Jun-ichi Yamaguchi

A genetic mechanism for female-limited Batesian mimicry in Papilio butterfly

In Batesian mimicry, animals avoid predation by resembling distasteful models. In the swallowtail butterfly Papilio polytes, only mimetic-form females resemble the unpalatable butterfly Pachliopta aristolochiae. A recent report showed that a single gene, doublesex (dsx), controls this mimicry; however, the detailed molecular mechanisms remain unclear. Here we determined two whole-genome sequences of P. polytes and a related species, Papilio xuthus, identifying a single ∼130-kb autosomal inversion, including dsx, between mimetic (H-type) and non-mimetic (h-type) chromosomes in P. polytes. This inversion is associated with the mimicry-related locus H, as identified by linkage mapping. Knockdown experiments demonstrated that female-specific dsx isoforms expressed from the inverted H allele (dsx(H)) induce mimetic coloration patterns and simultaneously repress non-mimetic patterns. In contrast, dsx(h) does not alter mimetic patterns. We propose that dsx(H) switches the coloration of predetermined wing patterns and that female-limited polymorphism is tightly maintained by chromosomal inversion.

siRNAs induce efficient RNAi response in Bombyx mori embryos.

Short interference RNA (siRNA) is widely used in mammalian cells. In insects, however, reports concerning the suitablility of siRNA in vivo is very limited compared with that of long dsRNA, which is thought to be more effective. There is insufficient information on the essential rules of siRNA design in insects, as very few siRNAs have been tested in this context. To establish an effective method of gene silencing using siRNA in vivo in insects, we determined the effects of siRNA on seven target genes. We designed siRNAs according to a new guideline and injected them into eggs of Bombyx mori. At the mRNA level, the expression of most of these genes was successfully silenced, down to less than half the constitutive level, which in some cases led to the development of distinctive phenotypes. In addition, we observed stronger effect of siRNA both on the mRNA level and the phenotype than that of long dsRNA under comparable conditions. These results indicate that direct injection of siRNA is an effective reverse-genetics tool for the analysis of embryogenesis in vivo in insects.

Periodic Wnt1 expression in response to ecdysteroid generates twin-spot markings on caterpillars

Among various pigmentation patterns on caterpillars, sequential spot markings are often observed and used for aposematic colouration. In contrast to adult wings, caterpillar cuticle markings are repeatedly generated at each moult, but little is known about how the patterns are formed and maintained periodically. Here we focus on a silkworm mutant, multi lunar (L), with twin-spot markings on sequential segments. Positional cloning of L and expression analyses reveal that cis-regulatory change in Wnt1 is responsible for the spot patterning. The periodical upregulation of Wnt1 in response to ecdysteroid is detected only in epidermis within spot marking area. We verify by transgenic expression that the ectopic Wnt1 induces the additional pigmentation. Furthermore, the association of Wnt1 expression with spot markings is observed in the wild Bombyx species and swallowtail butterfly Papilio machaon. Taken together, we anticipate that periodic Wnt1 expression may contribute to natural variations of spot patterning on caterpillar cuticle.

Molecular basis of wing coloration in a Batesian mimic butterfly, Papilio polytes

Batesian mimicry protects animals from predators through resemblance with distasteful models in shape, color pattern, or behavior. To elucidate the wing coloration mechanisms involved in the mimicry, we investigated chemical composition and gene expression of the pale yellow and red pigments of a swallowtail butterfly, Papilio polytes, whose females mimic the unpalatable butterfly Pachliopta aristolochiae. Using LC/MS, we showed that the pale yellow wing regions in non-mimetic females consist of kynurenine and N-β-alanyldopamine (NBAD). Moreover, qRT-PCR showed that kynurenine/NBAD biosynthetic genes were upregulated in these regions in non-mimetic females. However, these pigments were absent in mimetic females. RNA-sequencing showed that kynurenine/NBAD synthesis and Toll signaling genes were upregulated in the red spots specific to mimetic female wings. These results demonstrated that drastic changes in gene networks in the red and pale yellow regions can switch wing color patterns between non-mimetic and mimetic females of P. polytes.

The transcription factor Apontic-like controls diverse colouration pattern in caterpillars

Genetic polymorphisms underlie the convergent and divergent evolution of various phenotypes. Diverse colour patterns on caterpillars, which are ecologically important, are good models for understanding the molecular backgrounds of phenotypic diversity. Here we show that a single evolutionarily conserved gene apontic-like (apt-like) encoding for a putative transcription factor accounts for the silkworm p locus, which causes at least 15 different larval markings involved in branch-like markings and eye-spot formation. The expression of apt-like and melanin synthesis genes are upregulated in association with pigmented areas of marking mutants Striped (p(S)) and normal (+(p)) but not in the non-marking allele plain (p). Functional analyses, ectopic expression, RNAi and TALEN, demonstrate that apt-like causes melanin pigmentation in a cell-autonomous manner. These results suggest that variation in p alleles is caused by the differential expression of the gene apt-like which induces targeted elevation of gene expressions in the melanin synthesis pathway.

Transformation of acyloin O-acyl derivatives to ketones using tetrabutylammonium fluoride-thiol system

Abstract The treatment of acyloin (α-hydroxyketone) O -acyl derivatives with tetrabutylammonium fluoride hydrate (TBAF·xH 2 O)-1-3,propanedithiol system gave the corresponding ketones. Further, it was found that N -methylmorpholine added could prevent the concomitant of unidentified compounds to give the pure products in high yields.

Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at −2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.

Protruding structures on caterpillars are controlled by ectopic Wnt1 expression.

Spine-like or protruding structures, which may be aposematic for predators, are often observed in multiple segments of lepidopteran larvae (caterpillars). For example, the larvae of the Chinese wheel butterfly, Byasa alcinous, display many protrusions on their backs as a warning that they are toxic. Although these protrusions are formed by an integument lined with single-layered epidermal cells, the molecular mechanisms underlying their formation have remained unclear. In this study, we focused on a spontaneous mutant of the silkworm, Bombyx mori, Knobbed, which shows similar protrusions to B. alcinous and demonstrates that Wnt1 plays a crucial role in the formation of protrusion structures. Using both transgene expression and RNAi-based knockdown approaches, we showed that Wnt1 designates the position where epidermal cells excessively proliferate, leading to the generation of knobbed structures. Furthermore, in the B. alcinous larvae, Wnt1 was also specifically expressed in association with the protrusions. Our results suggest that Wnt1 plays a role in the formation of protrusions on the larval body, and is conserved broadly among diverse species in Lepidoptera.

Toll ligand Spätzle3 controls melanization in the stripe pattern formation in caterpillars

Significance A stripe pattern is widely observed among animals and often used for warning or camouflage in caterpillars. However, its genetic background is largely unknown. This study showed that the Toll ligand Spätzle3 (Spz-3) is responsible for the silkworm Zebra locus, which causes black stripes on the anterior margin of each segment. Exhaustive knockdown experiments of spz- and Toll-related genes clarified that spz-3 and Toll-8 are involved in the melanin pigmentation of Zebra and another mutant. The Spz-3/Toll-8 signaling pathway is suggested to induce Zebra stripe-specific expression of the pigmentation gene yellow. This study sheds light on not only the unique aspect of Spz/Toll functions but also, stripe pattern pigmentation in caterpillars through co-option of a Toll signaling pathway. A stripe pattern is an aposematic or camouflage coloration often observed among various caterpillars. However, how this ecologically important pattern is formed is largely unknown. The silkworm dominant mutant Zebra (Ze) has a black stripe in the anterior margin of each dorsal segment. Here, fine linkage mapping of 3,135 larvae revealed a 63-kbp region responsible for the Ze locus, which contained three candidate genes, including the Toll ligand gene spätzle3 (spz-3). Both electroporation-mediated ectopic expression and RNAi analyses showed that, among candidate genes, only processed spz-3 induced melanin pigmentation and that Toll-8 was the candidate receptor gene of spz-3. This Toll ligand/receptor set is also involved in melanization of other mutant Striped (pS), which has broader stripes. Additional knockdown of 5 other spz family and 10 Toll-related genes caused no drastic change in the pigmentation of either mutant, suggesting that only spz-3/Toll-8 is mainly involved in the melanization process rather than pattern formation. The downstream pigmentation gene yellow was specifically up-regulated in the striped region of the Ze mutant, but spz-3 showed no such region-specific expression. Toll signaling pathways are known to be involved in innate immunity, dorsoventral axis formation, and neurotrophic functions. This study provides direct evidence that a Toll signaling pathway is co-opted to control the melanization process and adaptive striped pattern formation in caterpillars.