Molecular Insights into Structural and Ligand Binding Features of Methoprene-Tolerant in Daphnids.

Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.

Autodissemination of Insect Growth Regulator, Methoprene, with Two Formulations Against Aedes albopictus.

Recent autodissemination studies have popularly favored pyriproxyfen, but methoprene is already established and widely used in control programs. This study demonstrated that methoprene could also autodisseminate through contaminated gravid females. Arenas of oviposition cups were arranged around methoprene-contaminated bait stations. Gravid female Aedes albopictus were released to allow oviposition. All cups were then removed and tested in an insect growth regulator bioassay. Liquid formulations did not result in adequate autodissemination among treatment groups. Granular formulations pulverized into a fine powder resulted in successful autodissemination. Emergence inhibition was recorded as high as 85% in individual assays. Pooled inhibition across the granular trials was 56.7%. Methoprene has benefits similar to pyriproxyfen use and has the added advantage of already being established and readily available in existing mosquito control programs throughout the USA. Methoprene is a good candidate for continued investigation of autodissemination.

Juvenile hormone (JH) esterase of the mosquito Culex quinquefasciatus is not a target of the JH analog insecticide methoprene.

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

Non-selective toxicological effects of the insect juvenile hormone analogue methoprene. A membrane biophysical approach.

The Gram-positive bacterium, Bacillus stearothermophilus, was used as a model organism to identify the non-selective toxic effects of the currently used insecticide methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate). A significant decrease of the yield of bacterial cultures and a premature appearance of ultrastructural abnormalities in cells cultured in the presence of the insecticide were taken as indicators of cytotoxicity. A putative correlation of this cytotoxicity with methoprene-induced perturbations on membrane lipid organization was investigated, using differential scanning calorimetry and the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH-PA). The membrane physical effects depended on the lipid bilayer composition and packing. The most striking effect was a progressive broadening and shifting to lower temperatures, with increasing methoprene concentrations, of the main transition phase of the dimyristoyl- or dipalmitoylphosphatidylcholine bilayers and of the lateral phase separation of liposomes reconstituted with the lipid extracts of B. stearothermophilus.

Electrochemical impedance spectroscopy for the study of juvenile hormones-recombinant protein interactions.

The interactions of recombinant juvenile hormone binding protein (His8-rJHBP) with juvenile hormones (JHs), methoprene and farnesol have been studied with electrochemical impedance spectroscopy (EIS). The protein was immobilized on the dodecanethiol (DDT) modified gold electrodes. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The conformation changes of His8-rJHBP upon JHs and methoprene binding have been presented. The EIS determined association constants in the JHs analogs-immobilized His8-rJHBP system indicate that lack of the epoxide moiety in methoprene molecule is not critical for observed high affinity of this compound to the binding region of the His8-rJHBP protein.

Identification and characterization of a juvenile hormone response element and its binding proteins.

Juvenile hormones (JH) regulate a wide variety of developmental and physiological processes in insects. Comparison of microarray data on JH-induced genes in the fruit fly, Drosophila melanogaster, L57 cells and in the honey bee, Apis mellifera, identified 16 genes that are induced in both species. Analysis of promoter regions of these 16 D. melanogaster genes identified DmJHRE1 (D. melanogaster JH response element 1). In L57 cells, the reporter gene regulated by DmJHRE1 was induced by JH III. Two proteins (FKBP39 and Chd64) that bind to DmJHRE1 were identified. FKBP39 and Chd64 double-stranded RNA inhibited JH III induction of a reporter gene regulated by DmJHRE1. FKBP39 and Chd64 proteins expressed in yeast bound to DmJHRE1. Two-hybrid and pull-down assays showed that these two proteins interact with each other as well as with ecdysone receptor, ultraspiracle, and methoprene-tolerant protein. Developmental expression profiles and JH induction of mRNA for FKBP39 and Chd64 proteins and their interaction with proteins known to be involved in both JH (methoprene-tolerant protein) and ecdysteroid action (ecdysone receptor and ultraspiracle) suggest that these proteins probably play important roles in cross-talk between JH and ecdysteroids.

Methoprene.

A brief overview is presented of the discovery and development of s-methoprene and some other juvenile hormone mimics. The identification of the natural juvenile hormones is described along with an outline of the part they play in the hormonal control of insect development. The properties and commercial applications of s-methoprene are presented with emphasis on its use in mosquito control and its minimal impact on the environment.

Effects of retinoids and juvenoids on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus.

Retinoic acid and insect juvenile hormone (JH) are structurally related terpenoids which are widespread in nature and are involved in many biological events such as morphogenesis, embryogenesis and cellular differentiation. Here, we investigated the effects of the retinoids 9-cis retinoic acid (9cisRA), all trans retinol (atROH), all trans retinoic acid (atRA) and the juvenoids methoprene (Met) and JH injection on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus. Overall, we observed that injection of retinoids or juvenoids (120 pmols) in the hemocoel of 4th instar nymphs reduced the percentage of insects which appeared normal in morphology upon moult. Noteworthy, insects exposed to 9cisRA or JH underwent profound morphological changes upon moult, generating abnormal 5th instar nymphs and also markedly increased the death of insects during the moulting process. In addition, reduction in the percentage of insects that moult without any morphological alteration, induced by retinoids or juvenoids treatment, was negatively correlated with insects that both display abnormal moult and those that die during moult. Hemolymphatic phenoloxidase activity in adult male insects injected with 9cisRA, Met and JH were significantly reduced after a bacterial challenge. Together, these results indicate that not only juvenoids but also retinoids play an important role on morphogenesis and on immune response in R. prolixus, suggesting that the molecular mechanisms involved in these events recognize the terpenoid backbone as an important structural determinant in insects.

Comparison of radioimmunoassay and liquid chromatography tandem mass spectrometry for determination of juvenile hormone titers.

This paper compares the results of juvenile hormone (JH) titer determinations in two insect species, Melanoplus sanguinipes, a migratory grasshopper, and Acyrthosiphon pisum, the pea aphid, using a chiral-specific JH radioimmunoassay (RIA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), after extraction of JH with either hexane or isooctane-methanol. We compared results of JH titer determinations done on extracts of M. sanguinipes hemolymph taken from animals flown to exhaustion in tethered flight tests or unflown controls and from whole body extracts of A. pisum raised at two different temperatures. In each case the two different treatments experienced by the experimental animals were expected to result in widely differing JH titers. Methoprene and precocene II were used as internal standards. Samples were split and titers determined simultaneously with both the LC-MS/MS and RIA procedures. Unambiguous detection of JH III by LC-MS/MS was done by identification of its specific parent ion and its mass fingerprint (m/z 289, 267, 249, 235, 217, and 189). We conclude that isooctane-methanol-extracted JH samples can be accurately analyzed by LC-MS/MS, but not by RIA without further separation of JH from contaminating lipids. Hexane extracted JH samples from hemolymph can be analyzed accurately by both RIA and LC-MS/MS. However, the RIA results from whole body extracts of aphids reared at two different temperatures were initially obscured with excess lipids even when hexane was the extraction solvent. Thus samples were further purified by Waters Sep-Pak C18 column, but contaminating phospholipids continued to cause problems with the RIA assay. The detection limit of JH III standard for RIA was 13.75+/-2.39 pg whereas that for LC-M/MS was 8.25+/-1.44 pg in our experimental conditions.

The antifouling activity of some juvenoids on three species of acorn barnacle, Balanus.

Analogues of insect juvenile hormones (juvenoids) have been tested for settling inhibition of cyprids from three species of barnacle, Balanus balanoides (L.), Balanus improvisus Darwin and Balanus amphitrite Darwin. Some 3-alkoxypyridine derivatives exhibited strong activity at mg litre(-1) concentrations; 3,7-dimethyloctyl 2-methyl-5-pyridyl ether (8) gave an EC(50) of 0.006 mg litre(-1) when tested on B. balanoides. When compound 8 and similar juvenoids were incorporated into paints, test panels kept in seawater for 8 months were free of barnacles.

Thermal behaviour, biological activity and conformational study of a [methoprene/beta-cyclodextrin] complex in a smoke generating formulation.

Methoprene, an insect growth regulator, was complexed with beta-cyclodextrin, yielding a stable inclusion complex. TGA, X-ray powder diffraction and conformational analysis have been used to confirm the nature of this inclusion complex. The interaction between methoprene and beta-cyclodextrin was investigated by means of Molecular Mechanics. The results account for the formation of a 1:1 inclusion complex stabilised by Van der Waals forces and hydrogen bonds. The [methoprene-beta-cyclodextrin] complex included in smoke generating formulations and protected from thermal decomposition by the foaming agent azodicarbonamide was shown to be stable enough to release methoprene in fumes with good yields. The improved stabilty of the methoprene complex showed a correlation with increased biological activity against Musca domestica.

Activity of new formulations of methoprene against midges (Diptera: Chironomidae) in experimental ponds.

The IGR, methoprene, in 3 solid (Altosid Pellet, Altosid XR Briquet and a granular) and a liquid formulation (A.L.L.) was evaluated against chironomid midges in experimental ponds. The A.L.L. was applied at 0.293 liters/ha (0.015 kg AI/ha) and at 5.86 liters/ha (0.28 kg AI/ha). The granules, pellets and briquets were applied at 13 kg/ha (0.17 kg AI/ha), 5.6 kg/ha (0.22 kg AI/ha) and one briquet/8m2 (0.82 kg AI/ha), respectively. The low rate of A.L.L. was ineffective, but the high rate produced 84-100% control of Tanytarsini and 30-100% of Chironomini during 2 wk posttreatment. The granular formulation gave 61-87% control of total midges in 2 wk posttreatment. Altosid Pellets gave initial and prolonged good control of Tanytarsini (64-99% for 7 wk), Chironomini (79-94% for 5 wk) and total midges (64-98% for 7 wk). The briquets at almost 4x the rate of pellets, yielded 38-98% control of midges for 7 wk posttreatment. Altosid Pellets have an excellent potential for midge control.

Efficacy and persistence of sustained-release methoprene pellets against Aedes mosquitoes in an irrigated pasture.

The efficacy and persistence of sustained-release methoprene (Altosid) pellets were evaluated at rates of 3.4 and 9.0 kg/ha against Aedes mosquitoes through 7 flood cycles (126 days) in an irrigated pasture. At both rates, the pellets provided greater than 98% control through 2 flood cycles, or 20 days posttreatment, and greater than 80% control through 5 flood cycles, or 69 days posttreatment.