Paralogous genes involved in juvenile hormone action in Drosophila melanogaster

Abstract

Juvenile hormone (JH) is critical for multiple aspects of insect development and physiology. Although roles for the hormone have received considerable study, an understanding of the molecules necessary for JH action in insects has been frustratingly slow to evolve. Methoprene-tolerant (Met) in Drosophila melanogaster fulfills many of the requirements for a hormone receptor gene. A paralogous gene, germ-cell expressed (gce), possesses homology and is a candidate as a Met partner in JH action. Expression of gce was found to occur at multiple times and in multiple tissues during development, similar to that previously found for Met. To probe roles of this gene in JH action, we carried out in vivo gce over- and underexpression studies. We show by overexpression studies that gce can substitute in vivo for Met, alleviating preadult but not adult phenotypic characters. We also demonstrate that RNA interference-driven knockdown of gce expression in transgenic flies results in preadult lethality in the absence of MET. These results show that (1) unlike Met, gce is a vital gene and shows functional flexibility and (2) both gene products appear to promote JH action in preadult but not adult development.

Figure 1.—

Northern hybridization analysis of gce transcripts. RNA was isolated from whole bodies at various developmental ages. Five micrograms total RNA was electrophoresed and transferred to nylon membranes. The membranes were hybridized with DIG-labeled gce, Rp49, and rRNA probes. The identity of the 7.5-kb minor band is undetermined.

Figure 2.—

RT–PCR analysis of Met and gce transcripts from selected tissues. The first-strand cDNA was synthesized, and PCR was performed with primer pairs for gce, Met, and Rp49 genes in adult brain, ovary, male accessory gland (MAG), and third instar larval fat body. Values are standard error of the mean (SEM) of three separate determinations expressed relative to those of Rp49. The Met PCR primer sequences are given in Barry et al. (2008).

Figure 3.—

(A) Expression of gce and rp49 in UAS-gce overexpressing strains. Expression was measured by RT–PCR in both larvae and adults. (B) Expression values for gce and rp49 in gce-overexpressed larvae and adults. Values are normalized to those of +; UAS-gce/TM6 sibs. Each value is the standard error of the mean (SEM error bar) of three determinations of larvae or adults of the indicated genotype.

Figure 4.—

Enhanced methoprene pupal toxicity of gce-overexpressing strains. gce refers to UAS-gce driven by GAL4-actin, GAL4-tubulin, or GAL4-LFB (larval fat body) promoters. Each value is the standard error of the mean (SEM error bar) of 30 pupae, three determinations. Pupal survival was defined as eclosion. Methoprene doses are in micrograms/vial.

Figure 5.—

Rescue of resistance to methoprene-induced failure of male genitalia rotation during pupal development. (A) Normal-appearing genitalia phenotype in w Met²⁷; UAS-gce/TM6 male exposed to 5.4 μg/vial methoprene during larval development. The absence of Met⁺ protects the fly from methoprene-induced malrotation, and the appearance is normal, having the anal plates posterior and the penis apparatus anterior. (B) Abnormal-appearing genitalia in Met²⁷; UAS-gce/tubulin-GAL4 adult exposed to 0.027 μg/vial methoprene. The genitalia has failed to complete the normal 360° rotation by ∼100°, an effect expected in methoprene-treated flies carrying Met⁺.

Figure 6.—

Rescue of eye phenotype in Met^W3 flies. (A) Nonconditional eye phenotype of disrupted posterior facets (evidenced by darkening) in w Met^W3; UAS-gce/TM6 adult. (B) Complete rescue of eye phenotype in w Met^W3; UAS-gce/tubulin-GAL4 adult. The light-red eye color in A results from the w⁺-carrying UAS-gce transgene, and the darker-red color in B from the additional w⁺-carrying tubulin-GAL4 transgene.

Figure 7.—

Fertility of adults overexpressing gce. (A) Eggs laid per female during a 4-day window of optimal oviposition. Each value is the mean of 30 females of the indicated genotype. (B) Males fertilizing newly encountered wild-type females during the indicated time periods; values are cumulative. Met⁺/Y; UAS-gce/tubulin-GAL4 males were tested only at the 0- to 1-hr period. Each value is the mean of 20 males, five replicates.

Figure 8.—

(A) Expression of gce and rp49 in UAS-dsRNA gce larvae having the designated GAL4 promoter or TM6 balancer chromosome as measured by RT–PCR. O-RC, Oregon-RC wild-type strain. (B) Expression values for gce and rp49 in Met²⁷; UAS-gce-dsRNA larvae. Values are normalized to those in Oregon-RC wild-type larvae. Values are standard error of the means (SEM error bars) of three determinations of larvae of the indicated genotype.