 µL of enzyme was added to 180 µL of reaction mixture (140 µL of buffer, 40 µL of 10 mM L-DOPA) under different pH conditions to assay the PO activity (as full activity). Another 20 µL of enzyme was added to 140 µL of different buffers, and incubated for 30 min at 4 °C before 40 µL of 10 mM L-DOPA was added and the remaining PO activity was assayed immediately at room temperature. The specific value of the remaining activity to full activity was used to evaluate PO stability.
Assay of PO Thermal Stability
To test the thermal stability of PO, 20 µL of enzyme was incubated at different temperatures (25°C, 30°C, 35°C, 40°C, 50°C, 60°C, 80°C) for 5 min and 15 min, and then added to 180 µL of reaction mixture (containing 40 µL of 10 mM L-DOPA, and 140 µL of PBS, pH 6.5) to assay the PO activity immediately. The thermal stability was evaluated by the ratio of remaining PO activity.
The responses of the fourth instar larvae of P. xylostella to butene-fipronil in different generations of the susceptible and resistant strains after selection are presented in Fig. (1). The resistance of the fourth instar larvae of Plutella xylotella (L.) diamondback mothsto butene-fipronil increased 83.80-fold in comparison with the susceptible strain until the election for the tenth generation. The resistance ratio of the parental generation increased 78.02-fold after nine generations of selection. In the experimens the eighth generation was not selected by butene-fipronil. Results showed that at the first three generations and the last three generations, the resistance increased slowly.
The resistance curve of diamondback moths to butene-fipronil in various generations.
Determination of Kinetic Parameters of PO
Under the conditions employed in the present study, the oxidization of L-DOPA by P. xylostella PO followed Michaelis-Menten kinetics. The kinetic parameters for the enzyme were determined using a Lineweaver-Burk plot, with the results shown in Fig. (2). The Km and Vmax values for PO from the butane-fipronil resistant strain (POr) were 1.108 mM and 153.84 µmol/L·min-1·mg-1 protein, respectively, while the Km and Vmax values for the PO from the susceptible strain (POs) were 1.114 mM and 67.114 µmol/L·min-1·mg-1 protein, respectively.
Lineweaver-Burk plot for the determination of Km and Vmax for phenoloxidase oxidation of L-DOPA at room temperature. Assay conditions were 20 mM PBS (pH 6.5), containing 0.3, 0.6, 1.2, 2.4, 4.8 mM L-DOPA, and 20 µL of enzyme, total volume was 200 µL. ■: Lineweaver-Burk plot of phenoloxidase from the susceptible strain. □: Lineweaver-Burk plot of phenoloxidase from the resistant strain.
Effect of pH on Enzyme Activity and pH Stability
The optimum pH for PO was determined to be about 6.5 (Fig. 3). The enzyme activity decreased rapidly when the pH was higher or lower than 6.5. The optimum pH of POr was the same as POs. Interestingly, when POs was added to the pH 4.4 and pH 5.3 buffers, some floccules appeared; but only in pH 4.4 buffer did POr turn into floccules.
Optimum pH for phenoloxidase activity.
After the enzyme was incubated in different pH buffers, the remaining activity was assayed, and the specific value of remaining activity to full activity was calculated (Fig. 4). The maximum stability of POr and POs was at pH 7.0 (the specific values were 0.94 and 0.88, respectively), with no obvious differences between them. As much of the PO became insoluble, the PO activity (from susceptible larvae) at pH 4.4 and pH 5.3, and the PO activity from resistant larvae at pH 4.4 were not determined.
pH stability of phenoloxidase.
Thermal Stability of PO
After the enzyme was incubated at different temperatures (25°C, 30°C, 35°C, 40°C, 50°C, 60°C, 80°C) for 5 min and 15 min, the remaining PO activity was assayed. The results showed that both POr and POs are relatively stable in the range from 25 °C to 40 °C. However, when the temperature was going greater than 40 °C the enzyme activity decreased rapidly (Fig. 5). When the temperature reached 80 °C the enzyme was completely denatured and no enzyme activity was detected (data not shown).
Stability of phenoloxidase at different temperatures. The ratio of remaining PO activity after a 15 min incubation to PO activity after a 5 min incubation was calculated.
DISCUSSION AND CONCLUSION
Using microtitration, the PO activity of P. xylostella selected for resistance to butane-fipronil was investigated. The resistant strain was found to have significantly higher PO activity than the susceptible strain, but the difference in PO activity between the two strains could not be explained by variations in total protein content (POr:POs was 1.5:1 for a 20 µL enzyme sample). This result is similar to data obtained with Sydney rock oysters Saccostrea glomerata [27Newton, K; Peters, R; Raftos, D Phenoloxidase and QX disease resistance in Sydney rock oysters (Saccostrea glomerata) Dev. Comp. Immunol, 2004, 28, 565-569. [http://dx.doi.org/10.1016/j.dci.2003.10.004] [PMID: 15177110] ] in which resistance was selected for QX disease for three generations, and its PO activity was found to be higher than in the non-selected populations. In addition, a novel form of PO protein in selected oysters was detected by native PAGE. In the present paper, POr and POs had the same Km values, indicating that they have the same catalysis ability but whether they are the same proteins is still unknown.
Armitage and Siva-Jothy [28Armitage, S.; Siva-Jothy, M.T Immune function responds to selection for cuticular colour in Tenebrio molitorHeredity, 2005, 94, 650-656. [http://dx.doi.org/10.1038/sj.hdy.6800675] [PMID: 15815710] ] reported that Tenebrio molitor beetle adults with darker cuticular color have higher PO activity. Also, the black larvae of Bombyx mori silkworms have higher PO activities than white larvae [29Xu, HR; Ling, EJ Determination of phenoloxidase activity from fifth instar larvae of Bombyx moriNewslett. Sericult. Tea, 1997, 90, 17-19.]. In the present study, the insect P. xylostella was selected for ten generations, and the cuticles of larvae were darker than in the non-selected larvae. We deduced that the darkened cuticle has more PO content which can catalyze more phenolic compounds to quinones, and the melanin content is also increased during this time. This is beneficial for defense against the penetration of butane-fipronil or infection of microorganisms.
On the other hand, insects have an unusual innate immune system and PO is the key enzyme responsible for melanization [30Hoffmann, J.A Innate immunity of insects Curr. Opin. Immunol, 1995, 7, 4-10. [http://dx.doi.org/10.1016/0952-7915(95)80022-0] ]. For instance, microorganism-infected locusts can form many black nodules to eliminate invaders [31Mullen, L.M; Goldsworthy, G.J Immune responses of locusts to challenge with the pathogenic fungus metarhizium or high doses of laminarin J. Insect Physiol, 2006, 52, 389-398. [http://dx.doi.org/10.1016/j.jinsphys.2005.10.008] [PMID: 16413931] ]. Undoubtedly, PO plays a major role in the innate response of insects towards infection. Some chemicals, such as the pesticide monocrotophos, can cause the PO activity to increase in the hemolymph of Penaeus chinensis [32Bai, J.; Li, Y.Q.; Li, K.R Study on the effect of monocrotophos on activity of PO in haemolymph of Penaeus chinensis Marine Sci, 1998, 22, 35-37.]; chlorfluazuron can also increase the PO activity in the cuticle of Ostrinia furnacalis [33Wu, G; Shang, Z.Z Effect of chlorfluazuron on the phenoloxidase and chitinase activity in Ostrinia furnacalisActa Entomol. Sin, 1992, 35, 306-310.]. In our previously work [34Liu, S.Z; Xiao, T; Xue, C.B.; Luo, W.C Physiological effect of quercetin on phenoloxidase from Tenebrio molitorActa Entomol. Sin, 2007, 50(12), 1201-1206.], the physiological effect of quercetin, an inhibitor of PO, was determined in vivo on Tenebrio molitor, results suggested that quercetin at low concentration can cause immune response to the tested insect. The response of PO to chemicals suggests that PO may participate in the detoxification of insects by immune response. With more PO content, insects will easily survive from all kinds of invaders, including pesticides. The fact that butane-fipronil selected P. xylostella has higher PO activity supports this view.
So, the contribution of PO to the resistance of P. xylostella to butane-fipronil can be attributed to two aspects: lower cuticle penetration and higher immune ability (Fig. 6).
Two pathways of phenoloxidase contributed to P. xylostella resistance to butane-fipronil.
Although the enzyme activity of POr was higher than POs they have similar Km and optimum pH values or thermal stabilities. The only notable difference was that POr was stable under pH 5.3 conditions, while POs were unstable under the same conditions. Maybe this phenomenon is due to different protein content in the reaction mixtures.
It was be concluded that the resistance-selected diamondback moths P. xylostella have higher PO activity than the susceptible ones. Consequently, PO may play an important role in the increasing resistance of pests to pesticides, the detailed mechanism of which needs further study.
This work was supported by a Grant from the National Natural Science Foundation of China (NO. 30571237 to Wanchun Luo) and the Expert National ministry of education Foundation of China (NO. 20070434006 to Wan Chun Luo).
Harcourt, DG Biology of the diamondbackmoth, Plutella maculipennis (Curt) (Lepidoptera: Plutellidae) in eastern Ontario. I Distribution, economic history, synonomy and general description. 37th Report of the Quebec Society for the Protection of Plants, 1956, 155-160.
Sayyed, AH; Omar, D; Wright, DJ Genetics of spinosad resistance in a multi-resistant field-selected population of Plutella xylostellaPest Manag. Sci, 2004, 60, 827-832. [http://dx.doi.org/10.1002/ps.869] [PMID: 15307676]
Mohan, M; Gujar, GT Local variation in susceptibility of thediamonback moth Plutella oxylostella Linnaeus to insecticides and role of detoxification enzymes Crop Prot, 2003, 22, 495-504. [http://dx.doi.org/10.1016/S0261-2194(02)00201-6]
Riberio, C; Simòns, N; Brehélin, M Insect immunity: the haemo-cytes of the armyworm Mythimna unipuncta (Lepidoptera: Noctuidae) and their role in defence reactions.In vivo and in vitro studies J. Insect Physiol, 1996, 42, 815-822. [http://dx.doi.org/10.1016/0022-1910(96)00048-0]
Liu, NN; Zhu, F; Xu, Q; Pridgeon, JW; Gao, XW Behavioral change, physiological modification, and metabolic detoxification: mechanisms of insecticide resistance Acta Entomol. Sin, 2006, 49, 671-679.
Plapp, FJ; Hoyer, RF Insecticide resistance in the housefly: decreased rate of absorption as the mechanism of action of a gene that act as an intensifier of resistance J. Econ. Entomol, 1968, 61, 1298-1303. [PMID: 5680754]
Sawicki, R Interaction between the factor delaying penetration of insecticides and the demethylation mechanisms of resistance to organophosphorus-resistant houseflies Pestic. Sci, 1970, 1, 84-87. [http://dx.doi.org/10.1002/ps.2780010302]
Wang, SD; Luo, WC; Xu, SJ; Ding, Q Inhibitory effects of 4-dodecylresorcinol on the phenoloxidase of the diamondback moth enzymatic properties of Plutella xylostella (L) (Lepidoptera plutellidae) Pestic. Biochem. Physiol, 2005, 82, 52-58. [http://dx.doi.org/10.1016/j.pestbp.2004.12.005]
Ismail, F; Wright, DJ Cross-resistance between acylurea insect grossth regulators in a strain of Plutella xylostella (L)(Lepidoptera: Yponomeutidae) from Malaysia Pestic. Sci, 1991, 33, 359-370. [http://dx.doi.org/10.1002/ps.2780330309]
FAO. Method for diamond back moth (Plutella xylostella L.) FAO Plant Prot, 1979, 27, 44-46.