DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:

ANDO, Toshiya
NAKAMURA, Taro
Specially Appointed Assistant Professor:
MORITA, Shinichi
Technical Staff:
MIZUTANI, Takeshi
Postdoctoral Fellow:
MATSUOKA, Yuji
SAKURA, Kazuki
JSPS Postdoctoral Fellow: YANO, Koki
Research Staff:
CHIKAMI, Yasuhiko
KAWAGUCHI, Haruka
MORITA, Junko
TAGUCHI, Rie
SOKENDAI Graduate Student: KITAZAWA, Yurina
Visiting Undergraduate: ODA, Hiroki
Visiting Scientist:
TAKENAKA, Masaki
Technical Assistant:
YOKOYAMA, Michiko
SUGIYAMA, HirokoAdmin Support Staff:
SAITO, EikoI. Origin and diversification of insect wings

The flight organs of insects have uniquely evolved whencompared to that of other various flying animals on earth.
Despite more than two centuries of debate, the evolutionary origin of insect wings is still an enigma; one which weare trying to decipher by the use of evo-devo methods. In
Drosophila melanogaster, the wing master gene vestigial(vg) and its interaction partner scalloped (sd) play pivotal
roles in the formation of wing field identity. For this reason,these genes are ideal research candidates in the investigationof wing origin and evolution.

BAFigure 1. The firebrat, Thermobia domestica (A). The two-spottedcricket, Gryllus bimaculatus (B).

The Division of Evolutionary Developmental Biology wasstarted in June 2015. We focus on the evolutionary novelties
acquired by insects in order to elucidate the molecular andevolutionary mechanisms that lead to the large variety oftraits that they display. Among this wealth of exciting traits,
our lab is currently focused on promoting research into (1)the origin and diversification of insect wings, (2) wing color
patterns and mimicry of ladybird beetles, and (3) the acquisition and diversification of beetle horns.

Visual overview of this lab’s work.

One way to identify the structure from which insect wings
first evolved is to explore the function of “wing” genes inancestral wingless (apterygote) species. To achieve this end,
we chose the firebrat, Thermobia domestica, as a model(Figure 1A). T. domestica belongs to Thysanura, whichis phylogenetically the closest extant relative of winged(pterygote) insects, thus making it ideal for elucidating wing
origin. We cloned vg and sd orthologs from T. domestica(Td-vg and Td-sd), and developed RNA interference (RNAi)
based methods for T. domestica to examine the functions ofthese genes. We are currently testing the functional effects ofaltered transcription for each of these wing genes in ancestrally wingless firebrats. Furthermore, we are performing
comparative analyses of the function of these same genes in“primitively winged” (hemimetabolous) insects (Figure 1B)
to obtain additional clues relevant to us understanding theorigin and evolution of insect wings.
Interestingly, our previous work showed that vg expressing epidermal tissue forms lateral outgrowths in non-winged
segments in the mealworm beetle (Ohde et al., 2013). Basedon this, we hypothesize that ancestral lateral body wall outgrowths evolved into functional wings. However, genetic
tools available for the analysis of basally branching winglessspecies are limited. To overcome these limitations, we established CRISPR/Cas9-based germline genome editing in T.
domestica. Heritable mutations were successfully introduced
in white locus, an evolutionarily conserved gene, encoding
the ATP-binding cassette (ABC) membrane transporter, ofT. domestica by using CRISPR/Cas9 system. This in turnresults in white-eyed firebrats. In addition to the RNAimediated gene knockdown (Ohde et al., 2009), germline
genome editing using CRISPR/Cas9 in T. domestica provides
a platform technology for creating new research opportunities concerning the evolution of insects, such as insect wing53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

origin. We are now conducting gene knock-out/in withinvarious “wing” genes to identify genetic details and celllineage analyses in T. domestica (Figure 1).

II. W ing color patterns and mimicry of
ladybird beetles

A tremendous range of diversity in wing color patterns has
evolved among insects, which in turn plays various ecologically important roles such as intraspecific sexual signaling,mimesis, mimicry, and is also used as a warning signal topredators. However, the molecular mechanisms responsible
for generating such color patterns in most ladybird species
remain elusive. To investigate the developmental mechanisms of color pattern formation, we have been focusing on
the multicolored Asian ladybird beetle, Harmonia axyridis,
which has conspicuous and variable wing color patterns
consisting of black and red pigments (Figure 2A). The ladybird’s vivid wing color pattern acts as a warning signal
to predators that they taste bad. At the same time, various
other insect species utilize this ecological signal by mimicking the ladybirds’ wing color patterns. Mimicry provides
us with an exciting opportunity to study how independent
lineages of insects have evolved convergent color patterns.
To explore color pattern formation mechanisms in mimicry,
we are currently focusing on the leaf beetle, Argopistes coccinelliformis, which has color patterns similar to Harmonia,
and is thought to be a Batesian mimicry of ladybird beetles(Figure 2B). To elucidate the molecular mechanisms underlying these wing color patterns, we established a technique forgermline transformation using a piggyBac vector and RNAiin the ladybirds.

AB

Figure 2. The ladybird beetle, Harmonia axyridis (A) and the leaf beetle,
Argopistes coccinelliformis (B).

We recently identified a key gene, pannier, which regulates intraspecific color pattern polymorphism in H. axyridis
using next generation sequencing technologies (RNA-seqand de novo genome assembly), and an RNAi-based screening method that we have established. pannier is expressedin specific regions in the wing, which synthesizes blackpigment, and suppresses red pigmentation. The expression
pattern of pannier is diversified according to the diverse
color pattern types in H. axyridis. These findings suggest that
regulatory shift, such as changes in enhancer activity, at thepannier locus may be crucial for the evolution of wing color
patterns in H. axyridis. We are currently trying to elucidate
the evolutionary origin of color patterns in ladybirds with afocus on regulatory shifts at the pannier loci.

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We are also attempting to apply genome-editing technologies such as TALEN and CRISPR/Cas9 to tackle this issue.
Thus far, we have achieved an efficient method of genedisruption using TALEN. Recently, we have begun successfully establishing the disrupting pannier gene function usingCRISPR/Cas9 system. Ribonucleoprotein (RNP) complexof Cas9 protein and guide RNA targeting pannier codingexon was microinjected into the fertilized eggs. As a result,over 60% of G0 founders produced pannier knockout progenies (Figure 3). CRISPR/cas9 mediated gene disruption
has advantages in terms of ease of designing gRNA andalso possesses a high KO efficiency compared to TALEN
mediated gene modification in Harmonia. To this end, weare continuing to develop CRISPR/Cas9 mediated geneknock-in method. To apply this approach, we are trying toattempt more complicated genome editing techniques such as
genomic insertion, inversion and duplication to identify thecrucial regulatory shift that may have driven the evolution ofwing color patterns in ladybird beetles.

Figure 3. Representative photographs of pannier mutant phenotypes in G0
wt-like (left side), G0 mosaic (middle side) and G1 KO mutant (right side).

We are also establishing cryopreservation methods for
germline cells in ladybird beetles to assist us with this. This isdue to the high risk of losing valuable genetic bioresources in
non-model insects. We recently established ovary transplantation and ovarian cryopreservation techniques in ladybird
beetles. We hope that the genetic tools and techniques that wehave established will further facilitate this research.
We plan to eventually analyze how the similar wing color
patterns of model and mimic are generated based on theknowledge obtained from H. axyridis. For example, do theyuse conserved or divergent mechanisms?

III. Acquisition and diversification of beetle horns

Insects show a tremendous range of diversity in “horns”;
rigid body outgrowths that function as weapons. Horns area subject of great potential for evo-devo studies because
they have arisen de novo multiple times as evolutionary“novelties”. However, the molecular mechanisms involved
in sexually dimorphic horn formation are still poorly understood. To investigate the developmental mechanisms ofhorn formation, we are focusing on the Japanese rhinoceros
beetle, Trypoxylus dichotomus (Coleoptera), which exhibits
remarkable sexual dimorphisms in head and thoracic horns.
The male-specific horns of T. dichotomus are among the bestmodels for studying how an extreme, sex-specific morphological structure is formed (Figure 4 A).

We first investigated the process of horn primordium formation to understand the developmental mechanism of hornformation. The insect body is covered by a rigid exoskeleton
mainly consisting of chitin, so changes in body size andshapes are produced by molting. Beetle horns first appear
at the pupal period (Figure 4 A). During the prepupal period(about 130 hours) which is an intermediate stage between thelast larval instar stage and pupation, the head horn primordia
are packed folded inside the larval head capsule (Figure 4B). This folded horn primordium develops into its final 3D
shape (pupal horn) during pupation. To properly make thehorn during pupation, the folding pattern (Figure 4 B) andsurface micro furrows (Figure 4 C) are precisely formed on
the horn primordium and packed within the confined spaceof the prepupal head. We recently attempt to use micro-CT toanalyze 3D pattern formation of head horn primordia during
the prepupal period. We found that horn 3D morphogenesisis accomplished in only 72 hours from the prepupal initiation(Figure 4 B, C).

Publication List:〔Original papers〕

S., Chikami, Y., Kawaguchi, H., Krylov, A.A., Niimi, T., and• Hashiro,Yasueda, H. (2021). Efficient production of long double-stranded RNAs•••

applicable to agricultural pest control by Corynebacterium glutamicum
equipped with coliphage T7-expression system. Appl. Microbiol.
Biotechnol. 105, 4987–5000. DOI: 10.1007/s00253-021-11324-9
Miyazaki, S., Fujiwara, K., Kai, K., Masuoka, Y., Gotoh, H., Niimi,T., Hayashi, Y., Shigenobu, S., and Maekawa, K. (2021). Evolutionarytransition of doublesex regulation from sex-specific splicing to malespecific transcription in termites. Sci. Rep. 11, 15992. DOI: 10.1038/s41598-021-95423-7
Ohde, T., Mito, T., and Niimi, T. (2022). A hemimetabolous wingdevelopment suggests the wing origin from lateral tergum of a winglessancestor. Nat. Commun. 13, 979. DOI: 10.1038/s41467-022-28624-x
Ylla, G., Nakamura, T., Itoh, T., Kajitani, R., Toyoda, A., Tomonari,S., Bando, T., Ishimaru, Y., Watanabe, T., Fuketa, M., Matsuoka, Y.,
Barnett, A.A., Noji, S., Mito, T., and Extavour, C.G. (2021). Insights
into the genomic evolution of insects from cricket genomes. Commun.
Biol. 4, 733. DOI: 10.1038/s42003-021-02197-9〔Review article〕

T., and Ando, T. (2021). Evo-devo of wing colour patterns• Niimi,in beetles. Curr. Opin. Genet. & Dev. 69, 97–102. DOI: 10.1016/j.
gde.2021.02.007

Figure 4 Morphological change of horn primordia in Trypoxylus dichotomus.
(A) Developmental process of Trypoxylus dichotomus male. (B, C)
Micro-CT images of the male head horn primordium during pupal development. (Adapted from Morita and Niimi, Sanshi Konchu Biotec, 86:
145–151, 2020.)

We are currently focusing on this developmental stage
because crucial regulatory factors for horn formation anddifferentiation are supposed to be activated at this stage in T.
dichotomus. The present study provides a good starting point
to unveil the gene regulatory network for sexually dimorphic
horn formation and to pursue the evolutionary origin of sucha regulatory system.

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