Research ArticleORGANISMAL BIOLOGY

Prepatterning of Papilio xuthus caterpillar camouflage is controlled by three homeobox genes: clawless, abdominal-A, and Abdominal-B

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Science Advances  10 Apr 2019:
Vol. 5, no. 4, eaav7569
DOI: 10.1126/sciadv.aav7569
  • Fig. 1 Sampling strategy in RNA-seq experiment.

    (A) Body color patterns shown in the fourth and fifth instar stage. The dorsal view of the fourth instar larva (black/brown mimetic pattern) is shown on the left. The dorsal view of the fifth instar larva (green pattern) is shown on the right. The enlarged side views in the middle represent the areas where the fourth and fifth instar larvae are framed by black dashed lines, respectively. The colored lines indicate the sampling regions in the fourth instar stage. The red oval shows the eyespot region (denoted as E), the purple box shows the white region (denoted as W), and the orange box shows the black region (denoted as B). The colored dashed lines are the corresponding regions in the fifth instar after the body color switch. A3, A4, and A5: the third, fourth, and fifth abdominal segments. Scale bars, 5 mm. (B) Sampling time during the JH-sensitive period. The third, fourth, and fifth instars indicate the larval development stage of P. xuthus. The JH-sensitive period is the first 20 hours of the fourth instar. The blue line indicates the presumptive JH titer (16). The red dots represent the sampling time points at 0, 6, and 12 hours during the JH-sensitive period. The fourth intermolt period lasts for 2 to 3 days, and the molting period lasts for about 20 hours. The gray box indicates the pigmentation process. HCS, head capsule slippage. (C to E) DEG numbers in three sampling regions of different sampling time combinations. The central white number indicates the 20 candidate genes. Photo credit: H.J.

  • Fig. 2 Expressions of cll, abd-A, and Abd-B by qRT-PCR.

    (A) Relative expressions of cll, abd-A, and Abd-B in larval epidermis using the same sampling regions and time points as in the RNA-seq experiment. n = 3. Error bars indicate SD. Tukey’s test, *P < 0.05 and **P < 0.01. (B) Relative expressions of abd-A and Abd-B in larval segments T3 to A6. The samples were dissected as shown in fig. S5. The expression of a certain gene in a particular segment was calculated as the sum of the relative expressions of that gene within that particular segment. n = 3. Error bars indicate SD. (C) The sampling regions used for detecting the precise gene expression in different color patterns. (D) Relative expressions of abd-A and Abd-B by presumptive fifth color patterns during the fourth instar. The expression of a certain gene in a particular color pattern was calculated as the sum of the relative expressions of that gene within that color pattern. Error bars indicate SD. Student’s t test, *P = 0.0325. rpL3 gene was used as the inner control. D, the dorsal region that is black and lies between segments A2 and A3; W, the white region; M, the region that lies across the white region and the presumptive V-shaped marking across segments A4 and A5; G, the region that lies next to the posterior side of the presumptive V-shaped marking in segment A5; A, anterior; P, posterior.

  • Fig. 3 Electroporation-mediated RNAi.

    (A) Measurement of knockdown effect using qRT-PCR. After injection of siRNA and electroporation, the epidermal samples were dissected at hour 12 of the fourth instar. Relative expressions of cll (n = 6), abd-A (n = 5), and Abd-B (n = 5) were measured. Student’s t test, *P < 0.05. (B) Injection of siRNA was performed at the end of the third instar, followed by observation during the fourth and fifth instars. Knockdown of cll was performed on the T3 segment (black dotted box). The blue dashed oval indicates the corresponding region of the eyespots in the fourth and fifth instars. The asterisk indicates that the complicated lines around it have changed after RNAi. The hollow arrowhead shows the si-cll side where the presumptive eyespot changed in size after RNAi. The filled arrowhead shows the control side where no phenotypic changes were detected. (C) Measurement of the red area in the E region after RNAi of cll. The cuticular samples were collected (n = 3), and the size for each region was measured using ImageJ. Student’s t test, *P < 0.05. Photo credit: H.J.

  • Fig. 4 Electroporation-mediated RNAi of abd-A and Abd-B.

    Injection of siRNA was performed at the end of the third instar, followed by observation during the fourth and fifth instars. (A) Phenotype of the fourth instar larva after knockdown of abd-A gene (n = 4). (B) Phenotype of the fourth instar larva after knockdown of Abd-B gene (n = 17). (C) Knockdown of abd-A on the segments A4 and A5. The enlarged side views show segments A4 to A6 of the experimental and control side (black dotted box). The hollow purple arrowhead indicates the regions where formation of the V-shaped marking was completely inhibited after knockdown of abd-A, comparing the control side indicated by a filled purple arrowhead. (D) Knockdown of Abd-B gene on segments A5 and A6 (black dotted box). The dashed purple arrowhead indicates the regions where the ectopic V-shaped marking appeared after knockdown of Abd-B in segment A6. (E) Knockdown of Abd-B gene after the JH-sensitive period. No phenotypic change was observed. Photo credit: H.J.

  • Fig. 5 Effects of JHA addition on prepatterning genes.

    (A) The expression changes of cll, abd-A, and Abd-B after the addition of fenoxycarb (JHA) using RNA-seq (n = 2, denoted as fenoxycarb1 and fenoxycarb2). The black bar indicates the control group. The green bar indicates JHA-treated samples. Krüppel homolog 1 (Kr-h1) gene, a known JH response gene, was used to compare the expression before and after the addition of JHA. RPKM, reads per kilobase million. (B) Phenotype of the fifth instar larvae after JHA addition on the T3 segment. Fenoxycarb (5 μg per individual) was artificially applied around the T3 segment at the fourth instar stage (n = 3). The purple arrowhead shows the corresponding red area. (C) Measurement of cll gene expression after JHA addition using qRT-PCR. The epidermal samples around the E region were dissected at hour 12 of the fourth instar (n = 3). Student’s t test, **P < 0.01. Photo credit: T.S.

  • Fig. 6 A schema of color pattern switching in P. xuthus larva.

    The fourth instar larva displays bird-dropping mimetic color patterns. The fifth instar larva displays a greenish cryptic color pattern. Two processes are necessary for the body color pattern switch, and there is a 2- to 3-day developmental interval between the two processes. First, decline of the JH titer [dotted line; the JH titer is from (16)] in the hemolymph starts the prepatterning process in the first 20 hours of the fourth instar. During this process, spatial-specific expressions of prepatterning genes such as cll, abd-A, and Abd-B are detected, and RNAi of those genes inhibits normal pattern formation in the fifth but not the fourth instar (red arrow). The eye mark indicates the timing for phenotype observation. However, once the fifth color patterning is determined (at least beyond the JH-sensitive period), RNAi of prepatterning genes could not change the color patterning (blue arrow). Second, a pigmentation process occurs during the molting period as regulated by the ecdysteroid cascade [dash-dotted line; the 20E titer is from (17)]. RNAi of pigmentation genes, such as Lac2, inhibits melanization in both the fourth and fifth instars (black arrow). During this process, pigmentation genes, such as melanin synthesis–related genes, carotenoid-binding protein (CBP) genes, and bilin-binding protein (BBP) genes, are expressed in the prepatterned regions, which contribute to the visible color transition after the fourth molt.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaav7569/DC1

    Fig. S1. Pairwise comparisons of gene sets among sampling regions and time points.

    Fig. S2. Heat maps of DEGs in color patterns.

    Fig. S3. Spatial-temporal expression of candidate genes from RNA-seq.

    Fig. S4. Phylogenetic analysis of abd-A, Abd-B, and cll.

    Fig. S5. The basal data and detailed sampling regions for qRT-PCR.

    Fig. S6. Knockdown effect of cll.

    Fig. S7. Knockdown effect of Abd-B.

    Fig. S8. Knockdown of the pigmentation-related gene Lac2.

    Table S1. Detailed BLASTx results of 20 candidate genes.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Pairwise comparisons of gene sets among sampling regions and time points.
    • Fig. S2. Heat maps of DEGs in color patterns.
    • Fig. S3. Spatial-temporal expression of candidate genes from RNA-seq.
    • Fig. S4. Phylogenetic analysis of abd-A, Abd-B, and cll.
    • Fig. S5. The basal data and detailed sampling regions for qRT-PCR.
    • Fig. S6. Knockdown effect of cll.
    • Fig. S7. Knockdown effect of Abd-B.
    • Fig. S8. Knockdown of the pigmentation-related gene Lac2.
    • Table S1. Detailed BLASTx results of 20 candidate genes.

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