Research Article Received: 9 October 2014 Revised: 12 December 2014 Accepted article published: 13 January 2015 Published online in Wiley Online Library: 30 January 2015 (wileyonlinelibrary.com) DOI 10.1002/ps.3975 Effects of methoprene, a juvenile hormone analog, on survival of various developmental stages, adult emergence, reproduction and behavior of Asian citrus psyllid, Diaphorina citri Kuwayama Gurpreet S Brar, Wendy Meyer and Lukasz L Stelinski* Abstract BACKGROUND: The Asian citrus citrus psyllid, Diaphorina citri Kuwayama, transmits a bacterium that causes huanglongbing in citrus. Frequent and repeated use of neurotoxic insecticides against D. citri has resulted in the development of insecticide resistance. We evaluated the effects of the juvenile hormone analog methoprene on egg hatch, nymphal development, adult emergence, reproduction and behavior of D. citri. RESULTS: Methoprene significantly reduced the viability of eggs that were between 0 and 4 days old. Egg hatch of 0–48-h-old and 49–96-h-old eggs was 8 and 9%, respectively, when treated with 320 gmL−1 of methoprene. Methoprene caused significant mortality of first-, third- and fifth-instar D. citri nymphs and reduced adult emergence as compared with controls. Methoprene caused less than 5% adult emergence when first- and third-instar stages were treated, respectively, and less than 40% adult emergence when fifth instars were treated. Reduced fertility of females was observed when they emerged from methoprene-treated fifth instars. CONCLUSION: Methoprene was effective in reducing egg hatch, suppressing nymphal development and decreasing adult emergence of D. citri under laboratory conditions. Treatment of fifth instars reduced the fertility of females. Methoprene might be a possible tool for integrated management of D. citri. © 2015 Society of Chemical Industry Keywords: insect growth regulator; juvenile hormone analog; Asian citrus psyllid; citrus greening; huanglongbing 1 INTRODUCTION their lifetime on young foliar growth. Upon hatching, the nymphs The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: undergo five nymphal instars to emerge as adults. The life cycle Liviidae), is a serious pest of citrus crops worldwide. It trans- duration ranges from 15 to 47 days, depending upon temperature mits a phloem-restricted, gram-negative bacterium, Candidatus regime. The nymphs and adults feed on plant sap and excrete hon- Liberibacter asiaticus (Las), that causes huanglongbing (HLB), also eydew rich in sugars that leads to growth of sooty mold on leaves, referred to as citrus greening.1 – 3 HLB is a devastating disease that which can affect photosynthesis. The pathogen can be acquired causes heavy leaf drop, dieback of stems, premature fruit drop, by second-, third-, fourth- and fifth-instar nymphs, as well as adults, 9 out-of-season flushing and blossoming and eventual tree death during feeding on infected trees. However, the pathogen can only within 5–10 years of initial infection.1 The fruits produced on be transmitted by fourth- and fifth-instar nymphs and adult D. 1,6,10,11 infected trees are small and distorted and have a bitter taste.4,5 citri. Infected adults play a major role in the spread of the In the United States, D. citri was initially reported in June 1998 in pathogen because of their dispersal capabilities. Once infected, 9,11 Florida, and since then it has been reported in Alabama, Arizona, adults remain infective throughout their lives. The pathogen California, Georgia, Louisiana, Mississippi and Texas.6 HLB was multiplies within D. citri and has been reported in hemolymph 9,11 – 13 reported initially in southern Florida in August 2005 and has subse- and salivary glands of adults. Acquisition of Las can also quently spread to all citrus-growing regions of the state. In the past 5 years, HLB has caused approximately $US 3.63 billion in revenue loss and 6611 lost jobs in agriculture and related sectors in Florida.7 ∗ Correspondence to: Lukasz L Stelinski, Entomology and Nematology Depart- Adult D. citri are small (2.7–3.3 mm in length) with mottled ment, University of Florida, 700 Experiment Station Rd, Lake Alfred, FL 33850, USA. E-mail: stelinski@ufl.edu brown wings. There are three adult morphs which differ in abdom- 8 inal color (gray/brown, blue/green and orange/yellow) . D. citri EntomologyandNematologyDepartment,UniversityofFlorida,LakeAlfred,FL, 1657 females are very prolific and can lay approximately 800 eggs in USA Pest Manag Sci 2015; 71: 1657–1665 www.soci.org © 2015 Society of Chemical Industry www.soci.org GS Brar, W Meyer, LL Stelinski occur transovarially from infected females to eggs, but at a low Center, University of Florida. The culture was established in 2000 frequency.14 from a field population collected in Polk County, Florida (28.0′ N, Currently, the spread of HLB is managed by removing infected 81.9′ W), prior to discovery of HLB in Florida. The colony is con- trees, applying fertilizers to maintain tree health and suppressing tinuously maintained on sweet orange [Citrus sinensis (L.) Osbeck] populations of D.citri.7 Multiple applications of a limited number of plants at 27–28 ∘C, 60–65% RH and a 14:10 h (L:D) photope- insecticides of various modes of action are made to reduce D. citri riod. Analytical-grade methoprene (purity 99.2%), pyriproxyfen populations during each growing season. Repeated use of limited (purity 99.0) and imidacloprid (purity 99.9) were procured from available insecticides has resulted in the development of insecti- Sigma-Aldrich (St Louis, MO). The insecticides were diluted in tap cide resistance in some D. citri populations.15 There is an imme- water for spray and dip treatments and in acetone for topical diate need for additional insecticides with novel modes of action treatments. Pyriproxyfen and imidacloprid were used as positive for incorporation into integrated management programs for D. controls. The concentrations of positive controls were selected on citri. These additional insecticides would not only serve as possible the basis of effective concentrations recorded against D. citri in new tools for D. citri management, they could also improve exist- previous studies.6,22 ing rotation with conventional insecticides to impede evolution of insecticide resistance. Insect growth regulators (IGRs) are reduced-risk insecticides 2.2 Effect of methoprene on egg hatch with various modes of action that differ from neurotoxic insecti- The objective of this experiment was to determine the effect of cides. IGRs cause hormonal and biochemical imbalances during methoprene on egg hatch. Forty-five potted ‘Swingle citrumelo’ the development of the target insect, resulting in various mor- plants (Citrus paradisi MacFaden × Poncirus trifoliata L. Raf.), 2–3 phological abnormalities and reduced development of immature months old and with new flush as defined by Hall and Albrigo,23 stages, which leads to reduced emergence of adults. IGRs are were placed into four fine-mesh screen cages (61 × 61 × 91 cm; less harmful to beneficial insects and humans than neurotoxic Bioquip, Ranch Dominguez, CA). Within each cage, approximately insecticides.16 They have been successfully used to control 400 D. citri adults of mixed gender were released for mating and ∘ insect pests of medical and veterinary importance.16 Juvenile oviposition for 48 h at 25 ± 2 Cand50± 5% RH and a 14:10 h hormone agonists, ecdysteriod agonists and chitin synthesis (L:D) photoperiod. After 48 h, the adults were removed from the inhibitors are three commonly used insect growth regulators.17,18 plants. After 48 h or 72 h, leaf flush with eggs was excised to obtain Reduced egg hatch, suppressed nymphal development and 0–48-h-old and 49–96-h-old eggs. Only one flush per plant was decreased adult emergence were observed when various removed. The excised flush was placed individually into 1.5 mL developmental stages of D. citri were treated with pyriproxfen microcentrifuge tubes (Fisher Scientific, Pittsburg, PA) containing [4-phenoxyphenyl (RS)-2-(2-pyridyloxy)propyl ether – a juvenile a 2% agar–water solution. Eggs per individual flush were counted hormone mimic], buprofezin (2-tert-butylimino-3-isopropyl- under the stereomicroscope. The flush samples were then dipped 5-phenyl-3,4,5,6-tetra-hydro-2-thiadiazine-4-one – a chitin in solutions of methoprene at various concentrations: 10, 20, synthetase inhibitor) and diflubenzuron [1-(4-chlorophenyl)-3-(2,6- 40, 80, 160 and 320 μgmL−1 prepared in tap water. Pyriproxyfen difluorobenzoyl) urea – a chitin synthetase inhibitor].5,6 (128 μgmL−1) and imidacloprid (100 μgmL−1) were both included Methoprene [1-methylethyl (E,E)-11-methoxy-3,7,11-trimethyl- as positive controls. Water alone served as the negative control. 2,4-dodecadienoate] is a juvenile hormone analog that has The flush was dipped for 30 s and allowed to air dry. The entire been widely used for insect pest management. It has been used experiment was repeated twice, and each treatment, including the as a mosquito larvacide, as a grain protectant against stored controls, was replicated 12–18 times. The number of eggs per flush insect pests and for control of a wide range of pests, including ranged between 7 and 95. Treated flush samples were placed in Coleoptera, Diptera, Hemiptera and Siphonoptera.16 Methoprene a rearing chamber maintained at 25 ± 2 ∘Cand50± 5% RH and a has been shown to reduce adult emergence of several eco- 14:10 h (L:D) photoperiod. After 5 days, the numbers of hatched nomically important insect pests such as Rhyzopertha dominica, and unhatched eggs were counted using a stereomicroscope. Tribolium castaneum, Triboleum confusum, Aedes aegypti and In the second experiment, contact toxicity of methoprene to Cimex lectularius.17 – 21 External application of methoprene to eggs was assessed by allowing D. citri to oviposit on plants that eggs during blastokinesis is lethal to embryo development, were previously treated with methoprene. Potted ‘Swingle cit- and application during metamorphosis leads to faulty devel- rumelo’ plants with new flush were sprayed until run-off with opment of immature stages.