doublesex Controls Both Hindwing and Abdominal Mimicry Traits in the Female-Limited Batesian Mimicry of Papilio memnon

Abstract

Papilio butterflies are known to possess female-limited Batesian mimicry polymorphisms. In Papilio memnon, females have mimetic and non-mimetic forms, whereas males are monomorphic and non-mimetic. Mimetic females are characterized by color patterns and tails in the hindwing and yellow abdomens. Recently, an analysis of whole-genome sequences has shown that an approximately 160 kb region of chromosome 25 is responsible for mimicry and has high diversity between mimetic (A) and non-mimetic (a) alleles (highly diversified region: HDR). The HDR includes three genes, UXT, doublesex (dsx), and Nach-like, but the functions of these genes are unknown. Here, we investigated the function of dsx, a gene involved in sexual differentiation, which is expected to be functionally important for hindwing and abdominal mimetic traits in P. memnon. Expression analysis by reverse transcription quantitative PCR (RT-qPCR) and RNA sequencing showed that mimetic dsx (dsx-A) was highly expressed in the hindwings in the early pupal stage. In the abdomen, both dsx-A and dsx-a were highly expressed during the early pupal stage. When dsx was knocked down using small interfering RNAs (siRNAs) designed in the common region of dsx-A and dsx-a, a male-like pattern appeared on the hindwings of mimetic and non-mimetic females. Similarly, when dsx was knocked down in the abdomen, the yellow scales characteristic of mimetic females changed to black. Furthermore, when dsx-a was specifically knocked down, the color pattern of the hindwings changed, as in the case of dsx knockdown in non-mimetic females but not mimetic females. These results suggest that dsx-a is involved in color pattern formation on the hindwings of non-mimetic females, whereas dsx-A is involved in hindwing and abdominal mimetic traits. dsx was involved in abdominal and hindwing mimetic traits, but dsx expression patterns in the hindwing and abdomen were different, suggesting that different regulatory mechanisms may exist. Our study is the first to show that the same gene (dsx) regulates both the hindwing and abdominal mimetic traits. This is the first functional analysis of abdominal mimicry in butterflies.

Keywords: Batesian mimicry; Papilio memnon; abdominal mimicry; doublesex (dsx); electroporation-mediated gene knockdown; female-limited polymorphism; supergene; swallowtail butterfly.

Figure 1

(A) Wing and abdominal patterns of Papilio memnon males and non-mimetic and mimetic females. (B) Schematic of doublesex (dsx) isoform in P. memnon. Female isoforms are divided into three types by start to stop codon sequences (F1, F2, F3), and F2 is divided into two types by 3’-untranslated region (UTR) sequences (F2, F2’). There is one male isoform type. The region shown in black is the UTR, and the region shown in blue is the open reading frame (ORF). Red bars indicate the position of the stop codon. The numbers above indicate the exon numbers (exon 1 to exon 6). Exons 3 and 4, which are highly variable among isoforms, are highlighted with a green background. (C) Alignment visualization of the dsx F1 isoform and the target positions of the small interfering RNA (siRNA) and quantitative PCR (qPCR) primers used in this study. There are few SNPs from exon 1 to exon 5, and dsx-A and dsx-a are diversified in exon 6. In exon 6, only some individuals had a 531 bp insertion. The top image shows the sequence similarity between dsx-A and dsx-a visualized by Geneious Prime (2022.0). The middle shows F1 from dsx-a, and the bottom shows F1 from dsx-A. Areas in gray indicate the sequence identity between dsx-a and dsx-A, black indicates single nucleotide polymorphisms (SNPs), and horizontal bars indicate deletions.

Figure 2

(A–D) Gene expression levels in mimetic (dsx genotype: Aa) and non-mimetic (dsx genotype: aa) Papilio memnon females and males (dsx genotype: Aa) at two (P2), five (P5), and ten (P10) days after pupation. The expression levels of dsx-A (A) and dsx-a (B) in the hindwing and dsx-A (C) and dsx-a (D) in the abdomen were estimated by reverse transcription quantitative PCR (RT-qPCR) using RpL3 as an internal control. (E–G) Gene expression levels in mimetic (dsx genotype: Hh) and non-mimetic (dsx genotype: hh) Papilio polytes females and males (dsx genotype: Hh) at P2, P5, and P10. The wing and abdominal color patterns of mimetic and non-mimetic females. dsx-H (E) and dsx-h (F) expression levels in the abdomen were estimated by RT-qPCR using RpL3 as an internal control. (A–F) Yellow, gray, and black bars show the expression levels of mimetic females, non-mimetic females, and males, respectively. Error bars represent standard errors. Different letters indicate significant differences (Tukey’s post hoc test, P<0.05).

Figure 3

(A, B) dsx-A and dsx-a expression levels in Papilio memnon hindwings of mimetic (dsx genotype: Aa; (A) and non-mimetic (dsx genotype: aa; (B) females at two (P2) and seven days after pupation (P7). The mean fragment per kilobase of transcript per million mapped reads (FPKM) values by RNA sequencing (RNA-seq) are shown with SE. Different letters indicate significant differences (Tukey’s post hoc test, P<0.05). Yellow and gray bars show the dsx-A and dsx-a expression levels, respectively. (C–F) Expression levels of each dsx isoform in P. memnon mimetic (dsx genotype: Aa; (C, D) and non-mimetic (dsx genotype: aa; (E, F) females. The mean FPKM values by RNA-seq at P2 and P7 are (C, E) and (D, F), respectively. Orange bars indicate the expression levels of dsx isoforms from the mimetic (A) allele, and gray bars indicate the non-mimetic a allele. F1, F2, and F3 represent the female isoforms 1, 2, and 3, respectively. There were no statistically significant differences between the isoforms.

Figure 4

dsx knockdown in the hindwings of Papilio memnon mimetic (A) and non-mimetic (B) females and males (C). Small interfering RNA (siRNA) targeted the sequence common to dsx-A and dsx-a, and all the female and male isoforms (dsx-common siRNA) were injected into the left pupal hindwing immediately after pupation and electroporated into the dorsal side. dsx knockdown changed the mimetic and non-mimetic female color patterns to resemble the male pattern (A, B), but no phenotypic change was observed in males (C). Pale yellow and red spots disappeared in the knockdown side of mimetic and non-mimetic females, resulting in a phenotype with blue scales on a black background (A, B). The red arrowheads indicate the area that originally had blue scales (untreated side) and where the knockdown appears to have expanded the area of blue scales (dsx siRNA side). Supplementary Figure S3 shows the other replicates. (D, E) dsx-a and dsx-A gene expression levels in the knockdown wings two days after pupation. When dsx was knocked down by dsx-common siRNA, dsx-a was significantly downregulated (D), and dsx-A was downregulated in all two individuals tested (E). White and gray bars show the expression in treated and untreated hindwings, respectively. We estimated the gene expression levels by reverse transcription quantitative PCR (RT-qPCR) using RpL3 as the internal control. (D) Error bars show the standard deviation of three biological replicates. *P<0.05 for Student’s t-test. (E) The expression levels are shown as relative values, with the expression level in the untreated wing taken as 1.

Figure 5

dsx knockdown in the abdomen of Papilio memnon mimetic females. Small interfering RNA (siRNA) targeted the sequences common to dsx-A and dsx-a, and all the female and male isoforms (dsx-common siRNA) were injected into the abdomen during the wandering stage of final instar larvae and electroporated into the fifth abdominal segment. Knockdown changed the yellow scales characteristic of mimetic forms to the black scales seen in non-mimetic forms and males. The red arrowheads indicate the changed area. Supplementary Figures S4 and S5 show the other replicates.

Figure 6

dsx-a knockdown in the hindwings of Papilio memnon mimetic (A) and non-mimetic (B) females, and the abdomen of mimetic females (C). Small interfering RNAs (siRNAs) targeting dsx-a (dsx-a siRNA) were injected into the left pupal hindwing immediately after pupation and electroporated into the dorsal side (A, B) and the abdomen during the wandering stage of the final instar larvae and electroporated into the fifth abdominal segment (C). No distinct phenotypic change was observed in mimetic females (A), but dsx-a knockdown changed the non-mimetic female color pattern to resemble the male pattern (B). As in the knockdown by dsx-common siRNA, pale yellow and red spots disappeared in the knockdown side of non-mimetic females, resulting in a phenotype with blue scales on a black background (B). (C) In the knockdown of the fifth abdominal segment in mimetic females, no phenotypic change was observed. Red dotted circles show the electroporated area. Supplementary Figure S7 shows the other replicates.

Figure 7

Model diagram of the evolution of mimetic dsx (dsx-A) and the acquisition of mimetic traits. Originally, dsx may have helped control sexual dimorphism in the hindwing, but its ancestral function concerning abdominal coloration is unknown, and it may not have been involved in determining abdominal coloration (1. Ancestral function). In P. memenon, the function of dsx in the male wing is not known, and we do not know if the dsx male isoform had any ancestral function in the wing. Thus, the male wing pattern may be a default and ancestral trait, and only female isoform may work to form sexual dimorphism in the wing. In the hindwing, dsx-A differentiation has resulted in the appearance of the mimetic form only in females, and ancestral dsx (i.e., dsx-a) is involved in phenotyping the non-mimetic form (2. Novel functions by evolving mimetic (A) allele). On the other hand, only dsx-A appears to be involved in abdominal coloration, while dsx-a appears to have no function.