1661 . 17 1 − b Asad Ali, b Adil Hussain, d Correspondenceto:HZhang,StateKeyLaboratoryofAgriculturalMicrobiology, Hubei Key Laboratory ofControl and Institute of Resource Urban and ApplicationScience Horticultural and Entomology, and College Technology, Sustainable of Plant Huazhong Hubei, Agricultural China. E-mail: University, [email protected] Wuhan 430070, State Key Laboratory ofInsect Agricultural Resource Microbiology, Hubei Application KeyUrban and and Laboratory Horticultural Entomology, Sustainable College of of Pest Plant ScienceHuazhong and Control Agricultural Technology, University, Wuhan, and Hubei, China Institute of Department of Agriculture,Pakhtunkhwa, Pakistan Abdul Wail Khan UniversityMolecular Biotechnology Mardan, Laboratory, College of Khyber Plant ScienceHuazhong and Agricultural Technology, University, Wuhan, Hubei, China USDA, Agricultural Research Service, SanCenter, Joaquin Parlier, CA, Valley USA Agricultural Sciences Flonicamid, a pyridine carboxamide, is a novel insecticide that is ∗ c a d b the first line of defence.chemical Currently, insecticides are several being conventional used and to control new this potential pest. in the selective -feeding blocker group (modegroup of action 9C; classificationdal by mechanism IRAC, appears irac-online.org). to The be insectici- starvation caused by irreversible A. , is one of the most destructive pests of cotton in Asia. This species Bacil- Cotton (Ishida) 2 China is one a* (Ishida), is by disruption 1 Farmers adopt Elaine A Backus, 9 c – 6 , 3 biguttula ; electrical penetration graph; electropenetrography; feeding behaviour of which insecticides are The introduction of Amrascabiguttula 3 5 Mah Noor, A. biguttula, a,b Piercing-sucking cause 40–50% of : 1661–1669 www.soci.org © 2016 Society of Chemical Industry cause damage to the crop by sucking cell sap 4 ) cotton reduced the problem of bollworms, 73 spp.) is a major commercial crop, designated as and Hongyu Zhang Bt ( a 2017; flonicamid; can reduce cotton yield by 24.45%. A. biguttula

Gossypium

The cotton or cotton jassid, (Hemiptera: Cicadellidae), isdestructive one pests of of theincluding cotton most China. The leafhopper in has multiple subcontinental hostsand such South-east as jute cotton Asia, andsuch several as potato, other , and plants brinjaladults (eggplant). from of Nymphs and the familyand Solanaceae injecting disruptive salivabiguttula into tissues. A severe attack of lus thuringiensis but unfortunately the population of piercing-suckinggradually insect pests increased. Pest Manag Sci the ‘king of fibres’, that has global importancefibre in and the seed, production of raw material for the textile industry. 1 INTRODUCTION Cotton ( of ingestion: an electropenetrography study Abstract BACKGROUND: The cotton leafhopper, is thought to causeresulting damage macerate. Flonicamid by is injecting a novel enzymatic systemic insecticideis saliva used unknown. to into control the various, cotton presently leafhopper; however, unknown, its mode cotton of action tissues andRESULTS: The ingesting mechanism of the action of flonicamidrecordings on cotton revealed leafhopper six was investigated waveforms, using i.e. electropenetrographyprobably (EPG). Np represent EPG (non-probing), active ingestion A1 with (channel (A2)are cutting), and presently without A2, unknown, (A3) but A3, simultaneous minor watery A4, in salivation. duration. A5strongly The Flonicamid inhibited and significantly meanings ingestion increased of by A6. the A4, treated mean Waveforms insects, A5 duration A2 which and ofdependent, resulted and A6 non-probing in and events the A3 near-complete and slow suppression death was of observed . Inhibition when of the ingestion flonicamid was concentration dose was increased to 10 000 mg L Kaleem Tariq, Wei Peng Amrasca biguttula (wileyonlinelibrary.com) DOI 10.1002/ps.4508 The toxicity of flonicamid to cotton leafhopper, Research Article Received: 29 July 2016 Revised: 26 October 2016 Accepted article published: 19 December 2016 Published online in Wiley Online Library: 13 February 20 of the largest cotton-producing countries inalmost the world 20% and meets of the annual worldwide cotton demand. plays a pivotal role intries. the Insect economic pests and development crop of diseases are many majorin yield-limiting coun- almost factors all cotton-growing countries. the damage to the cotton crop. various strategies to control Keywords: Supporting information may be found in the online version of this article. CONCLUSIONS: We propose that starvation caused byThis knowledge inhibition could of aid active in ingestion applicability is© and the 2016 use mechanism Society of of of this toxicity Chemical new for Industry flonicamid. insecticide for field management of leafhopper populations. . A et al. )and input 1 − Ω 9 : 1661–1669 73 were confined for the related on cotton was compared with 22 2017; feeding behaviour et al. biguttula were removed from the A. biguttula . A. biguttula A 4 ft, aluminium frame with a × 2 A. biguttula Pest Manag Sci × A. biguttula C and 60–65% RH under continuous light for ∘ 2 m diameter) with silver conductive glue (Pelco μ ± on insecticide-treated and control leaves were were chilled on ice for 2–3 min. The immobilised insects Briefly, ten third-instar nymphs of biguttula 19 . A Third-instar nymphs of Insect starvation treatment was given as described by Yueping Voltage fluctuations representing feeding behaviours of nymphs water-treated control plants. EPGrecorded of using one Giga-4 DC EPG amplifier with a 10 petiole was cutwere to dipped into a freshly prepared length insecticide solutionshung of for vertically 5 approximately s for and 5 cm. 5–10petiole min The was in passed leaves through the a opencup small bottom. shade hole The to on outer cup the dry. containing sideprevent The water overdrying of was leaf and the placed to inner inside keep the to leaf in an upright position. culture with a fine camel hair brush, andinto ten the nymphs test were released cup. The cup was coveredof with 50 a perforated nymphs lid. (five A total cups)control were treatment tested contained a for cotton each leaf dipped concentration. inEach The distilled treatment water. was replicated 3cups times, were including maintained in the laboratory conditions control. as The described above, and mortality was assessed daily. et al. 8 h using PROBE 3.4 software (Wageningen AgriculturalWageningen, University, The Netherlands) operatingfreshly treated in cotton plants Windows-XP. and New, insectscation. were A used total for of each ten repli- 8centration h of EPG flonicamid, readings which were were recorded used for for each final data con- analysis. 2.5 Statistical analyses Six primary types ofprobing waveform cotton produced leaves by were cotton determinedtheir (Figs leafhoppers similarities 1A to to those D), based described on by Jin inside a plastic cup containing aleaf). water-saturated cotton A swab total (no oftreatment. 50 nymphs The (five experiment cups) wasof were the tested repeated for data 3 starvation on times,bioassay mortality experiment and were was conducted recorded the for as 7 rest days. described above. The 2.4 Electropenetrography of The objective of this studycamid was on to the determine probing the effects behaviour of of floni- resistance and less thanWageningen, The 1 Netherlands). pA Treatments input weretions five bias of flonicamid concentra- current (20, 200, (EPG 1000, 5000 Systems, and 10 000 mg AI L distilled water as the control. Theseedlings leaves were of treated 40–50-day-old by dipping cotton inof the flonicamid. After different being concentrations starved for 2 h, third-instar nymphs of steel base) at 30 colloidal silver paint; Ted Pella, Inc., Redding, CA).trode The ground was elec- placed intoplants the to provide soil the voltage, near and the theelectrical insect was circuit stems connected by of to gluing the the treated golddiameter) cotton wire with to conducting a silver copper paint, wireto (0.5 which a mm brass was nail then inserted into soldered thefier. input The lead insect of attached the to head the stage gold ampli- on wire the was leaf then of carefully the placed treated cotton plant. of recorded onto a computer hard diskconversion through for an further analog-to-digital analysis. To reducenal interference electrical from noise, exter- the entire experimental set-upinside was a performed large Faraday cage (2 biguttula were individually connected, via the thoracic notum, to(2 a cm gold wire length, 18 A. www.soci.org K Tariq Among 14 Flonicamid A. biguttula 11 reported that © 2016 Society of Chemical Industry 13 and may provide 18 et al. A. biguttula Ghelani 12 Flonicamid inhibits the feeding A. biguttula 10 used in this experiment were collected and was statistically as effective as dinote- C with a 14:10 h light:dark photoperiod and In EPG, an insect and feeding substrate are ∘ a leaf dip bioassay was conducted with 4 17 – ± 21 . To minimise any chemical decomposition, the , 1 15 − has not yet been examined. This study characterised but the effect of flonicamid on the feeding behaviour A. biguttula et al. A. biguttula 20 , In the bioassay, two plastic cups were used, one as the 19 , 15 A. biguttula Several previous studies have been conducted to evaluate the Although laboratory and field experiments provide useful infor- the effects of flonicamid onon the cotton feeding plants behaviour treated of with theof systemic insecticide. this The research results willtoxicity help mechanism of the flonicamid scientific to community interpret the 2.2 Insecticide The insecticide flonicamid 96% TC (Hangzhou Tialongogy Biotechnol- Co., Ltd, Hangzhou,to China) make was a diluted series10 with 000 mg of distilled AI concentrations: water L 0, 20, 200, 1000, 5000 and 60–65% RH. TheZhong 206) 25–30-day-old were infested with potted insectsconfined from on cotton the plants field. in Insects cages; plants the were leafhoppers (variety weregenerations. cultured for four from a cotton field attures Huazhong were Agricultural University. maintained Theinsecticides in cul- at 30 the laboratory without exposure to of valuable information for field application of flonicamid. effects of insecticides oninsects, the feeding behaviour of sap-sucking biguttula. inner test chamber andFresh, the uncontaminated other cotton ascotton leaves the field were outer and were water collected cleaned reservoir. from with the a wet cotton swab. The leaf wileyonlinelibrary.com/journal/ps 22.1 MATERIALS AND METHODS Insects Colonies of inhibition of feeding activity. 2.3 Bioassay methods with solutions were used immediately after preparation. As developed and recommendedAction Committee by (IRAC method the No. Insecticide 8),by and Resistance previously Preetha described incorporated into an electricalwhen circuit. the insect Changes begins to in feed and voltagetuations completes the occur in circuit. biopotentials The fluc- (voltage insideproduce the plant) waveforms and resistance thatof correspond the to insect, the suchingestion as feeding and cell stylet behaviour location puncturing, within stylet plant insertion, tissue. salivation, activity of insects immediately afterinsecticide treatment against and sap-sucking is insects an such effective leafhoppers as (jassids), whiteflies, aphids, thrips, scales and mealybugs. furan and imidacloprid. mation on mortality causedcomprehensive by the information application on offeeding an how insecticide, behaviour an of insecticide insects requires affects other the methods. the application of flonicamidmortality in of cotton fields caused significant inhibited the feeding activity ofwithout aphids showing within symptoms of 0.5 h neuronal intoxication, of andactivity treatment, feeding was inhibited until death. those methods, electropenetrography (EPG) ismethod the for most examining rigorous theing effects behaviour of of piercing-sucking an insect pests insecticide andto on on the plant the damage tissues. feed-

1662 1663 ; -axis y (B) (F) (D) values were 50 biguttula . The least significant A . wileyonlinelibrary.com/journal/ps -axis indicates time in seconds. Note that the x probed on cotton leaves, six different types of biguttula . A EPG recording data were analysed with SAS v.9.2 for Windows A3, after being treated with different concentrationsand of water flonicamid (CK), were also calculated. (SAS Institute, Cary, NC)difference statistical (LSD) software test was usedEPG variables to between control evaluate (CK) and the treatments differencescentrations at various of in con- flonicamid. the Regression analyses weredetermine conducted to the relationship betweentrations EPG of variables flonicamid (log and transformed).dose–response concen- The bioassays mortality and the data calculations from of LC 3.1 Characterisationnymphs of feeding waveforms on from cotton leaves When waveform (Fig. 1 and Table 1)characterisation were of identified identified waveforms in is the as EPG follows. out. The 3.1.1 Np When insects were notflat probing, baseline the (0 recording V always reference showed level) a (Fig. 1A). The output voltage of 3RESULTS determined using PoloPlus software (PoloPlus: probit and logit). Time (s) 24 22 (C) , (E) (A) et al. on cotton leaves. (A) non-probing (Np) and waveform A1 (including subtypes) early in et al. A. biguttula Biological meanings of these : 1661–1669 © 2016 Society of Chemical Industry vitis. 73 -axis indicates the amplitude (voltage) at standardised gain for all examples. The The number of events for each waveform was the Np y 23 2017;

et al.

Characteristic EPG waveforms recorded from Voltage (V) Voltage indicates that waveforms have very different amplitudes despite being portrayed as similar heights. number of times that a waveformtime. The appeared percentage during of each the waveform recording was calculatedthe by dividing total duration of each waveformthe total over recording the time. whole The total recording waveform by durationwaveform for each was EPG calculated by summingform the events durations of of all all wave- time. insects The average duration that of each occurred EPG waveform duringmean represented the duration the of observation each waveform perof insect. EPG Sequential waveforms variables were also analysed similarly to Sarria Pest Manag Sci (E) A5; (F) A6. The including the following: the duration ofstart non-probing of (A) the from EPG the recording to thethe first first probe, and second the probes duration of between A1, thefirst number probe of containing probes A3, before the the duration of the firstA3 probe and containing the interval from theevent. start Furthermore, of the the percentages first of probe to the the insects first that A3 reached waveforms were hypothesised, again on the basis of Jin Each EPG waveform wasvariables: summarised the using number four of non-sequential tion events and and the average percentage, durationrecording total time. of dura- These each variables were waveform calculatedin during similarly Backus to the those entire leafhopper species Figure 1. a probe (only a portion of A1 is shown; A1-C continued after this excerpt); (B) A2, performed after A1; (C) A3 after A2; (D) A4, interspersed with A2 and A3 Flonicamid disrupts ingestion of cotton leafhopper www.soci.org et al. : 1661–1669 73 Electrical origin feeding behaviour, 2017; was characterised by was also a highly regular b biguttula . A biguttula . was always preceded by A1 and Pest Manag Sci A biguttula . A A. biguttula Repetition rate Voltage level waveform (Fig. 1C) andevent; always the appeared amplitude after ofin an A3 depth was A2 much compared waveform higher withA3 with A2 was less waveform. 8–10 variation The Hz.at frequency A3 times was range consistently varying of of inover the time amplitude. into same higher amplitude Usually, shape, and A3 except lower frequencymean (Fig. would 1C). voltage The slowly level evolve ofhigher A3 than was all always the positive otherappeared (extracellular) frequently detected and and lasted waveforms. more briefly The than A3 A2. Theorigin waveform electrical of A3 was emf. 3.1.5 A4 The A4 waveform(Fig. 1D). was A4 appeared regular occasionallythe and and A1 usually waveform. A was complex rapid preceded rate by amplitude of with repetition (0.5–0.7 (4–5 tall V) Hz) with spikes was aevent, higher although recorded not at at the theover end. time beginning This with waveform of a evolved lower gradually an amplitudespike A4 (0.2–0.3 repetition V) (0.5–1 and Hz). afollowed slower The A4. rate Like A1 of the and/or A3 waveform, A5emf the (Table electrical waveform 1). origin usually of A4 was 3.1.6 A5 The A5 waveform produced by mostly followed by A3. However, A2 appearing alone, withoutsequent sub- A3, was also observed. Thealways mean positive (extracellular), voltage similarly level to of waveform A2 A1.ally, was Addition- when the applied substratetive voltage to was negative, changed no from change posi- inwas seen. characteristics This of result the demonstrates A2 thatwas waveform electromotive the force main (emf) component of the A2 waveform (Table 1). 3.1.4 A3 Waveform A3 produced by regular intervals, producing a ‘ruffle’.narrow Consequently, A2 spectral showed band a of frequencyThe A2 ranging waveform between of 5 and 6 Hz. a high-amplitude sequence of(Fig. 1E). peaks A with higher negative frequencyning (0.8–1.4 of deflections Hz) this occurred waveform,over at time. with the During a the begin- recording tendency of to decline (0.4–0.6 Hz) a nymphs feeding on cotton leaves during ten recordings of 8 h each www.soci.org K Tariq biguttula Low 0 0 . © 2016 Society of Chemical Industry was composed recordings was A biguttula biguttula . . A A A1-B 30–60 6–12 e R A1-C 100 Mixed e R A1-ns Unknown emf Electrical characteristics of waveforms from non-probing. extracellular voltage level. = = c e Voltage deflection (minimum to maximum) is a percentage of the maximum waveform amplitude. Np All the subtypes of the A1 waveform were extracellular (above A2A3A4A5A6 20–60 80–100 3–20 5–18 4–30 5–6 8–10 4–5 0.4–1.4 1–6 e e e e e emf emf emf emf emf A1 A1-A 20–40 Mixed e R c a b EPG waveformNp Subtypes Relative amplitude Table 1. wileyonlinelibrary.com/journal/ps 3.1.2 A1 The voltage level changed suddenly to a positive valueupon (depending the applied voltageinserted. Stylet to penetration the always started electrode)(Fig. with 1B). as The the A1 waveform A1 the produced by waveform stylets were all the detected waveforms was higher thanthe the baseline, baseline and was thus used toactivities differentiate of probing insects. from The non-probing baselinerelatively with flat; however, small irregularities were alsowere detected that due to the(Fig. 1A). movement of leafhoppers on the leaf surface 3.1.3 A2 The A2 waveform was highlywave regular, with resembling a variable loosewas amplitude square characterised (Fig. by 1B, aof compressed repetitive square view), and peaks and regular ofshowed overlying high pattern amplitude. a Moreover, sequence the A2 of waveform underlying, short spikes appearing at of waveform subtypes A1-A, A1-B, A1-Cspikes) and A1-ns (Fig. (brief 1A). negative to Usually, or the higher median thanwas voltage the lower of median than A1 voltage theof was of A3 the the equal waveform A1 A2 (Table waveform waveformFourier 1). (including and transform subtypes) The (FFT) was repetition spectrumspectral variable. of rate band of the The low A1 frequency fast between waveform 0 andfrequencies showed 20 between Hz; a 0.4 however, the and 0.8 Hz predominated. 0 V) and originatedalways as generated just R after component.cates initial The stylet that penetration, waveform this which A1-Aleaf indi- subtype epidermis. was Among appeared the when A1some extent) waveform stylets regular subtypes, in A1-C frequency, contacted with was6–12 a the (to wide Hz frequency (Table band 1). of ThereA1-C, was and alternating the occurrence A1 ofA1-ns event A1-B always always and ended appeared with withinpotential A1-C. the drops. Brief A1-B Although events waveform, A1-ns of as seemedsimilar to very to that have of brief the an drop caused appearance be by used a calibration to pulse distinguish (which probing can activitiesties), from there non-probing were differences activi- in the details betweenworthy them. that It A1 is always note- occurred inin every alternating probe moments and of also two appeared different waveforms.

1664 1665 50 df 002, . 0001, 0 . (Fig. 2 A. 0 1 of floni- < − = 1 P 17.07 min − P ± 0.85; = 99 and 2 . ) of flonicamid was 2b R 1 (Fig. 2 and Table 2). 0 𝜒 − , which was 3.8-fold 96 respectively) (Figs 1 . = 1 − 0 − 2 77; exponential decay: . ) of flonicamid. The LC 76 respectively) (Figs 3B R . = 0 1 0001, . 0 − 2 0 = R = 2.4 min and 46.2 2 < value of 153.96 mg L 2 R 0001, ± P . R 50 0 0001, = . a wileyonlinelibrary.com/journal/ps ) of flonicamid was approximately 0 P 05) (Fig. 3A and supporting informa- N 1 . 0.035, − 0.035, = 0 = P = < P P P value was 358.11 mg L 91 and 50 . 35) (supporting information Fig. S2A). Analysis of . 0 . 0 046 and 069 and . . = 0 0 = 2 ) R P = = 1 2 − 2 R value (239.22 mg L R 50 87 respectively) (supporting information Figs S1C and D). 0001, 35) (supporting information Figs S1B and E). A significant . . . A. biguttula 15, 21, . . 0.88 respectively). The duration of Np waveforms increased 0 0 0 0 0 ======2 2 P P R treatment reached 100%, whereas in the CK the mortalityafter was 96 7.3% h of flonicamid treatment. Afterity 96 for h the of highest treatment, concentration mortal- (10 000 mg L as the concentration of flonicamid increased (Fig.ing 3A information and Fig. support- S1A). The numberwere of not events significantly different of between Np the waveforms CK andtreatments the ( flonicamid the average duration revealed no significantthe correlation CK between and flonicamidthe treatment fit for to A1 either a and linearP A4 or waveforms, an and exponential model was poor (linear: difference in the total durationfound of among the the treatments ( A2 and A3 waveforms was R value at this time point was 155.27 mg L and E). However, a significant difference wasage observed duration in the of aver- the A2( and A3 waveforms among the treatments half the value atoccurred in 72 h. the At CK,the whereas 168 h highest mortality concentration after of (10 000 86.8% treatment, mg L was 8.8% recorded mortality at camid reached 92.9%, with an LC P 75.2%, and the LC After 120 h, less thanthe 10% mortality LC was observed in the CK, and 3C and D). Noduration significant of difference the was( A1 observed and in A4 the waveforms total among the treatments lower than the valuein at the 72 h. CK was After 10.7%, 216 whereas h mortality of at treatment, 10 mortality 000 mg L and Table 2). Bioassays demonstrated that flonicamid was slowing act- and was significantly slower than starvationity in of causing mortal- 3.3 EPGbiguttula and feeding behaviour in flonicamid-treated During the 8 h recording period, thetheNpwaveformoftheCKwere8.2 average and total duration of respectively. The average and total durationsof of the the CK Np were significantly waveform shorter thantreated those with on flonicamid ( plants that were tion Fig. S1A). Regression analysis showed ationship significant between linear flonicamid rela- concentrations and thetotaldurationoftheNpwaveform( average and (95% CL)(mg L 50 A. of floni- 0.05. 1 < − treated with P values of floni- 50 SE) LC after different time periods A. biguttula 0.055) 7510.94 (3764.78–20125.65) 150 2.12 2 0.049) 974.10 (478.50–2057.10) 150 0.86 2 0.050) 595.71 (283.71–1181.8) 150 2.08 2 0.050) 358.11 (166.54–677.48) 150 3.71 2 0.051) 239.22 (108.08–446.78) 150 5.22 2 0.051) 155.27 (60.16–309.85) 150 5.02 2 0.052) 153.96 (63.47–279.20) 150 7.30 2 ± ± ± ± ± ± ± ± A. biguttula A. biguttula tested. : 1661–1669 © 2016 Society of Chemical Industry at 24, 48 and 72 h were 7510.94, 974.10 and 73 A. biguttula respectively (Table 2). Mortality in the starvation 2017; 1 − Toxicity of flonicamid to was compared with the control (CK: 0 mg L Mortality of third-instar nymphs of A. biguttula : number of Chi-square testing linearity of dose–mortality response: N a b 24 0.39( Table 2. Time after treatment (h) Slope ( 48 0.31( 72 0.33( 96 0.36( 120 0.37( 168 0.35( 216 0.38( Pest Manag Sci 3.2 Activity of flonicamid against 3.1.7 A6 The A6 waveformhighly was variable characterised amplitude by andwaveform frequency upward was very (1–6 spikes stereotypical, Hz) appearing with with (Fig.which the was 1F). a same easily shape This distinguished from otherdetected waveforms. A6 in all was tested not leafhoppers, but whenrelatively it short happened, duration it (1.5–2 was min). of Also, A6by was always A1, preceded and usually followed by A2. flonicamid or starvation. * indicates 0%. A5 generally followed anally. A4 Events event, of the but A5 occurred waveform2–3 only often min) had occasion- than shorter events of duration A1, A2 (upindicated and A3. to an The extracellular positive level position of of voltage stylets during A5. The toxicity of flonicamidbiguttula 96% TC to third-instar nymphs of Figure 2. Flonicamid disrupts ingestion of cotton leafhopper www.soci.org camid) and starvation (water only, no cotton leafments for (Fig. feeding) treat- 2 and Table 2).nymphs After to 24 h, flonicamid the at mortality10%, different of whereas third-instar concentrations mortality of was 0and less and starvation than 13.16% treatment was respectively. observed Aftertested 48 in h, concentrations the mortality of CK for flonicamid all mortality was was less zero than and 30%, 31.7%respectively whereas in (Fig. the 2). CK After 72 and h, starvationCK mortality treatment and was starvation 5.6 treatment and respectively. 71.7% The in LC the camid to 595.71 mg L et al. : 1661–1669 SE. Asterisks *, 73 ± 2017; 001) respectively, and ‘ns’ denotes . 0 Pest Manag Sci < P 01 and . 0 < P 05, . 0 < P uration of each EPG waveform, including the average duration of the www.soci.org K Tariq © 2016 Society of Chemical Industry 05). . 0 > P Relationship between flonicamid concentrations (log) and the average d non-significance ( wileyonlinelibrary.com/journal/ps Figure 3. Np waveform (A), A1 waveform** (B), and A2 *** waveform by (C), a A3 data waveform point (D) show and a A4 significant waveform difference (E). between Each the data CK point and these is values presented ( as the mean

1666 1667 A. . Previous has never was due to E. fabae A. biguttula leafhoppers survived feeding behaviour waveforms E1-A, E1-B, A. biguttula and 22 E. vitis A. biguttula E. vitis The A. biguttula wileyonlinelibrary.com/journal/ps 25 , on flonicamid-treated cotton plants or in our study. 22 22 suggest channel cutting in either mes- 25 et al. The present study revealed that the feed- could not survive long on water alone (thus found that flonicamid induced starvation in However, both of these were minor waveforms We propose that waveforms A2 and A3 identi- 26 , 28 A. biguttula 22 12 22 E. fabae et al. E2 waveform represents watery salivation combined et al. values of flonicamid were associated with a decrease We hypothesise that similar waveforms detected in this may have preferred to lacerate and ingest from phloem, A. biguttula 50 22 and is translocated in the xylem and phloem. Our bioassay E. vitis 27 A significant negative correlation was observed between total Flonicamid is a systemic insecticide that circulates within the The longer when compared withsise starved that leafhoppers. Wethese hypothe- insects may be unlikely tohypothesise ingest that from the xylem concentration on of plants). flonicamidmesophyll/parenchyma We may cells be lower than in inbiguttula vascular cells. If so,but then may have switched their ingestionon to flonicamid-treated leaves mesophyll/parenchyma to survive slightly longer than starved insects. Cho and/or non-vascular tissues. in the durationperiod; these of findings support phloem the resultsstudy. ingestion obtained in and the present increase in the Np revealed that flonicamid-treated fied in this study (Figswith 1B and and without C) watery alsoleaf salivation represent tissue(s) active in respectively. which ingestion, At such present,tion channel occur the are cutting, unknown salivation for both and inges- 4.2 Effects of flonicamid on ing behaviour of was significantly inhibited.observed A between Np dose-dependent percentage and correlationwhich flonicamid concentration, indicated was that insectsmore on time flonicamid-treated in plants Np spent those or resting on and the were notdetected CK. as between active A active in ingestion dose-dependent feedingflonicamid. (A2 as Furthermore, negative and a correlation A3 significant waveforms)the was decrease number and of was test observed insects able in to(A3 reach the waveform) active ingestion as phase the flonicamidindicated clearly concentration that increased, flonicamid toxicity which to inhibition of ingestion. and average durations of A2 and A3of waveforms and concentration flonicamid. However, thewaiting number times to of first Npand probe, second and the probes time A1 and intervals the events,A3 number between were the of the not A1 significantly first probes altered by before flonicamid. the first plant detected for both the species, and theyacterised were not either biologically by char- Jin with active ingestion, whileingestion alone. the E3 waveform represents active study, A1-A, A1-B, A1-Cchannel and cutting by A1-ns stylets. (respectively), also represent ophyll/parenchyma or general phloem tissues. aphids with thelower inhibition LC of phloem sap ingestion, and that EPG studies of Flonicamid isHemiptera-feeding blocker, a withanism the stated novel being insecticidalfeeding starvation mech- activity. insecticide caused by that irreversible is inhibition of a selective E1-C and E1-ns have beenstylet shown movements and to secretion be of stronglycutting. watery correlated with saliva during channel been observed to ingestA4, from A5 and xylem A6 cells. were only Here,and occasionally E6 the observed, in Jin similarly waveforms to E4, E5 84 . 99) 34) . . 0 0 0 = = = EPG 2 2 P R R were bio- 0001, . 0001, . 0 0 = = A. biguttula biguttula P 92) (Fig. 4). Addition- P . ), rather than the sali- . 0 A 25 . All previously studied = , a species in the same 2 R E. vitis biguttula 05) among the treatments for the . . 0 0.0001, A > = P P 82) (Fig. 5E). . 83 respectively) (supporting information . 0 : 1661–1669 © 2016 Society of Chemical Industry 0 = 73 = 2 2 R R 2017; of waveforms from 022, . 22 001, 0 . Percentage of insects reaching the A3 waveform after being 0 = P = P Thus, the EPG results showed that, as the concentration of floni- A significantly negative correlation was observed between the The number of events of Np, A1 and A4 waveforms were sta- camid increased, the percentageby of Np recording increased, time while represented Accordingly, the the percentage effect of of flonicamid A2 was significantlyA2 and negative and A3 on A3. decreased. However, nowaveforms (Fig. significant 6). effects were detected on A4 ally, five sequential variables were analysed (Figs 5A toicant E). difference No was signif- found ( transition time from the starting point to thefor first A1 the event interval (Fig. from 5A), the first tothe the number second of A1 probes probe before (Fig. the 5B) first A3 orsignificant event for (Fig. exponential 5C). However, regression a was observed betweendose the of log flonicamid and the first A3 period ( Figs S2C and D). concentration of flonicamid and thethat percentage reached of the tested A3 insects event ( tistically similar in the CK and flonicamid treatments ( (supporting information Figsicant S2A, differences B werebetween and the found CK E). and for flonicamid However, treatments the ( signif- A2 and A3 waveforms Pest Manag Sci waveforms Different EPG waveforms produced by 44.1 DISCUSSION Hypothesised biological meanings of Figure 4. treated with water (CK) and different concentrations of flonicamid. Flonicamid disrupts ingestion of cotton leafhopper www.soci.org and vary sheath feeding strategyrupture typical feeding, of only a other partial leafhoppers. salivaryinsect In sheath actively is cell moves produced its and stylets the through manynel cells cutting’), (termed actively ‘chan- ingesting from multiple cell types in vascular (Fig. 5D). The analysis from A1nificant to linear the relationship first between A3 event theevent revealed log ( a dose sig- and the first A3 subfamily () as typhlocybine leafhoppers use the(formerly known cell as the rupture lacerate-and-flush feeding strategy logically interpreted oncorrelation the basis of the previously defined et al. 05). . 0 : 1661–1669 > 73 P 2017; Pest Manag Sci SE. Asterisks *, ** and *** by a data point show a ± 001) respectively, and ‘ns’ denotes non-significance ( . 0 < as injection andther verification. topical In applicationdetermine addition, could any further effect be studiesspeculation on are used is the necessary forto plant that to insects. fur- from flonicamid-treated flonicamid, plants and are the distasteful P 01 and . 0 www.soci.org K Tariq < P 05; . 0 < © 2016 Society of Chemical Industry P Relationship between flonicamid concentrations (log) and sequential variables of each EPG waveform, including the time from the start of In the present study, after leaf dipping application with floni- camid, a difference betweeningestion the was effects observed, of and flonicamidonly thus on effects active to should not thefore, be translocation related in of addition flonicamid to within the plant. leaf There- dipping, other methods such wileyonlinelibrary.com/journal/ps Figure 5. the EPG recording to theduration first of probe (A), the the first time A3 interval (D) from and the first the probe time to from the the second first probe (B), probe the to number the of first probes A3 before the (E). first Error A3 bars (C), indicate the significant difference between the CK and these values (

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Pestology Agric Res Performance of novel vs. traditionalAmrasca insecticides biguttula for biguttula the control of Pak J Agric Sci Directorate of Economics and Statistics In conclusion, our EPG data showed that flonicamid significantly 2 Wu KM and Guo YY, The evolution of cotton pest management 8 Rafique MA and Shah HA, Cotton pest scouting of farmers’ fields at 1 3 Luttrell RG, Fitt GP, Ramalho FS and Sugonyaev ES, Cotton pest 4 Mohan S and Nandini S, A promising entry for cotton leafhopper. 7 Patel Z and Patel JR, Re-surveyed of Jassid ( 5 Karar H, Babar TK, Shahazad MF, Saleem M, Amjad Ali and Akram M, 6 Borah RK, Insect pest complex in brinjal ( increased the duration of Npperiod and (A2 and decreased A3 the waveforms). It active mayto ingestion also have stimulated change insects fromingestion before dying. phloem ingestion to mesophyll/parenchyma Pest Manag Sci REFERENCES SUPPORTING INFORMATION Supporting information may be found inarticle. the online version of this This research was supported by theAgriculture earmarked fund Research for the System China tal No. Research CARS-27 Funds and forEfforts the the by Central Fundamen- EA Universities Backus (No.the were 2014PY005). Crop supported Diseases, Pests by and in-houseAgricultural Genetics Research Research funding Service. Unit Mention from of of trade the names orcial USDA commer- products in thisviding publication specific is information solely and for doesor the not endorsement purpose imply by of recommendation the US pro- Departmentequal of opportunity Agriculture. provider USDA and is employer. an ACKNOWLEDGEMENTS time after being treatedflonicamid. with water (CK) and different concentrations of Figure 6. Flonicamid disrupts ingestion of cotton leafhopper www.soci.org