Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness

Abstract

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt^-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox^-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox^-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

Keywords: Aedes aegypti; corpora allata; juvenile hormone; methyl farnesoate; reproduction.

Fig. 1.

Characterization of jhamt^−/− and epox^−/− mutants. (A) The last two steps of JH biosynthesis in mosquitoes. (B) Schematics of the A. aegypti jhamt and epox genes. Exons are represented by boxes (open parts depict coding regions) connected by introns (solid lines); the dashed line represents the upstream genomic region of jhamt. The large arrows depict the DNA constructs with marker genes inserted via CRISPR/Cas9-mediated HDR into the jhamt (magenta) and epox (turquoise) loci, respectively. The insertion sites are indicated. Small arrows show the orientation and approximate positions of primers used for genotyping WT (black) and mutant (colored) alleles; the amplicon sizes are given. The scheme is not drawn to scale. (C) Brightfield and fluorescent images of L4-instar larvae expressing the dsRed (jhamt^−/−) and eCFP (epox^−/−) markers under the 3xP3 enhancer. (Magnification, 20×.) (D) Examples of PCR genotyping using genomic DNA of WT (^+/+), heterozygous (^+/−), and homozygous (^−/−) mutant larvae. M, molecular size marker; NTC, no template control (arrowheads indicate 1 kb). (E) RT-qPCR analysis. For jhamt, RNA was from five independent samples of individual L2-instar larvae; epox RNA was quantified from three independent samples, each with five CA complexes dissected from 5-d-old adult females. Levels of mRNAs are expressed as copy number per 10,000 copies of rpL32 mRNA. Columns represent mean ± SEM (***P ≤ 0.001, unpaired t test).

Fig. 2.

MF and JH synthesis and hemolymph titers. (A) Average amounts of JH III synthesized by 10 individual CA complexes from L4-instar WT and jhamt^−/− larvae. (B and C) Average amounts of MF synthesized by 10 individual CA complexes from L4-instar jhamt^−/− larvae (B). Average amounts of MF (B) or JH III (C) synthesized by three CA complexes dissected from four independent groups of sugar-fed WT or epox^−/− adult females aged 5 to 6 d. (D) JH III titers in the hemolymph (four independent replicates) pooled from eight sugar-fed WT or epox^−/− adult females aged 5 to 6 d. n.d., levels not detectable (below 0.5 fmol). Columns represent mean ± SEM in all panels. Significant differences are marked with asterisks (***P ≤ 0.001, unpaired t test); letters above columns indicate in B, P ≤ 0.05 (one-way analysis of variance with Tukey’s post hoc comparison).

Fig. 3.

Phenotypes of wild-type (WT) and null mutant larvae. (A) Light microscope images of live, late L4-instar larvae. (Scale bar, 1 mm.) (B) Average larval body size at the L4 S stage (see also SI Appendix, Figs. S6, S8, and S9). ImageJ software was used to measure the body length of individual larvae, represented by the data points. Lines denote mean values, letters above them indicate significant differences (P ≤ 0.05, one-way ANOVA with Tukey’s multiple post hoc comparison).

Fig. 4.

Developmental expression of JH-response genes in jhamt and epox null mutants. (A) Kr-h1, (B) E93, and (C) Br-C. Expression of the mRNAs was determined by RT-qPCR in whole body of larvae of the indicated instars; the final-instar stages were day 1 (L4 d1), day 2 (L4 d2), and L4-instar larvae with visible siphons 6 to 7 h before pupation (L4 S). Levels of mRNAs are expressed as copy number per 10,000 copies of rpL32 mRNA. Each data point is average of three to seven independent biological replicates, each from three larvae. Columns represent mean ± SEM in all panels. Significant differences are marked with letters above columns (P ≤ 0.05, one-way ANOVA with Tukey’s multiple post hoc comparison; ns, nonsignificant).

Fig. 5.

Reproductive fitness cost in EPOX-deficient females and the effect of Met. (A) Examples of mature previtellogenic follicles from WT and epox^−/− females that had been treated with Met (meth) or solvent (ac) at 1 to 4 h after adult eclosion. Note lipid accumulation (white arrows) except in the oocytes of untreated epox^−/− females. (Scale bars, 100 μm.) (B and C) Lengths and numbers of mature primary previtellogenic follicles from females of the indicated genotypes with or without the prior Met treatment. Each point represents an average of independent length measurements of 10 follicles from an ovary (n = 10 females) (B), or the number of follicles per ovary (n = 10 to 20 females) (C). (D) Number of eggs laid by WT and epox^−/− females during two gonotrophic cycles. Each point represents the number of eggs laid by a female (n = 35 to 50 females). Lines in the scatter plots represent mean values, letters above them mark significant differences (P ≤ 0.05, one-way ANOVA with Tukey’s multiple post hoc comparison).

Fig. 6.

Reproductive fitness cost in male epox^−/− mutants. (A) Numbers of eggs laid by WT females inseminated by epox^+/+ (WT) males or their epox^−/− siblings. Each point represents the number of eggs laid by a female (n = 20, line represents the mean). ns, nonsignificant. (B and C) Mating competition between epox^+/− and epox^−/− males. Five males of each genotype were allowed to mate with five WT females in a mating cage. After genotyping of the larval progeny by the eCFP transgene that marks the epox null mutant allele, the number of females inseminated (B) and the numbers of larvae fathered (C) by males of either genotype were evaluated. Columns represent mean ± SEM of 10 independent assays (***P ≤ 0.001, unpaired t test).