Juvenile Hormone Regulates Butterfly Larval Pattern Switches

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Science  22 Feb 2008:
Vol. 319, Issue 5866, pp. 1061
DOI: 10.1126/science.1149786


Insect color patterns can be very diverse. This variation is also seen among many larval instar stages, which can take on vastly different phenotypes. Young caterpillars of the swallowtail butterfly, Papilio xuthus, are mimics of bird droppings, whereas the fifth larval instar is camouflaged among the leaves of host plants (cryptic pattern). We find that juvenile hormone (JH) titers decrease during the fourth larval instar. Furthermore, treatment with JH analog at the beginning of the fourth instar stage resulted in reproducing the mimetic pattern instead of the usual cryptic one and likewise altered gene expression patterns to that associated with the mimetic pattern. These findings suggest that JH regulates the progressive larval pattern switch of this insect.

The spectacular diversity of adult insect color patterns can also extend to differences among sequential larval instars within some species. The swallowtail butterfly, Papilio xuthus, represents such an example: Young caterpillars (from the first to the fourth instars) are mimics of bird droppings, whereas the larger, final larval instar (the fifth) has a completely different pattern that is well camouflaged among the leaves of the host plant (Fig. 1A). Here, we show that this developmental switch is regulated by juvenile hormone (JH), which is known to regulate the overall black or green forms caused by differing environmental conditions in some larvae of Lepidoptera and some adults of Orthoptera (1, 2). We applied a JH analog (JHA) to the dorsal surfaces of fourth instar larvae and observed the color pattern of the fifth instar larvae. Treatment at the beginning of the fourth instar stage resulted in a high proportion (67%) of treated larvae reproducing the mimetic pattern instead of the usual cryptic one (Fig. 1B and table S1). No effects were observed when JHA was applied to larvae 20 hours after the appearance of the fourth instar (there are about 96 hours in the total fourth instar stage), indicating that there is a JH-sensitive period (circa 0 to 20 hours after the third ecdysis, Fig. 1B, gray box). A 100-fold lower dose of JHA (50 ng of fenoxycarb) had no effect (table S1). We analyzed JH titers in P. xuthus by liquid chromatography–mass spectrometry and found that JH titers decrease during the fourth larval instar (Fig. 1B and fig. S1A). These results suggest that JH regulates the larval pattern switch.

Fig. 1.

(A) Mimetic and cryptic pattern of P. xuthus larva. (B) Model of JH titer and its determination of larval body pattern (table S1). Gray box indicates a JH-sensitive period. HCS indicates head capsule slippage, the clear sign of molting period. 20E, 20-hydroxyecdysone. Scale bars indicate 1 mm. (C) Model of JH effect on expression of genes associated with mimetic and cryptic pattern (fig. S1).

The major differences between the mimetic and the cryptic larval color patterns include the green coloration of the cryptic pattern, specific tubercle structures (arrowheads in fig. S1D) as components of the mimetic pattern, and the distribution of black pigment. We examined the effects of JHA treatment on gene expression associated with these three differences. By using cDNA subtraction methods, we cloned the hard cuticle protein genes (HCP1 and HCP2) associated with the specific tubercle structures and the bilin-binding protein gene (BBP) that is only expressed at the final molt [supporting online material (SOM) text]. JH treatment induced the expression of tubercle-associated cuticle protein genes and inhibited BBP expression at the fourth molt, as is the case for the normal third molt (fig. S1, B and C). Larval black patterning is regulated by co-localization of melanin synthesis genes tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) (3). The spatial expression patterns of these genes are changed to the mimetic pattern in the JH-treated specimen (fig. S1D). These results suggest that JH regulates the stage-specific gene expression pattern in P. xuthus (Fig. 1C) that is required to modulate the larval pattern from mimetic to cryptic.

Our results suggest that a high titer of JH induces the expression of genes associated with the mimetic pattern and that a decrease in JH titer causes a switch to the cryptic pattern. In addition to overall coloration, JH also regulates exoskeletal structures and pigment distribution at specific markings. Because progressive changes of green and black coloration and exoskeletal structures are frequently found in lepidopteran larvae, our findings imply that JH regulation on progressive larval pattern switches may commonly exist.

Supporting Online Material

Materials and Methods

SOM Text

Fig. S1

Table S1

References and Notes

References and Notes

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