1657 D. 015 The pathogen 11 , 9 Acquisition of Las can also 13 – However, the pathogen can only 9 11 , 9 of methoprene. Methoprene caused and has been reported in hemolymph * 1 − Diaphorina citri D. citri gmL has resulted in the development of insecticide 𝛍 Infected adults play a major role in the spread of the 11 , D. citri 10 , 6 , 1 . Correspondence to: Lukasz L Stelinski,ment, Entomology University and of Florida, Nematology 700 Depart- USA. Experiment E-mail: Station stelinski@ufl.edu Rd, Lake Alfred, FL 33850, EntomologyandNematologyDepartment,UniversityofFlorida,LakeAlfred,FL, USA ∗ their lifetime on young foliar growth.undergo Upon hatching, five the nymphal nymphs instars toduration ranges emerge from 15 as to adults. 47 days, Theregime. depending The life upon nymphs and temperature cycle adults feed on planteydew sap rich and excrete in hon- sugars that leads towhich growth can of sooty affect mold photosynthesis. on leaves, Theby second-, pathogen third-, fourth- can and fifth-instar be nymphs, as well acquired during as feeding adults, on infected trees. pathogen because of theiradults dispersal remain capabilities. infective Once throughout infected, their lives. multiplies within and salivary glands of adults. be transmitted by fourth- and fifth-instar nymphs and adult citri Kuwayama, transmits a bacterium that causes huanglongbing 7 5 , 4 nymphs and reduced adult emergence as compared with controls. D. citri . . 8 HLB was D. citri 6 Candidatus D. citri . Diaphorina citri D. citri Kuwayama (Hemiptera: The fruits produced on 1 HLB is a devastating disease that 3 – 1 was initially reported in June 1998 in Diaphorina citri under laboratory conditions. Treatment of fifth instars reduced the fertility of females. Methoprene might : 1657–1665 www.soci.org © 2015 Society of Chemical Industry D. citri 71 are small (2.7–3.3 mm in length) with mottled D. citri 2015; insect growth regulator; juvenile hormone analog; Asian citrus psyllid; citrus greening; huanglongbing D. citri

Adult brown wings. There are three adult morphs whichinal differ in color abdom- (gray/brown, blue/green and orange/yellow) Pest Manag Sci In the United States, Liviidae), is amits serious a pest phloem-restricted, of gram-negativeLiberibacter asiaticus citrus bacterium, (Las), that crops causes huanglongbing worldwide.referred (HLB), to also as It citrus greening. trans- 1 INTRODUCTIONThe Asian citrus psyllid, Abstract BACKGROUND: The Asian citrus citrus psyllid, behavior of Asian citrus psyllid, Gurpreet S Brar, Wendy Meyer and Lukasz L Stelinski stages, adult emergence, reproduction and analog, on survival of various developmental Effects of methoprene, a juvenile hormone (wileyonlinelibrary.com) DOI 10.1002/ps.3975 Kuwayama in citrus. Frequent and repeated use of neurotoxic insecticides against Research Article Received: 9 October 2014 Revised: 12 December 2014 Accepted article published: 13 January 2015 Published online in Wiley Online Library: 30 January 2 resistance. We evaluated the effectsemergence, of reproduction the and behavior juvenile of hormone analog methoprene on egg hatch, nymphal development, adult females are very prolific and can lay approximately 800 eggs in reported initially in southern Florida in August 2005 andquently has spread subse- to all citrus-growing regions of the state.5 In years, the HLB past has caused approximately $USloss and 3.63 6611 billion lost in jobs in revenue agriculture and related sectors in Florida. infected trees are small and distorted and have a bitter taste. causes heavy leaf drop,out-of-season dieback flushing of and stems, blossoming prematurewithin and fruit eventual 5–10 drop, tree death years of initial infection. Florida, and since then itCalifornia, has been Georgia, reported in Louisiana, Alabama, Mississippi Arizona, and Texas. RESULTS: Methoprene significantly reduced the viabilityand of eggs 49–96-h-old that were between eggs 0 was and 4 8 days old. and Egg hatch 9%, of 0–48-h-old respectively, when treated with 320 Keywords: © 2015 Society of Chemical Industry Methoprene caused less than 5%40% adult emergence adult when emergence first- and whenmethoprene-treated third-instar fifth fifth stages instars. were instars treated, were respectively, and treated. less Reduced than fertility ofCONCLUSION: females Methoprene was was observed effectiveemergence when of in they emerged reducing from be egg a possible hatch, tool for suppressing integrated management nymphal of development and decreasing adult significant mortality of first-, third- and fifth-instar 23 in D. N, 5% ′ ± 91 cm; × D. citri 61 : 1657–1665 (L.) Osbeck] L. Raf.), 2–3 5% RH and a × 71 ± Cand50 ∘ 2 2015; ± adults of mixed gen- ) were both included 1 5% RH and a 14:10 h ), diluted in tap water. − 1 ± − Cand50 ∘ ), as well as pyriproxyfen Citrus sinensis to oviposit on plants that 1 2 D. citri − gmL ± μ gmL Poncirus trifoliata μ × gmL Pest Manag Sci D. citri μ Cand50 ∘ prepared in tap water. Pyriproxyfen 2 1 ± − MacFaden gmL μ C, 60–65% RH and a 14:10 h (L:D) photope- 22 , ∘ 6 ) and imidacloprid (100 ) or imidacloprid (100 1 1 adults of mixed gender were released for mating and − − Citrus paradisi gmL gmL W), prior to discovery of HLB in Florida. The colony is con- μ μ D. citri ′ were removed from the plants. After 96 h, one flush with eggs In the second experiment, contact toxicity of methoprene to months old and with new flush as defined by Hall and Albrigo, Center, University of Florida. Thefrom culture a was field established population in collected 2000 in Polk County, Florida (28.0 plants at 27–28 tinuously maintained on sweet orange [ 2.2 Effect ofThe methoprene on objective egg of hatch thismethoprene experiment on was egg to hatch. determine Forty-fiveplants the potted ( effect ‘Swingle of citrumelo’ were placed into four fine-mesh screen cages (61 (L:D) photoperiod. After 48 h,plants. the After 48 adults h or were 72 removed h, leaf0–48-h-old from flush the with and eggs was 49–96-h-old excised to obtain removed. eggs. Only The one excised flush flush permicrocentrifuge was tubes plant (Fisher was placed Scientific, individually Pittsburg,a PA) into 2% containing agar–water 1.5 mL solution. Eggs perunder individual the flush stereomicroscope. were The counted flush samplesin were then dipped solutions of40, methoprene 80, 160 at and various 320 concentrations: 10, 20, Water alone served as thedried. Each control plant treatment. was The infested with foliageder 20 was and air individually coveredcitri within mesh screens. Afterwas 48 h, removed from eachcentrifuge plant tube containing and a placed 2%then agar–water into counted using a solution. a Eggs stereomicroscope, 1.5 and mL were within the micro- flush a was placed rearing chamber maintained at 25 RH and a 14:10 hexperiment was (L:D) replicated photoperiod twice over to time,per allow with treatment 11–12 for and egg replicates with 7–98 hatch.of The eggs treatment, per the flush numbers sample. ofcounted. After hatched 5 and days unhatched eggs were (128 81.9 (128 as positive controls. Water aloneThe served flush as was the dippedexperiment negative for was repeated control. 30 twice, s and each and treatment, including allowedcontrols, the was replicated to 12–18 air times. The dry. number ofranged The eggs per between entire flush 7 and 95.a rearing Treated chamber flush maintained samples at were 25 placed in 14:10 h (L:D) photoperiod. Afterand 5 unhatched eggs days, were the counted using numbers a of stereomicroscope. hatched eggs was assessed by allowing riod. Analytical-grade methoprene(purity (purity 99.0) 99.2%), and pyriproxyfen imidaclopridSigma-Aldrich (purity (St Louis, 99.9) MO). were The insecticideswater procured were from for diluted in spray tap treatments. and Pyriproxyfen dip and treatments imidacloprid werecontrols. and used The concentrations in as of positive positive acetone controlsthe were for selected basis on topical of effectiveprevious concentrations studies. recorded against Bioquip, Ranch Dominguez, CA). Within each400 cage, approximately oviposition for 48 h at 25 were previously treatedrumelo’ with plants methoprene. with Potteda ‘Swingle new cit- flush handheld were atomizerprene sprayed using (20, until 40, various run-off 80, 160 concentrations with and of 320 metho- , 18 D. D. , 17 and D. citri Juvenile 6 16 , 5 Methoprene www.soci.org GS Brar, W Meyer, LL Stelinski © 2015 Society of Chemical Industry 16 There is an imme- Aedes aegypti 15 , Rhyzopertha dominica -butylimino-3-isopropyl- tert )-11-methoxy-3,7,11-trimethyl- were treated with pyriproxfen E , populations. E D. citri D. citri External application of methoprene to Triboleum confusum Multiple applications of a limited number of )-2-(2-pyridyloxy)propyl ether – a juvenile 7 , 21 . management, they could also improve exist- RS – Because of its low mammalian toxicity, selec- were collected from a laboratory colony 17 17 , They have been successfully used to control D.citri 16 D. citri 16 14 . The objectives of this study were to quantify the effects . These additional insecticides would not only serve as possible Methoprene [1-methylethyl ( Currently, the spread of HLB is managed by removing infected Insect growth regulators (IGRs) are reduced-risk insecticides 5-phenyl-3,4,5,6-tetra-hydro-2-thiadiazine-4-one – asynthetase inhibitor) and diflubenzuron [1-(4-chlorophenyl)-3-(2,6- difluorobenzoyl) urea – a chitin synthetase chitin inhibitor]. hormone mimic], buprofezin (2- 2,4-dodecadienoate] isbeen a widely used juvenile for insectas hormone pest a management. analog It mosquito hasinsect that been larvacide, pests used has as and for aColeoptera, control Diptera, grain Hemiptera of and a protectant Siphonoptera. wide against range stored of pests, including [4-phenoxyphenyl ( insect pests of medical and veterinary importance. trees, applying fertilizers to maintain treepopulations of health and suppressing insecticides of various modes of action are made to reduce populations during each growing season. Repeated useavailable of insecticides limited has resulted incide the resistance development in of some insecti- tivity and uniquecandidate mode for of integration action, into methoprene is management a programs potential for citri of methoprene on:adult (1) emergence; (2) egg fecundity hatch, andfemales fertility emerging nymphal of from development treated treated females fifth and of or instars; adults (3) feeding on behavior treated treated plants. leaves; (4) settling behavior of adults on maintained in a greenhouse at the Citrus Research and Education wileyonlinelibrary.com/journal/ps 22.1 MATERIALS AND METHODS Insect cultureDiaphorina and insecticides citri Reduced eggdecreased hatch, suppressed adultdevelopmental nymphal emergence stages development of were and observed when various occur transovarially from infectedfrequency. females to eggs, but at a low hormone agonists, ecdysteriodinhibitors are three agonists commonly used and insect growth chitin regulators. synthesis eggs during blastokinesisand application is during lethalopment metamorphosis of to immature leads stages. embryocause to Application sterility. development, to faulty the devel- adult stage can diate need for additional insecticidesfor with novel incorporation modes into of action integratedcitri management programs for new tools for ing rotation with conventional insecticides to impedeinsecticide evolution of resistance. with various modes of actioncides. that IGRs differ cause from hormonal neurotoxicthe and insecti- development biochemical of imbalances the during phological target abnormalities and insect, reduced resulting development ofstages, in immature which various mor- leadsless to harmful reduced to emergence beneficialinsecticides. of insects adults. and IGRs humans are than neurotoxic has been shownnomically to important insect reduce pestsTribolium adult such castaneum as emergence of several eco- Cimex lectularius.

1658 1659 ) ), C 1 1 ∘ − − 2 ± adults gmL gmL μ μ to quantify D. citri 24 5% RH with a ± ) and 50% first-instar 50 Cand50 ∘ 2 ± adults settling on plants treated settling on plants. ‘Swingle cit- wileyonlinelibrary.com/journal/ps of mixed gender were placed into D.citri ) or water alone for 30 s and then air 1 − ) and the corresponding 95% confidence 40 cm). All six treatments were randomly 50 D. citri D. citri × gmL across different treatments at the two different μ 40 cm × D. citri 5% RH and at a 14:10 h (L:D) photocycle. The number of 10 mm petri dishes (Fisher Scientific) upon a bed of agar ± × 14:10 h (L:D) photoperiod. Filterreplaced papers and were then removed again after collected 48 72filter h, h papers post-treatment. Collected were subjected to the ninhydrin test and 50 adults settling on plantsmethoprene and treated the with control were various recorded after concentrations 24, of 48 and 72 h. 2.7 Statistical analyses Percentage data from the experiments werethe tested assumptions to ensure of that homogeneity ofmet variance before and data were normality analyzed. were Percentage egg hatchof and survival first, third, and fifthgence, instars, were as subjected well to as(ANOVA) separate and percentage Fisher’s one-way adult protected analysis LSD emer- means ofcentrations separation variance causing tests. 50% Con- egg inhibition (LC intervals (CIs), at both agesing of the eggs, data were to determined probit by analysis.by The subject- numbers the of females eggs that laid developed per fromous plant treated concentrations fifth of instars methoprene with and vari- thefor percentage these egg hatch treatments were subjected toand separate Fisher’s protected one-way LSD ANOVA tests. Thelaid data per for female the and number the of percentage egg eggs adults hatch at for 2, topically treated 7 andone-way 14 ANOVAs and days Fisher’s protected were LSD analyzed tests. by Thein differences conducting feeding of separate time intervals were determined byANOVAs conducting and Fisher’s protected separate LSD one-way tests for each time interval.differences The in the number of with various concentrations of methoprene48 or or 72 h the after control release were atFisher’s determined protected 24, by LSD one-way tests ANOVAs and for each time interval. Proc GLM was citrus leaves collected from insecticide-free Valenciawere orange cut trees into discs 34 mmin in diameter. The solutions leaf discs of were dipped methoprene (20, 40, 80, 160 and 320 honeydew droplet production as anThere indirect were at measure least of six feeding. replicates (individualdisc) petri per dish treatment. with treated 2.6 Settling behavior The objective ofmethoprene this affects experimentrumelo’ was plants to ofheight the determine and same whether vigor agecentrations were (12–14 sprayed of weeks until methoprene old) run-off (20, and with 40, same various 80, con- 160 and 320 pyriproxyfen (128 nymphal mortality (LC dried in a fume hood.35 The mm treated leaf discs wereto then maintain placed moisture on for thewere leaf lined with disc. Whatman The filter lids paper (Whatman ofKent, International the Ltd, UK). petri dishes Five adult each dish. The lids were sealed with parafilm (Beemisage, Flexible Neenah, Pack- WI) and heldScientific, upside down Waltham, in MA) an set incubator (Thermo at 25 diluted in waterwere or allowed to water air dry alonecages and (40 as were cm randomly a placed into negative Plexiglas control. Plants of mixed gender were releasedcages into were the placed in center the of rearing each chamber maintained cage. at The 25 arranged within each cage asset a up choice test. for There choice were tests six cages with each treatment. Fifty Lof μ Fresh adults 24 D. citri (male:female in ) or water alone as ) diluted in acetone 1 1 on treated plant sur- − D. citri − gmL gmL μ D. citri μ of mixed gender were released , and subsequently first-, third- ). Two pairs of 1 − D. citri D. citri ), as well as a set sprayed with pyriprox- , and honeydew excretions were quanti- adults obtained from nymphs, as well as 1 gmL − . Ten-day-old adults obtained from treated μ : 1657–1665 © 2015 Society of Chemical Industry nymphs were obtained from two cages for 5% RH and a 14:10 h (L:D) photoperiod. Adults 71 ± ), imidacloprid (100 gmL D. citri 1 D. citri μ D. citri − nymphs following treatment with methoprene dur- D. citri 2015; The numbers of each nymphal instar were counted . gmL μ Cand50 ∘ D. citri 2 were evaluated following topical treatment of adults with L Hamilton syringe (Hamilton Co., Reno, NV). After treatment, ± μ In a second experiment, the effects of methoprene on fertility of under a stereomicroscope, and theious plants were concentrations sprayed of with methoprene var- 64, diluted 128 in and tap 256 water (16, 32, into a cage withabove. Females potted were allowed ‘Swingle to citumelo’ layand eggs plants on then the as all plants described for adultscages 5 were days, were aspirated infested from with theor fifth-instar cage. A totaleach of instar six ing various instars. Adult were allowed to lay eggseggs for per 72 h. plant After was 72 counted h,was excised, using the inserted a total into number stereomicroscope. 1.5 mL The of microcentrifugeagar flush tubes and filled placed with into a rearinghatched chamber and for unhatched 5 eggs days, were after counted. which There werecates the 6–9 per treatment. repli- D. citri various concentrations (128 and 256 Pest Manag Sci Effects of methoprene on development of Asian citrus psyllid2.3 Effectand of adults methoprene on development of nymphs The objective of thisment of experiment was to quantify the develop- www.soci.org 1:1) were released onto untreated ‘Swinglenew flush. citrumelo’ The plants plants were with placed in a rearingat chamber 25 maintained or a negative control treatment of acetone alone. A total of 0.2 fied to assess the amount of feeding using a ninhydrin test. 2.5 Effect ofThe methoprene on objective adult of feeding thismethoprene experiment on was feeding to behavior determine of the effect of were removed from plantsof after eggs 2, per plant 7 wasThe and numbers counted 14 2, of days. 7 eggs and thatcounted The at 14 hatched number 6, days and 11 and after first-instar 18 treatment. nymphs days after were treatment. faces. Surfaces treated with various concentrationswere of insecticides exposed to three pairs of adults of the‘Swingle citrumelo’ same plants. Treatments age were replicated were 3 times, placed and ontothe entire untreated experiment was repeated twice over time. the treatment solution was applied to the abdomen ofa10 adults using yfen (128 2.4 Effect ofThe methoprene objective on reproduction of this experimenttive was potential to determine of the reproduc- a negative control treatment.using Plants a were handheld sprayed atomizerCA) until and (Chemical were run-off Guys left to MFG.vidual air screen Co., dry. sleeve. Each Gardena, The plant experiments wastime, were covered with replicated with twice each an over treatment indi- replicatedreceiving 7–104 7–11 nymphs. To times determine and acute toxicity,of each the nymphs number plant surviving to the72 subsequent h, instar and adult was emergence recorded was after quantified for each treatment. adults, topically treated withity methoprene. of Fecundity eggs and were viabil- tive measured potential to of ascertain thefifth-instar effects on nymphs, reproduc- described incollected the and sexed previous for experiment, each concentration were (0, of 32, methoprene 64, 128 tested and 256 , 1 − )and 1 gmL − μ : 1657–1665 gmL μ 71 eggs laid after treat- eggs of (A) 0–48 h or 2015; concentrations), followed 1 − Diaphorina citri Diaphorina citri Pest Manag Sci gmL μ 0.0001). The greatest reduction in )(Fig.4B). 1 < 0.05). − < = P P gmL μ 7, 60; = . The highest concentration tested, 256 1 − Mean percentage hatch of Mean percentage hatch of 23.36; df gmL 0.05). μ = < Survival of fifth-instar nymphs was significantly reduced after F P adult emergence occurred with imidacloprid (100 by pyriproxfen (128 Figure 1. (B) 49–96 h age priorprene, to pyriproxyfen or treatment imidacloprid. with Bars various representSEM. concentrations treatment of means Treatment metho- with barsdifferent not from labeled one with( another the according same to letter an are LSD significantly mean separation test Figure 2. 32 ment of plants with various concentrations ofBars methoprene or represent treatment pyriproxyfen. means with SEM. Treatment barsthe not same labeled letter with are significantly different fromLSD one mean another according separation to test an ( treatment with methoprene, pyriproxyfen or imidacloprid as caused 77% mortality ofimidacloprid caused third-instar 48 nymphs. and Pyriproxyfen 93%Each and treatment mortality tested respectively significantly (Fig. reduced 3B). from emergence of treated adults third-instar nymphs( as compared with the control methoprene (at 256 and 128 7, 90 = )was 30.73; 1 − 0.0001) = and LC < F 50 = respectively gmL 0.0001). The P values calcu- μ 18.01; df 1 www.soci.org GS Brar, W Meyer, LL Stelinski < © 2015 Society of Chemical Industry − ). Furthermore, = 90 1 = F − P 7, 70; ) after 0–48 h and gmL 1 μ = − gmL and LC μ 6, 76; 50 gmL = μ 35.56; df = F egg hatch was 23 and 38% follow- 57.16; df 0.0001) (Fig. 1). Similarly, methoprene 0.0001). There was no emergence of = < < F = = did not indicate a statistical difference in P P D. citri ). 90 1 25 − 0.0001) (Fig. 1). We observed 74 and 77% hatch 7, 49; < 8, 129; = = = gmL and LC μ P 50 0.0001). The lowest concentration of methoprene that < = 8, 114; 42.87; df 52.47; df P = = = Treatment of third instars with various concentrations of metho- F F adults from first-instartrations nymphs of treated methoprene atrecorded tested, at the and the higher lower less concentrationsnymphs concen- than treated tested with (Fig. pyriproxyfen 2% 4A). or imidacloprid First-instar survivalto did emerge not was as survive adults (Fig. 4A). prene, pyriproxyfen orreduction in imidacloprid survival resulted of in third-instar a nymphs significant ( 60; lowest concentration of methoprenepercentage tested egg resulted hatch intested a of resulted mean 53%, in and aPyriproxyfen mean resulted the in percentage highest a egg mean concentration were hatch egg of hatch no of 7% eggs 22% (Fig.because (Fig. 2). all observed 2). of There the on adults diedtoxicity. plants There before were egg sprayed no laying significant owing differences with to in contact the imidacloprid LC for 0–48-h-old and 49–96-h-oldconcentration eggs, of respectively, at methoprene the tested lowest (10 caused significant mortality of third-instar nymphs (34%) was susceptibility of eggs, based on age (Tableiment, 1). In contact the toxicity second exper- ofegg methoprene resulted hatch in in a awith reduction in concentration-dependent the manner water as control compared ( wileyonlinelibrary.com/journal/ps 3.2 Effectand of adults methoprene on development of nymphs There was anymphs significant to reduction the in second the instar ( survival of first-instar df values of methoprene when appliedbased before on and the after overlapping of egg 95% laying, CIs (Table 1). after treatment with methoprene.nymphs Acute was mortality observed ofThe first-instar in lowest concentration a tested concentration-dependenthighest caused manner. 46% caused mortality, 93% andPyriproxyfen mortality the and of imidacloprid caused first-instarfirst 76 instars nymphs respectively and (Fig. (Fig. 98% 3A). 3A). The mortality LC of significantly reduced the hatch of 49–96-h-old eggs ( 3RESULTS 3.1 Effect ofMethoprene methoprene significantly on reduced egg the hatch hatch of 0–48-h-olda concentration-dependent eggs in manner as compared with the( control used to conduct one-way ANOVAs and Fisher’s protected LSD tests, and Proc Probit was used for probitInstitute, analysis Cary, with NC). SAS software (SAS 49–96 h of ageinghatched respectively following treatment (Fig. with imidacloprid 1). (100 The percentage of eggs (Table 1). There was reduced development of methoprene-treated first-instar nymphs to( adults as compared with the control 8 and 9%hatched of following 0–48-h-old methoprenetested and treatment (320 49–96-h-old at the eggs, highest respectively, rate lated for methoprene were 27.2 and 371.8 51 and 52% for(Fig. 1). 0–48-h-old Although 0–48-h-old and eggs 49–96-h-oldprene were more treatments sensitive eggs to than metho- respectively 49–96-h-oldCIs for eggs, LC the overlapping 95% ing treatment with pyriproxfen (128

1660 1661 , c D. 5.92; (df) 0.46) 0.26) man- 0.046) 2 D. citri = = = 𝜒 = F P P 0.35) and P 0.46), 48 h = D. citri = P 4, 32; It is necessary P 5, 30; 2, 7; 0.0039) (Fig. 6). = = 15 = = 0.93) (Fig. 7). SE) 0.10 15.1 (4) 0.14 11.2 (3) 0.170.12 31.1 (4) 10.5 (4) 2, 11; P ± = ± ± ± ± 5, 30; = P respectively (Fig. 8). = 1 1.36; df 0.96; df − 4.90; df = 4, 26; Slope ( = in Florida. = F 2, 14; F = F 1.15; df gmL = μ 0.95; df = 0.0005) and 49–72 h ( F = D. citri b = F wileyonlinelibrary.com/journal/ps P 5.03; df adult feeding during the 0–48 and 0.07; df = did not differ between the methoprene = F F 0.45) or 72 h ( ) (95%CI) 7, 53; 1 D. citi = − = P D. citri gmL μ populations by frequent application of available, ( 0.0001) of feeding. The lowest concentrations that 5, 30; 90 4.57; df ) reduced egg hatch as compared with the control. = = 1 LC P = − F D. citri gmL 7, 56; 0.96; df μ a = = Topically applied methoprene significantly reduced the hatch F df significantly reduced 49–72 h intervals were 40 and 160 after release. 14 days after treatment ( 3.4 Effect ofThere methoprene on adult were feeding significantdew droplets differences recorded per inpyriproxfen filter the paper treatments, for number as the0–48h methoprene of compared ( and with honey- the controls, after ( for the higher dosages ofno methoprene significant tested (Fig. differences 7). in Therecontrols egg were for hatch eggs between laid treatments at and 7 days ( (Fig. 5). However, eggcantly hatch from of the treated controlOnly females ( the differed higher signifi- concentrations128 of methoprene tested (256 and of eggs laid 2 days after treatment ( 4 DISCUSSION Currently, management of HLB in citrusment relies heavily of on manage- registered insecticides for usemodes of in action citrus available, the crops. repetitive andinsecticides Given frequent use has the of resulted these limited in thetance development in of certain insecticide populations resis- of 3.3 Effect ofThere methoprene were on no reproduction significant differences between the numberoviposited of by eggs female adults that emerged fromnymphs treated as fifth-instar compared with the control ( 3.5 Settling behavior Settling behavior of and control treatments 24 h ( to prevent or delay insecticide resistance development in and inclusion of additionalintegrated management insecticide program is modes one possible of approach. Insectgrowth action regulators into are an characterizedthat by differ several significantly modes from ofthat that can action of be incorporated neurotoxic into insecticides, the current and rotation for agement. The objective of thisefficacy investigation of was the to juvenile evaluate hormone the analog, methoprene, against ) (95% CI) Diaphorina citri 1 − 7, 68; 0.0001) = gmL μ < ( = 50 P LC concentration. ), as well as the 3.29; df 1 1 − − = F 7, 68; gml = gmL μ n μ 7.83; df = . . F 90 50 0–48 2773 41.91 (32.71–52.77) 336.16 (227.72–590.13) 1.41 49–9649–96 2006 2228 56.11 (34.78–91.28) 56.81 (48.74–66.59) 645.17 (289.62–3711) 211.21 (166.08–298.48) 1.20 2.24 when treated with various concentrations of : 1657–1665 © 2015 Society of Chemical Industry 71 0.05). < P 2015; Toxicity of methoprene to various developmental stages of Diaphorina citri Mean percentage survival of (A) first-, (B) third- and (C) fifth-instar d d e 0.0045) as compared with the controls. The higher concentra- 95% confidence intervals for LC Leaf-dip bioassay was conducted after egg laying. Plants underwent different treatments before egg laying. 95% confidence intervals for LC Chi-square goodness-of-fit test and degrees of freedom. Developmental stage Age (h) Egg Egg First-instar nymph 1909 27.19 (13.38-40.8) 371.84 (194.7–1569) 1.12 a b c d e Egg Table 1. = tions of methoprene tested (256 and 128 P Pest Manag Sci methoprene, pyriproxyfen or imidacloprid. Bars notletter labeled are with significantly different the from one same another according toseparation an LSD test mean ( compared with the controls ( Figure 3. nymphs of (Fig. 3C). The highest reduction in survivalprid, occurred with followed imidaclo- by methoprene at the 256 Effects of methoprene on development of Asian citrus psyllid www.soci.org Application of methoprene,nificantly pyriproxyfen reduced the or emergence imidacloprid of adults sig- ( pyriproxfen and imidacloprid treatments, reduced the emergence of adults from fifth-instar nymphs(Fig. 4C). as compared with the control . 1 − D. citri Diapho- Popillia gmL μ : 1657–1665 for younger Diaphorina citri 71 1 Helicoverpa zea − resulted in similar 2015; 1 In the present study, gmL − 6 μ . gmL μ resulted in similar egg inhi- D. citri 1 − Pest Manag Sci gmL μ ), it prevents these compounds from ow K Similarly, the mortality of 29 . 1 from interfering with embryogenesis. Metho- − 30 gmL μ 0.05). 0.57) < Given that the lipid layer of the insect chorion acts as Mean number of eggs oviposited per plant by = Newman eggs in soil treated with concentrations ranging Mean percentage hatch of eggs oviposited per plant by P females emerging from fifth-instar nymphs treated with various 0.05). 31 , ow < 30 K P Treatment with pyriproxyfen at 128 reaching the embryo. We hypothesizeeggs that the in chorion the(log may case of have selectively prevented imidacloprid Figure 6. rina citri concentrations of methoprene. Barssignificantly not different labeled from one with another the accordingtion to same test an letter ( LSD are mean separa- egg mortality to thosehormone mimics. observed previously with other juvenile Figure 5. females emerging from fifth-instartrations nymphs of treated methoprene. with Bars variousicantly not concen- different labeled from with one thetest another same ( according letter to are an signif- LSD mean separation no oviposition was observed on plants treated with imidacloprid. egg hatch inhibition to methoprene applied at 160 bition to methoprene applications at 40 a barrier totioning hydrophilic coefficient compounds log (low octanol–water parti- eggs. In the present investigation,methoprene the juvenile and hormone analogs pyriproxyfenthan appeared imidacloprid, a to commonly beegg used survival. neonicotinoid, more Imidacloprid in effective had reducing nojaponica effect on egg hatchfrom of 0 to 2 prene exhibited residual effectiveness inconcentration-dependent reducing manner. egg Similarly, hatch residues in of a pyriprox- yfen resulted in reduced egg hatch of Imidacloprid applied at 100 Boddie eggs was notfield. affected by imidacloprid treatment in the The higher This trend is 28 , www.soci.org GS Brar, W Meyer, LL Stelinski © 2015 Society of Chemical Industry 27 26 Trialeurodes vapo- when treated with various 0.05). < P (Gennadius) and eggs, leading to reduced egg hatch Diaphorina citri Juvenile hormone analogs inhibit egg (Germar), are similarly more susceptible Also, eggs of glassy-winged sharpshooter, 27 6 D. citri Bemisia tabaci Mean percentage adult emergence from (A) first-, (B) third- (Westwood). . Both direct effects against immature stages and sublethal In the present investigation, application of methoprene resulted hatch by penetrating intoat the the egg and blastokinesis blocking stages.potent embryogenesis Juvenile if hormone they enter analogs the are egg prior more to blastokinesis. wileyonlinelibrary.com/journal/ps concentrations of methoprene,labeled pyriproxyfen with the or sameaccording imidacloprid. letter to an are Bars LSD significantly mean not different separation test from ( one another citri effects against adults were investigated. in acute toxicity to Figure 4. and (C) fifth-instar nymphs of of both younger andincreased in older a stages dose-dependent tested.Older-stage manner, irrespective Egg eggs of hatch egg inhibition wereyounger-stage age. eggs, less which is susceptibletions congruent for to with pyriproxyfen. previous methoprene observa- than Homalodisca vitripennis to pyriproxyfen at younger than at older stages. also true for rariorum probability of blockagehave of resulted in blastokinesis increased susceptibility in ofeggs. younger younger Methoprene than of eggs exhibited older may similar age-dependent effects on

1662 1663 6 D. D. and than D. citri during Acheta with 18 D. citri D. citri Pyriproxyfen D. citri 35 ), it appears that (L.) (Lepidoptera: 1 − are similar to those Transmission of the 12 , by targeting eggs and gmL 6 μ The present findings are D. citri 33 , and cyantraniliprole because of the demonstrated 32 D. citri , Methoprene could be a good The application of methoprene 17 17 , (L.), were more susceptible than Cydia pomonella 38 32 , 16 may require approximately 30 min wileyonlinelibrary.com/journal/ps 28 , D. citri 17 Deterring feeding may be one method is thought to require 5–7 h of feeding D. citri 37 , Similarly, first-instar nymphs of the German 12 6 , . imidacloprid 10 36 , 6 D. citri 6 Pyriproxyfen was also more effective in reducing D. citri These results suggest that methoprene treatment 6 Blattella germanica (L.) (Orthoptera: Gryllidae) only when applied during 34 methoprene could potentially be used in an insecticide 14 , nymphs. under laboratory conditions. In our laboratory investigation, citri The Las pathogen is acquired and transmitted by Methoprene was first registered by the US Environmental Protec- There was no effect of treating fifth-instar nymphs or females . applied to earlier thangence to following treatment later with instars. methoprene Inhibitionamong is well of several documented adult insect emer- species. cockroach, fourth instars whenfenoxycarb. treated with a juvenile hormone analog, citri methoprene did not affecttherefore does the not appear settling to act behavior as a of repellent to this insect. tion Agency in 1975 and hasvariety proven to of be effective insect against pests. apests, It wide as is well particularly as pests useful ofof for livestock stored-product its and domestic low animals, because mammalian toxicity. Juvenile hormone analogs are typically characterized by aof window maximum effectiveness, and inimmatures many exhibit cases greater sensitivity younger than stagesis older of variation stages, but among there species. to decrease the spreadtrations of of methoprene HLB tested in (80–320 citrus. At the higher concen- to two-day-old and six-day-old Torticidae) larvae did not result in reduction in fecundity,cation but to appli- four-day-old larvae did reduce fecundity. would be most effectiveearly instars against during applications.imidacloprid, Our was positive more control effective treatment, againstmethoprene all or stages pyriproxyfen. of directly with methoprenethere on were subsequent sublethal effectsfemales fecundity. that emerged observed However, from treated with fifth-instarthat nymphs reduced or were females fertility treated topically. The of percentageemerging egg hatch from for females treatedconcentration-dependent manner. fifth-instar Egg hatch nymphs was reducedto was for 1 up week reduced when females in wereThese treated results a topically with with methoprene methoprene. against observed with another juvenile hormone analog, pyriproxifen. survival and adultlater emergence instars of when applied to earlier than to rotation when nymphal populationsthe are high transmission in of order pathogenGiven to the reduce by effectiveness emerging of andther methoprene field in mobile testing is laboratory adults. warranted. Registration assays, ofenvironmentally fur- additional and friendly more insecticides formanagement is integration needed. into Las pathogen by on healthy plants. consistent with earlier studies conductedcitri with pyriproxfen on psyllid feeding was reduced. Similarwith effects pyriproxyfen, have been observed effects on nymphalpathogen development is and acquired more eggD effectively by hatch. nymphs As than by the adult HLB choice for management of feeding. Acquisition by of continuous feeding on infected trees. caused abnormalities in the development of embryos of domesticus a limited window of(pronymphal) molt. sensitivity, prior to the second embryonic . 1 − gmL μ Diaphorina 0.05). < P : 1657–1665 © 2015 Society of Chemical Industry 71 females topically treated with various concentrations of 2015; Mean percentage egg hatch per plant for eggs oviposited by Effect of methoprene or pyriproxfen on feeding by 0.05). nymphs. Methoprene was most effective when applied to < P adults at (A) 0–48 h and (B) 49–72 h after placing five adults on each citri In the present study, treatment with methoprene resulted in . the first-instar nymphs andinstars. least Methoprene and effective pyriproxfen resulted when in similar applied mortalitiesof to first, fifth third and fifth instars at concentrations of 128 acute mortality of early instars and delayedD mortality of late-instar Pest Manag Sci Figure 8. citri treated leaf disc. Thewas number quantified of after honeydew (A) droplets 48labeled h per and with filter (B) the paper 72 same disc haccording for letter to each an are concentration. LSD significantly mean Bars different separation not test from ( one another Diaphorina citri methoprene or control (acetoneBars alone) not labeled at with three theicantly different same different time letter from within intervals. one eachtest another time ( according interval to are signif- an LSD mean separation Figure 7. Effects of methoprene on development of Asian citrus psyllid www.soci.org Imidacloprid was highly effective, resulting in 98, 94tality and of 94% mor- first, third andthe fifth highest concentration instars tested respectively. caused 93, For 77of methoprene, and first, 53% third mortality and fifth instars respectively.were Methoprene treatments more effective in suppressing adult emergence when , Tri- Crop et al. :1–11 JEcon 12 Conotra- :395–399 :435–440 :380–384 Diaphorina Candidatus :1531–1541 69 67 (Coleoptera: : 1657–1665 100 :1–6 (1997). Pest Manag Sci J Econ Entomol 71 103 33 after acquisition ,ed.byRobinson :29–36 (2009). (Hemiptera: Cimici- JInsectSci (Coleoptera: Tenebri- 2015; . 155 Insect Control: Biological Pest Manag Sci J Econ Entomol :1066–1072 (2013). 69 J Econ Entomol (Homoptera: Cicadellidae). J Stored Prod Res Diaphorina citri Liberibacter asiaticus by Asian cit- Rhyzopertha dominica Ann Appl Biol Pest Manag Sci :29 (2008). 8 Cimex lectularius :217–224 (2012). 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Pest citrus Manag psyllid, Sci shoots in citrus with implicationswith on flush. monitoring insects associated diamide insecticide, against Asianandfieldconditions. citrus psyllid under laboratory lators, buprofezin and pyriproxyfen,shooter, to the glassy-winged sharp- Annu Rev Entomol imidacloprid on Japanese beetle (Coleoptera: Scarabaeidae)sition, ovipo- egg hatch, and100 larval viability in turfgrass. prene and hydroprene onof survival, six species development of or stored-product insects. reproduction future perspectives of thecontrol use of of stored insect product insects. growth regulators for the Bostrychidae) on wheat, rice and maize. (2011). ( Activity of selected neonicotinoids and dicrotophos on nontarget Enhanced proliferation and efficient transmission of Liberibacter asiaticus by adult feeding in the nymphal stage. sion parameters for rus psyllid (Hemiptera: Psyllidae). (2010). 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Unlike broad-spectrum neurotoxins, methoprene is stage specific andtal will require stages. targeting Field oflaboratory early efficacy results. developmen- testing is warranted, based on these wileyonlinelibrary.com/journal/ps REFERENCES ACKNOWLEDGEMENTS We thank K Addison,I J Neff, Jackson B for Holliday, T Addison, A Hoyte and assistance. The Citrusprovided funding Research for this and study to Development LLS. Foundation Methoprene reduced survival of 5 CONCLUSIONS and effectively reduceda the concentration-dependent survival of manner.reduced early-instar Methoprene adult nymphs emergence significantly in were from more treated sensitiveprene nymphs. to significantly Early methoprene reduced instars thanfrom treated late fertility fifth instars. of instars.reduced Metho- Treatment females fertility of of that adults with femalesless emerged methoprene for on 1 methoprene-treated week. a surfaces than concentration-dependent negative manner.methoprene controls There on in the were settling nosurfaces. behavior of effects The of presentshould investigation effectively suggests reduce that methoprene

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