Figure 1.
Chemical structures of juvenile hormone (JH) and JH analog (JHA) insecticides investigated in this study.
Seven forms of JH have been isolated from insects; all posses a methyl ester at one end of the molecule and epoxide at the other (A). JH III is the principal form of JH that is found in dipteran insects. JHA insecticides (B) are structural and/or biological mimics of JH.
Figure 2.
Effects of the juvenile hormone (JH) analog insecticide methoprene on mosquito development.
Under normal conditions 4th instar larval mosquitoes undergo a larval-pupal molt following a rapid reduction in hemolymph JH levels and concurrent spikes in molting hormone. When 4th instar Cx. quinquefasciatus are exposed to methoprene at exceptionally low levels (60 to 120 ng liter−1) unique morphologies are observed including larval-pupal monsters (A) and insects that are unable to complete pupal-adult eclosion (B).
Figure 3.
Phylogenetic relatedness of CqJHE and JHE sequences from six insect orders.
The phylogenetic analysis was performed using MEGA version 5.05 [49]. The tree was generated by the Neighbor-Joining method using a ClustalW generated alignment of 14 JHE or putative JHE sequences. The percentage of replicate trees in which the sequences clustered together in the bootstrap analysis (1000 replicates) is shown at the branch nodes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances (computed using the Poisson correction method) used to infer the phylogenetic tree. The double dagger (‡) and dagger (†) indicate proteins that show a specificity constant (kcat/KM) for JH III that is greater than or less than 106 M−1 s−1, respectively. The asterisk (*) indicates that the specificity constant of the protein is uncharacterized. The insect order, GenBank accession number, and key reference of the sequences are as follows. Diptera: CqJHE (JN251105), AaJHE (EAT43357) [23], DmJHE (AF304352) [24]; Orthoptera: GaJHE (EF558769) [31]; Coleoptera: PhJHE (AB259898) [50], TmJHE (AF448479) [51], TcJHE (NP_001180223) [30]; Lepidoptera: CfJHE (AF153367) [52], HvJHE (AF037197) [53], HaJHE (FJ997319) [54], MsJHE (AF327882) [55], BmJHE (AF287267) [56]; Hymenoptera: AmJHE (AY647436) [57]; and Hemiptera: NlJHE (EU380769) [58].
Table 1.
Kinetic properties of CqJHE for ρ-nitrophenyl acetate, α-naphthyl acetate, and JH IIIa.
Figure 4.
LC/MS/MS analysis of the metabolism of methoprene by CqJHE.
In these assays 25 or 500 pmol of methoprene was incubated with 0.4 µg (6.3 pmol) of CqJHE (an amount that was sufficient to form 7,200 pmol of JH III acid under the same incubation conditions) for 15 min at 30°C. At the end of the incubation period, the reaction was stopped by the addition of methanol, and the amount of methoprene remaining was analyzed by LC/MS/MS. CUDA (12-(3-cyclohexylureido)dodecanoic acid) was used as an internal standard for LC/MS/MS. The error bars indicate the standard deviation of the mean of three independent experiments. No metabolism of methoprene was detected under the conditions tested.
Figure 5.
Effect of methoprene and other JHAs on the specific activity of CqJHE.
The ability of methoprene and other JHAs to compete with JH III for the substrate binding pocket of CqJHE was determined in a reaction containing 2 ng (i.e., 32 fmol) of CqJHE and 5000 pmol of JH III or JHA (A) or 0.32 ng (i.e., 5.3 fmol) of CqJHE and 500 pmol of JH III and 5000 pmol of JHA (B). The error bars indicate the standard deviation of the mean of three independent experiments. Significant differences (P<0.001) in CqJHE specific activity between control reactions containing ethanol and experimental reactions containing a JHA are indicated by the asterisk (*).