Figure 1.
Focal stag beetle Cyclommatus metallifer.
(A) Intraspecific sexual dimorphism and male variation in Cyclommatus metallifer. Female (left), small male (center), and large male (right) are shown. Scale bar indicates 20 mm. (B) This species exhibits strong sexual dimorphism of mandibles between the sexes. Mandibles of female (left) and large male (right) are shown. This difference in size is a result of male-specific disproportionate mandibular growth. Scale bar indicates 10 mm. (C) Maxilla are not sexually dimorphic. Maxilla of female (left) and large male (right) are shown. Scale bar indicates 2 mm.
Figure 2.
Developmental staging chart of prepupal development from the larval-prepupal transition to pupation.
Mandibular growth (cell proliferation) is indicated on the y-axis over developmental time in days on the x-axis (hashmark = 1 day). Three distinct forms are recognized - the last instar larva, the prepupa, and the pupa. Known landmarks of mandibular proliferation in large males are indicated by the red circles and shown in diagram form below the graph [5]. The end of the larval period and the initiation of the prepupal period is indicated by Pupal Cell Construction (PCC) and is defined as the starting point of prepupal Stage 1. Outwardly the larva does not change its morphology. Stage 1 lasts approximately 2 days until the initiation of the first Gut Purge (GP) in which the prepupa begins to transform and empties out half of its gut contents. The time the prepupa spends in the first GP is known as Stage 2. Stage 3 is a quiescent phase where the prepupa undergoes massive adult imaginal tissue proliferation but outwardly appears suspended in the first GP. Stage 4 occurs over only a few hours and begins with the second GP in which the last remnants of the gut contents are egested and the prepupa completely metamorphoses into the pupa.
Figure 3.
Characterization of the Cyclommatus metallifer dsx transcript.
(A) Predicted gene models C. metallifer doublesex (Cmdsx) from transcripts. Four alternative splice variants were found and designated Cmdsx splice variants A, B, C and D. The coding sequence is in light gray, the conserved DM domain is in black, and the predicted dsx dimer formation site is in dark gray. Arrows indicate forward and reverse primer locations on the sequences and the region that was synthesized for dsRNA is indicated. (B) RT-PCR using exon-specific primers. Template cDNAs were derived from Stage 2 mandibles of both sexes. Transcripts A and B are male specific in expression and C and D are female specific in expression. (C) Temporal and spatial expression patterns of Cmdsx exon 1 in developing mandibular and maxillar tissues from small males, large males and females during the prepupal period. Total Cmdsx expression is shown by exon 1 as it is common to all four Cmdsx splice forms. Maxillae were used as a control trait as they show isometric growth both in males and females and do not show strong sexual dimorphism in C. metallifer. Relative expression of exons 4, 6, 8, & 9 are shown from Stage 2 male and female mandibles. The averages and 95% confidence intervals of three technical replicates are shown. For each exon, different small-case letters indicate significant differences (Tukey-Kramer test, P<0.05).
Figure 4.
Intersex phenotypes of dsxRNAi in females and males.
(A, E) Adult phenotypes of a dsxRNAi and control GFPRNAi individuals of both sexes. Scale bars indicate 10 mm. (B, F) The relationship between prothorax width (X-axis) and mandible length (Y-axis) for GFPRNAi individuals (closed circles) and dsxRNAi individuals (open circles) in females (pink) and males (blue). (C, G) Adult tibial phenotypes of dsxRNAi individuals. Arrowheads indicate female-specific tibial spines. Foreleg tibia of GFPRNAi female which has several female-specific spines. The foreleg tibia of a dsxRNAi female; note that the number of female-specific spines decreased in comparison to the GFPRNAi female. The foreleg tibia of a dsxRNAi male in which female-specific spines are seen. The foreleg tibia of a GFPRNAi male that does not show female-specific spines. Scale bars indicate 2 mm. (D, H) Adult genital phenotypes of dsxRNAi individuals. Dorsal view of genitalia of GFPRNAi female, dsxRNAi female, dsxRNAi male and GFPRNAi male. Schematic views of the genital plates are indicated next to the photographs. Genital plates that are homologous in males and females are indicated in the same color. Scale bars indicate 2 mm.
Figure 5.
Effects of JHA application on GFPRNAi and dsxRNAi individuals.
The relationships between pupal weight and mandible length were described. Sex is indicated by color of symbols (male: blue and light blue, female: pink and orange). Hormone treatments are indicated by shape of symbols (acetone treatment: circle, JHA treatment: square). RNAi treatments are distinguished by closed (GFP dsRNA injection) or open (dsx dsRNA injection). Scale bars indicate 10 mm (in males) or 5 mm (in females).
Figure 6.
Schematic view of developmental link between nutrition and sex via JH signaling for sex-specific exaggerated trait development in the stag beetle.
In addition to JH and Dsx, other possible factors were described which might be involved in integration of spatio-temporal information. Nutrition information is mediated by JH signaling and promote mandible enlargement in males. DsxM (CmDsxA and CmDsxB) might play a promoting role for JH-dependent mandible enlargement or recruiting other signaling pathway such as insulin signaling pathway (ISS). In females, DsxF (CmDsxC and CmDsxD) inhibit JH effect by reducing the JH sensitivity of mandible cell.