Accepted Manuscript Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae) Yudai Masuoka, Hajime Yaguchi, Ryutaro Suzuki, Kiyoto Maekawa PII:

S0965-1748(15)30029-1

DOI:

10.1016/j.ibmb.2015.07.013

Reference:

IB 2744

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 10 April 2015 Revised Date:

11 July 2015

Accepted Date: 11 July 2015

Please cite this article as: Masuoka, Y., Yaguchi, H., Suzuki, R., Maekawa, K., Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae), Insect Biochemistry and Molecular Biology (2015), doi: 10.1016/j.ibmb.2015.07.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Met expression

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Soldier differentiation

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ZnMet RNAi

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Knockdown of the juvenile hormone receptor gene inhibits soldier-

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specific

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nevadensis (Isoptera: Archotermopsidae)

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the

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damp-wood

termite

Zootermopsis

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morphogenesis

Yudai Masuoka, Hajime Yaguchi, Ryutaro Suzuki and Kiyoto Maekawa

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Graduate School of Science and Engineering, University of Toyama, Toyama,

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Japan

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Abstract

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The Methoprene-tolerant (Met) protein has been established as a juvenile

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hormone (JH) receptor. Knockdown of the Met gene caused precocious

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metamorphosis and suppression of ovarian development. However, the function

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of Met in caste development of social insects is unclear. In termites, JH acts as

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a central factor for caste development, especially for soldier differentiation,

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which involves two molts from workers via a presoldier stage. Increased JH titer

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in workers is needed for the presoldier molt, and the high JH titer is maintained

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throughout the presoldier period. Although presoldiers have the fundamental

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morphological features of soldiers, the nature of the cuticle is completely

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different from that of soldiers. We expected that JH signals via Met are involved

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in soldier-specific morphogenesis of the head and mandibles during soldier

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differentiation, especially in the presoldier period, in natural conditions. To test

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this hypothesis, we focused on soldier differentiation in an incipient colony of

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the damp-wood termite Zootermopsis nevadensis. Met homolog (ZnMet)

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expression in heads increased just after the presoldier molt. This high

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expression was reduced by ZnMet double stranded (dsRNA) injection before

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the presoldier molt. Although this treatment did not cause any morphological

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changes in presoldiers, it caused strong effects on soldiers, their mandibles

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being significantly shorter and head capsules smaller than those of control

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soldiers. Injection of ZnMet dsRNA throughout the presoldier stage did not

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affect the formation of soldier morphology, including cuticle formation. These

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results suggested that the rapid increase in ZnMet expression and subsequent

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activation of JH signaling just after the presoldier molt are needed for the

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formation of soldier-specific weapons. Therefore, besides its established role in

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insect metamorphosis, the JH receptor signaling also underlies soldier

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development in termites.

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37 Highlights

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・ Role of the Methoprene-tolerant (Met) gene in termite soldier differentiation

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・ RNAi knockdown of Met prior to presoldier molt suppressed morphogenesis of soldier-specific characters.

・ JH acts through its receptor Met to direct social cast polyphenism in termites.

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was examined.

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Keywords

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juvenile hormone, methoprene-tolerant, termites, soldier differentiation

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1. Introduction

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Juvenile hormone (JH) has pleiotropic functions and works as an important

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regulator of insect molting, metamorphosis, dormancy, reproduction and

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polyphenism, such as caste differentiation (Nijhout, 1994; Riddiford, 2008).

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Despite its important role, the JH signaling cascade is only partially understood.

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However, the methoprene-tolerant (Met) protein, which contains basic-helix-

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loop-helix (bHLH) and Per-Arnt-Sim (PAS) domains, has been established as a

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JH receptor in insects (Charles et al., 2011; Jindra et al., 2015). The function of

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Met, especially with regard to metamorphosis and reproduction, has been

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analyzed in some model insect species (Jindra et al., 2013; Smykal et al.,

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2014). For example, in the red flour beetle Tribolium castaneum and the firebug

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Pyrrhocoris apterus, knockdown of Met expression during the nymphal stage

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caused precocious metamorphosis (Konopova & Jindra, 2007; Konopova et al.,

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2011). Furthermore, ovarian development was repressed by Met knockdown in

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the viviparous cockroach Diploptera punctata (Marchal et al., 2014). In contrast,

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studies on other Met functions, including caste differentiation in social insects,

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are very limited. For termites, in particular, the only report in the literature is by

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Saiki et al. (2015), and it showed that Met was involved in fecundity via

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vitellogenin gene expression during reproductive caste differentiation in the

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rhinotermitid termite Reticulitermes speratus. However, the role of Met in caste

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differentiation and caste-specific morphogenesis is unknown.

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In termites, JH is the central factor in caste differentiation (Watanabe et al.,

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2014; Korb, 2015). Acquisition of a caste is the most important step for termite

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social evolution (Howard & Thorne, 2011). In particular, because a soldier is

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expected as the first sterile caste (Nalepa, 2011), clarification of the proximate

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mechanism of soldier differentiation is necessary to understand the whole

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picture of termite social evolution. In soldier differentiation, increased JH titer in

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workers is needed, and two molting processes (worker−presoldier and

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presoldier−soldier) always occur in all species examined (Noirot, 1985; Roisin,

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1996; Miura & Scharf 2011; Masuoka et al., 2013). Tens of studies confirmed

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that presoldier morphological changes were induced by JH (or JH analog)

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treatment of workers in some species (e.g. Scharf et al., 2003; Cornette et al.,

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2006; Tsuchiya et al., 2008; Toga et al., 2009; Maekawa et al., 2014).

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Moreover, based on JH quantification analyses, high JH titer was confirmed

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throughout the presoldier period in the damp-wood termite Hodotermopsis

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sjostedti (Cornette et al., 2008). Consequently, during soldier differentiation, JH

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may be involved not only in the determination of soldier differentiation, but also

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in other functions, such as in the morphogenesis of soldier-specific weapons

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(Watanabe et al., 2014). To clarify these roles of JH during soldier

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differentiation, functional analysis of Met should be performed. Although, in

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some species, analyses of the genes expressed via increased JH titer have

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been performed in the worker−presoldier molt using artificial induction methods

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(e.g. Cornette et al., 2006; Toga et al., 2012), the roles of Met during soldier

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differentiation remain a mystery. Here, we analyzed Met expression and

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function during soldier differentiation (worker−presoldier−soldier molts) of

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Zootermopsis nevadensis. Expression levels of the JH-inducible transcription

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factor Krüppel-homolog1 (Kr-h1) and the downstream gene Broad-complex (Br-

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C) were also analyzed to determine JH signaling activity in each individual. This

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species has a linear caste developmental pathway, in which late-instar larvae

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(engaged in worker tasks) differentiate into soldiers via a presoldier stage in the

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mature colony (Itano and Maekawa, 2008; Saiki et al., 2014). However, in the

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colony initiation stage (i.e. an incipient colony), early-instar larvae (the oldest

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3rd instar) always molt into presoldiers and soldiers (Itano and Maekawa, 2008;

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Maekawa et al., 2012; Fig. S1). Consequently, we focused on soldier

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differentiation in an incipient colony to analyze the two molting processes (3rd

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instar larva−presoldier, and presoldier−soldier) in natural conditions without JH

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(or JH analog) treatment.

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2. Materials & Methods

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2.1. Insects

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Several mature colonies were collected from laurel forests in Hyogo Prefecture,

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Japan, in May 2013 and 2014. Colonies were brought to the laboratory and kept

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in plastic cases at approximately 25°C in constant

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emergence of newly molted alates (winged adults). In accordance with previous

darkness until the

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studies (Itano & Maekawa, 2008; Maekawa et al., 2012), 120 incipient colonies

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were founded by male and female alates in petri dishes, and these dishes were

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kept in plastic cases at approximately 25°C in cons tant darkness. In the

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absence of a soldier in the incipient colony of this species, the oldest 3rd instar

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(Individual that first molted into L3; No. 1 larva) molts into a presoldier, and then

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a soldier (Fig. S1). However, in the presence of a soldier, second or third molted

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L3 (No. 2 or No. 3 larvae) molt into L4 larvae (Maekawa et al., 2012). Because

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L3 (except for the No. 1 larva) and L4 normally function as workers in the

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incipient colonies, we called them workers in this study. For experiments during

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worker−presoldier and presoldier−soldier molts, No. 1 larvae were used. For

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those during worker−worker molts, No. 2 or No. 3 larvae were used.

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2.2. cDNA synthesis and cloning

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Total RNA was extracted from workers and soldiers (whole bodies) using

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ISOGEN (Nippon Gene, Tokyo, Japan). cDNA was synthesized using a

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SMARTer RACE cDNA Amplification Kit (Clontech Laboratories, Mountain

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View, CA, USA). The cDNA fragments were amplified by 3’-rapid amplification

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of DNA amends (RACE) PCR. Gene-specific forward primers (Table S1) were

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designed against conserved regions of the Met and Kr-h1 homologs in D.

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punctata (GenBank accession Nos. KJ564130 and KJ564131, respectively) and

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R. speratus (GenBank accession No. LC017912 and LC017913, respectively).

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The PCR products were purified using a QIAquick gel extraction kit (Qiagen,

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Tokyo, Japan) and subcloned into a pGEM-T Easy Vector (Promega, Madison,

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WI, USA). The inserted DNA was amplified by PCR using primers SP6 and T7

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and purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA). The purified

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products (five clones per gene) were sequenced using a BigDye Terminator

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v3.1 cycle sequencing kit (Applied Biosystems, Waltham, MA, USA) and an

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automatic DNA sequencer 3130 Genetic Analyzer (Applied Biosystems).

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Obtained sequences were subjected to BLAST database searches using the

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NCBI server. Especially for Met, the deduced amino acid sequence was aligned

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with published sequences of some other taxa (termite, R. speratus; cockroach,

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Blattella germanica, D. punctate; beetle, T. castaneum; moth, Bombyx mori;

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water flea, Daphnia pulex) using MUSCLE with MEGA6 (Tamura et al., 2013).

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2.3. Gene expression

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For gene expression analysis during each molt, total RNA was extracted using

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ISOGEN (NipponGene, Tokyo, Japan) from the heads of individuals (n = 4)

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sampled at each time point (3, 2, and 1 days before molting and 0, 1, 3, 5, and

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7 days after the molt). Extracted RNA was treated with RNase-free DNaseI

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(Takara, Shiga, Japan) to remove genomic DNA. The purity and concentration

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of extracted RNA was confirmed using a NanoVue spectrophotometer (GE

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Healthcare Bio-Sciences, Uppsala, Sweden). Moreover, integrity of extracted

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RNA was checked by the Agilent 2100 bioanalyzer (Agilent Technologies,

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California, USA) using randomly selected samples (four samples from each

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molt process, total 12 samples). We confirmed that all RNA samples examined

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were not damaged and high quality. cDNAs were synthesized from equal

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concentrations of total RNA (2 µg) using a High Capacity cDNA Reverse

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Transcription Kit (Applied Biosystems). The relative quantification of transcripts

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was performed using a THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka,

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Japan) with Mx3005P Real-Time QPCR System (Agilent Technologies) and

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Mini Opticon Real-Time PCR System (Bio-Rad, California, USA). We designed

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gene-specific primers (Table S1) using Primer3 plus software (Untergasser et

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al., 2007). We confirmed the melt curve with one peak and amplified efficiencies

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of each primer pair (within 80-123%; Table S1). To select an endogenous

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control with constitutive expression, the suitability of six reference genes of this

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species, EF1-alfa (Accession No. AB915828), beta-actin (No. AB915826),

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NADH-dh (No. AB936819) and three ribosomal protein genes (RS49: GeneID:

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KDR21989, RPS18: KDR22651 and RPL13a: KDR22610; Terrapon et al.,

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2014) were evaluated using GeNorm (Vandesompele et al., 2002) and

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NormFinder (Andersen et al., 2004). Relative expression levels of target genes

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were calculated by adopting the standard curve method. Expression levels were

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calculated using technical triplicates, because of the limited numbers of

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individuals examined; L3 and presoldier were basically only one individual in

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each colony and it was very hard to collect many samples. Statistical analysis

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was performed using Tukey’s test for comparison among developmental stages.

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2.4. dsRNA synthesis and injection

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For dsRNA synthesis, the partial cDNA sequence (585 bp) of Met homologs of

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Z. nevadensis (ZnMet, see results) were amplified using gene-specific primers

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(Table S1 and Fig. S2), and subcloned into a pGEM-T Easy Vector (Promega).

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To avoid an off-target effect of the target sequence, we utilized the genome

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sequence data of this species (Terrapon et al., 2014). First, we searched the

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most effective siRNA in the obtained partial sequences using siDirect ver. 2.0

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(Naito et al., 2009). Next, we searched this sequence (CTG TTA TAA CAT CTT

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TAG TGT CT; see Fig. S2A for the position) in the published genome database

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(gene model OGSv2.2). Then, we confirmed that this sequence was only

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observed in the ZnMet sequence. A control GFP cDNA sequence (706 bp) was

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amplified from GFP vector pQBI-polII (Wako, Osaka, Japan). Primer sequences

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were as reported in Toga et al. (2012). ZnMet and GFP dsRNAs were

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transcribed using T7 RNA polymerase with a MEGA script T7 transcription kit

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(Ambion, Austin, TX, USA) from cDNA sequences purified using a QIAquick gel

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extraction kit (Qiagen). ZnMet or GFP dsRNA (500 ng in 136 nL) was injected

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into the side of the thorax of workers or presoldiers using a Nanoliter 2000

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microinjector (World Precision Instruments, Sarasota, FL, USA) fitted with a

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glass needle. To check for the knockdown of gene expression, individuals were

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collected 3 days after dsRNA injection (n = 4 per each treatment) and used for

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expression analysis as shown in the previous section. Expression levels were

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calculated using biological quadruplicates to consider the individual differences

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of RNAi effects. Statistical analysis was performed using Mann-Whitney’s U test

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for the comparison between ZnMet and GFP dsRNA treatments.

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After dsRNA injection treatment, newly molted presoldiers and soldiers were

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collected every 24 hours for the observation of phenotypic effects. Soldiers

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were maintained for 7 days in their original colonies to complete cuticular

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pigmentation (c.f. Masuoka et al., 2013) (n = 4−15 per treatment). Presoldiers

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were collected from gut purging to the soldier molt (n = 10 per treatment). They

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were preserved in the FAA solution (ethanol : formalin : acetic acid = 16 : 6 : 1)

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for 24 h and stored in 70% ethanol. Fixed individuals were placed on a

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microscope slide and images of their heads and bodies were taken using a

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SZX10 stereo microscope and 3CCD digital camera XD250-2D (Olympus,

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Tokyo, Japan). Measurements of nine soldier-characteristic traits (left mandible

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length, head capsule length and width, pronotum length and width, hind femur

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length and width, and hind tibia length and width; Koshikawa et al., 2002) were

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measured using XD Measurement software (Olympus). Principal component

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analysis (PCA) was performed based on these nine measurements using

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statistical software Mac Multivariate Statistical Analysis ver. 1.0a (Esumi, Tokyo,

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Japan). Left mandible and head capsule lengths of dsRNA-treated soldiers

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were compared, and two-way analysis of variance (ANOVA) and Scheffe’s F

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test were used for statistical analyses using Mac Statistical Analysis ver. 2.0

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(Esumi). Moreover, from the captured images of their mandibles and head

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capsules, ten points were chosen at random per individual using Adobe

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Photoshop CS software 7.0 (Adobe Systems Inc., San Jose, CA, USA). The

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color properties of each individual were evaluated as the average of values for

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brightness (c.f. Tsuchida et al., 2010; Masuoka et al., 2013). Statistical analysis

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was performed using Mann-Whitney’s U test for comparison between ZnMet

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and GFP dsRNA treatments.

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3. Results

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3.1. Cloning of JH signaling genes

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We performed RT-PCR using degenerate primers and 3’-RACE, and obtained

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two partial sequences (2,099 bp and 987 bp; Fig. S2A, B). One of the cDNA

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sequences encoded three domains (bHLH, PAS_A and PAS_B) that are well

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conserved in Met homologs of other species (Ashok et al., 1998). The deduced

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amino acid sequence (699 aa) was subjected to BLASTP searches, and was

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found to be 67-69% similar to the Met homologs of other insect species (D.

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punctata, GenBank accession no. AIM47235; B. germanica, CDO33887). The

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aligned sequences with some taxa showed the conservation of all domains

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shown above (Fig. S2C). The other cDNA sequences contained C2H2 Zn-finger

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domains that are well conserved in Kr-h1 homologs of other species (Minakuchi

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et al., 2009). The deduced amino acid sequence (328 aa) was subjected to

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BLASTP searches, and found to be 86-94% identical to Kr-h1 homologs in

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other insect species (R. speratus, GenBank accession no. LC017913; B.

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germanica, CCC55948). We regarded these two sequences as Met and Kr-h1

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homologs of Z. nevadensis, and named them ZnMet and ZnKr-h1, respectively.

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The presence of these sequences was confirmed in Z. nevadensis genomic

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sequences (gene model OGSv2.2; Terrapon et al., 2014). The determined

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nucleotide

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DDBJ/EMBL/GenBank databases (accession no. LC062714, LC062715). ZnBr-

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C sequence was obtained from the database (GeneID: KDR08439; Terrapon et

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3.2. Expression analysis during caste differentiation

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To understand the JH signaling activity during soldier differentiation, we

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compared ZnMet, ZnKr-h1 and ZnBr-C expression patterns among worker

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(L3)−presoldier, presoldier−soldier and worker−worker (L3−L4) molts in the

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incipient colony. First, we investigated the time schedule during each molting

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process (Fig. S1). Workers (L3) with yellowish-white abdomens, because of gut

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purging, emerged in the incipient colony with a soldier. They molted into next

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instar workers (L4) 3.4 ± 0.57 days after gut purging (mean ± S.D., n = 65). In

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the case of colonies without soldiers, gut-purged workers (L3) molted into

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presoldiers after 3.1 ± 0.51 days (n = 136), and gut-purged presoldiers emerged

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6.7 ± 0.75 days after the presoldier molt (n = 66). Then, they molted into

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soldiers after a further 3.5 ± 0.66 days (n = 56). Next, we performed gene

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expression analysis of the heads every 24 hours during each molt from 3 days

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before the molt (day −3) to 7 days after the molt (day 7). Both GeNorm and

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NormFinder software showed that the expression levels of RPL13a were the

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most stable (Table S2). In the worker−worker molt, gene expression levels of

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both ZnMet and ZnKr-h1 were up-regulated before the molt (day −1) and

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dropped just after the molt (day 0; Fig. S3). In contrast, during soldier

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differentiation, both gene expression levels increased just after each molt (day

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0; Fig. 1). Furthermore, ZnBr-C expression was also up-regulated just after the

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presoldier molt (Fig. 1), whereas this peak was not observed during the

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worker−worker molt (Fig. S3).

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3.3. Effects of ZnMet dsRNA injection in the presoldier period

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To elucidate the role of ZnMet in the presoldier period, we injected dsRNA

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every 24 hours in the presoldier period [total 9 days (day 0−9), n = 4−15 per

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treatment per day]. The expression level of ZnMet in individuals collected 3

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days after dsRNA injection (injected on day 1) was less than half of that in

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control individuals (Fig. S4). However, the morphology of soldiers after the molt

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was completely normal regardless of injection timing (Fig. 2). The brightness of

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the soldier cuticles in the head capsule had no significant differences compared

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with those of control individuals (Fig. S5). Because gene expression was not

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immediately reduced by dsRNA injection, there was a possibility that

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knockdown of high expression on day 0 (Fig. 1) could not be conducted

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efficiently. Thus, we performed dsRNA injection analysis 1−3 days before the

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presoldier molt to reduce the high expression level just after the molt (day 0 in

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the presoldier period; Fig. 1).

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3.4. Effects of ZnMet dsRNA injection before the presoldier molt

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Injection of dsRNA before the presoldier molt (after gut purging) caused an

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approximately 40% knockdown of ZnMet expression levels just after the molt,

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compared with those of the control treatment (Fig. S6A). The expression levels

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of ZnKr-h1 were also significantly lower than those of the control (Fig. S6B).

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Presoldiers that molted after dsRNA injection exhibited normal phenotypes in

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both treatments (Fig. S7). The mandibular and head-capsule lengths of molted

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presoldiers after ZnMet dsRNA injection were not significantly different from

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those of the control (Fig. S8). On the other hand, all molted soldiers that

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emerged after ZnMet dsRNA treatment before the presoldier molt were quite

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small (Fig. 3). PCA using measurements of nine characteristic traits of soldiers

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showed that these soldiers (solid triangles) were plotted completely separately

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from other soldiers, including control treatment (all squares and white triangles),

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but plotted near presoldiers (circles; Fig. 4). Moreover, the head-capsule and

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mandibular lengths of dsRNA-treated soldiers were compared. Two-way

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ANOVA was performed and interaction was detected between dsRNA injection

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timing and treatments in both measurements (head capsule: d.f. = 7, F = 3.76, p

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= 2.21E−3; mandible: d.f. = 7, F = 4.78, p = 3.19E−4). The result showed a

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statistical difference among dsRNA injection timings for both measurements

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(head capsule: d.f. = 7, F = 9.74, p = 9.93E−8; mandible: d.f. = 7, F = 11.37, p =

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1.07E−8). Significant differences were also found between ZnMet and GFP

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dsRNA treatments in both measurements (head capsule: d.f. = 18, F = 1.72, p =

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2.56E−8; mandible: d.f. = 18, F = 1.96, p = 9.13E−8). Both measurements of

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soldiers that emerged after ZnMet dsRNA treatment before the presoldier molt

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were significantly shorter than those of any other soldiers examined (Figs. 5A,

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B; two-way ANOVA followed by Scheffe’s F test, p < 0.05).

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320 4. Discussion

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In this study, we performed expression and function analyses of ZnMet during

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soldier differentiation of Z. nevadensis in an incipient colony without any artificial

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induction methods. The results clearly showed that ZnMet expression was

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involved in the formation of soldier-specific morphological characters (i.e.

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weapons). This is the first report on the function of Met during termite soldier

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differentiation.

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4.1. Expression pattern of ZnMet, ZnKr-h1 and ZnBr-C during soldier

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differentiation

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Expression levels of ZnMet and ZnKr-h1 increased abruptly just after the

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presoldier molt, but not after the soldier and worker molts. Generally in

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hemimetabolous insects, Met expression levels had low fluctuation during each

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molt in contrast to Kr-h1 expression (Konopova et al., 2011). These distinctive

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patterns observed after the presoldier molt were quite similar to those of

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pupation in the holometabolous red flour beetle T. castaneum, rather than those

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of a nymphal molt in the hemimetabolous firebug P. apterus and the German

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cockroach B. germanica (Parthasarathy et al., 2008; Minakuchi et al.,

339

2009; Konopova et al., 2011; Lozano and Belles, 2014). Furthermore, ZnBr-C

340

expression pattern was also similar to that of ZnMet during the presoldier molt.

341

Because Br-C is involved in the pupation via the synthesis of pupae-specific

342

cuticular protein in the holometabolous insects (Zhou & Riddiford, 2002), Br-C

343

may also have some presoldier-specific roles influenced by the JH signaling

344

pathway. All of these results obtained suggested that there is a developmental

345

similarity between the presoldier molt and pupation of holometabolous insects,

346

both of which are associated with drastic morphological changes. In fact, in

347

other termites with nasute soldiers (termitid subfamily Nasutitermitinae),

348

concentric circular structure similar to the imaginal disc of holometabolous

349

insects was constructed in the heads during soldier differentiation to form a

350

horn-like projection (Miura & Matsumoto, 2000; Toga et al. 2009). It has been

351

suggested

352

metamorphosis through the pupal stage (Miura & Matsumoto, 2000; Miura,

353

2001). Our results are not inconsistent with this hypothesis from a view point of

354

the gene expression pattern. To discuss this issue, physiological and gene

355

expression profiles should be clarified in the presoldier period in natural

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that

soldier

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resembles

holometabolous

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356

conditions, and compared with the pupation of T. castaneum and other

357

holometabolous insects.

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358 4.2. Function of ZnMet during soldier differentiation

360

Injection of ZnMet dsRNA throughout the presoldier period did not affect the

361

formation of soldier morphology. Expression of ZnMet in the presoldier period

362

after dsRNA injection might not be involved in soldier-specific external

363

morphogenesis, including cuticle coloration. However, there is a possibility that

364

specific internal morphological changes during soldier differentiation (e.g.

365

neuronal modification; Ishikawa et al., 2008) may be affected by JH signaling

366

activity via Met expression in the presoldier period. Further morphological and

367

histological observations of dsRNA-injected individuals should be performed to

368

evaluate this issue. Moreover, in some insects, it was suggested that there was

369

a developmental relationship between cuticle formation and ecdysteroid

370

signaling activity (Futahashi et al., 2007; Hiruma & Riddiford, 2009). In the

371

rhinotermitid termite R. speratus, Laccase2 (Lac2: a key gene of the tyrosine

372

metabolic pathway involved in cuticular tanning in insects; Arakane et al., 2005,

373

2009) was highly expressed during the soldier molt, compared with the worker

374

and presoldier molts (Masuoka et al., 2013). Because Lac2 expression was

375

suggested to be regulated by ecdysteroid signaling in the honey bee

376

Apis mellifera (Elias-Neto et al., 2010), termite soldier cuticle formation might

377

also be controlled by ecdysteroid as well as JH signaling. Although high JH titer

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is maintained during termite soldier differentiation (e.g. Cornette et al., 2008),

379

the cuticular nature changes extraordinarily during each molt (Masuoka et al.,

380

2013); from brown and hard (worker), then white and soft (presoldier), and

381

finally to brown-black and very hard (soldier). Further developmental analyses

382

are needed to understand the crosstalk between JH and ecdysteroid signaling

383

involved in soldier-specific cuticle formation.

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384

Expression levels of not only ZnMet but also ZnKr-h1 just after the presoldier

386

molt were reduced by ZnMet dsRNA injection before the presoldier molt,

387

suggesting that ZnMet expression in the worker stage is involved in JH

388

signaling activity just after the presoldier molt. Interestingly, ZnMet dsRNA

389

injection at this time point did not cause any morphological changes in

390

presoldiers, but caused strong effects on soldiers leading to short mandibles

391

and small head capsules. These results suggested that a rapid increase in

392

ZnMet expression and subsequent activation of JH signaling just after the

393

presoldier molt are needed for the formation of soldier-specific weapons.

394

Furthermore, these results also indicated that JH is the crucial factor for not only

395

the determination of soldier differentiation but also the growth of soldier-specific

396

traits during the presoldier period. Based on knockdown experiments of the

397

insulin receptor gene, Hattori et al. (2013) suggested that insulin signaling was

398

involved in mandibular formation during the JHA-induced presoldier molt in H.

399

sjostedti. A similar mechanism may exist in the soldier molt, and the rapid

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activation of JH signaling probably occurred first. In holometabolous insects,

401

such as the dung beetle Onthophagus taurus, JH and insulin signaling are

402

involved in the determination of body size and the formation of exaggerated

403

traits (e.g. horns) (Moczek & Nijhout, 2002; Emlen et al., 2006). Further

404

analyses on the crosstalk between JH and insulin signaling to form exaggerated

405

soldier weapons should be performed.

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406

In this study, there were no presoldier phenotypic effects when the dsRNA

408

injection was performed before the presoldier molt. This is probably because

409

the experimental time was insufficient for the RNAi to take effect, or alternatively

410

presoldier morphogenesis is independent of JH. Because presoldier formation

411

can be induced by JH or its analogs, it may not be independent of JH signaling.

412

Thus, the lack of phenotypic effects may just be due to insufficient time given for

413

the RNAi to take effect. There is a possibility that the injection in the earlier

414

stages (L2 larvae or L3 larvae before the gut purging) may affect presoldier-

415

specific morphogenesis or stop the presoldier molt. We are now expanding our

416

study to address this issue.

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4.3. Conclusion

419

The roles of JH in soldier differentiation have focused especially on the

420

presoldier molt, because it can be induced by artificial treatment. In this study,

421

however, using an incipient colony of Z. nevadensis, we could focus on not only

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the presoldier molt but also the soldier molt in natural conditions. The results

423

suggested that JH signaling activity caused by a rapid increase in ZnMet

424

expression just after the presoldier molt is involved in the formation of soldier-

425

specific weapons. These roles of JH during the presoldier period, i.e. not only a

426

determining factor of soldier differentiation but also as a growth factor for

427

soldier-specific traits, are clarified here for the first time. Soldier differentiation

428

with drastic morphological changes might be regulated by different temporal

429

roles of JH during the presoldier and soldier molts.

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430 Acknowledgement

432

We are grateful to Dr. Tsutomu Tsuchida (University of Toyama) who helped for

433

qPCR and bioanalyzer experiments. Thanks are also due to Akiko Fujiwara,

434

Takashi Sugimoto, Kouhei Toga, Ryota Saiki, Takaya Inoue, Syutaro Hanmoto

435

and Souichiro Kawamura for their help during both field and laboratory work.

436

This study was supported in part by Grant-in-Aids for Scientific Research (Nos.

437

24570022 and 25128705 to KM) from the Japan Society for the Promotion of

438

Science.

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References

441

Andersen, C.L., Jensen, J.L., Ørntoft, T., 2004. Normalization of real-time

442

quantitative reverse transcription-PCR data: a model-based variance estimation

ACCEPTED MANUSCRIPT

443

approach to identify genes suited for normalization, applied to bladder and

444

cancer data sets. Cancer Res. 64, 5245−5250.

RI PT

445 Arakane, Y., Muthukrishnan, S., Beeman, R.W., Kanost, M.R., Kramer, K.J.,

447

2005. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning.

448

Proc. Natl. Acad. Sci. U.S.A. 102, 11337−11342.

SC

446

449

Arakane, Y., Lomakin, J., Beeman, R.W., Muthukrishnan, S., Gehrke, S.H.,

451

Kanost, M.R., Kramer, K.J., 2009. Molecular and functional analyses of amino

452

acid decarboxylases involved in cuticle tanning in Tribolium castaneum. J. Biol.

453

Chem. 284, 16584−16594.

TE D

454

M AN U

450

Ashok, M., Turner, C., Wilson, G.T., 1998. Insect juvenile hormone resistance

456

gene homology with the bHLH-PAS family of transcriptional regulators. Proc.

457

Natl. Acad. Sci. U.S.A. 95, 2761−2766.

AC C

458

EP

455

459

Castle, G.B., 1934. The damp-wood termites of the western United States,

460

genus Zootermopsis (formerly, Termopsis). In Kofoid, G. A. (Ed.) Termite and

461

termite control. University of California Press, Berkeley, pp. 273−310.

462

ACCEPTED MANUSCRIPT

Charles, J.P., Iwema, T., Epa, V.C., Takaki, K., Rynes, J., Jindra, M., 2011.

464

Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant.

465

Proc. Natl. Acad. Sci. U.S.A. 108, 21128−21133.

RI PT

463

466

Cornette, R., Koshikawa, S., Hojo, M., Matsumoto, T., Miura, T., 2006. Caste-

468

specific cytochrome P450 in the damp-wood termite Hodotermopsis sjostedti

469

(Isoptera, Termopsidae). Insect Mol. Biol. 15, 235−244.

SC

467

M AN U

470 471

Cornette, R., Koshikawa, S., Miura, T., 2008. Histology of the hormone-

472

producing glands in the damp-wood termite Hodotemopsis sjostedti (Isoptera:

473

Termopsidae): A focus on soldier differentiation. Insectes Sociaux 55, 407−416.

TE D

474

Elias-Neto, M., Soares, M.P., Simoes, Z.L., Hartfelder, K., Bitondi, M.M., 2010.

476

Developmental characterization, function and regulation of a Laccase2

477

encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae). Insect

478

Biochem. Mol. Biol. 40, 241−251.

AC C

479

EP

475

480

Emlen, J.D., Szafran, Q., Corley, S.L., Dworkin, I., 2006. Insulin signaling and

481

limb-patterning:

482

diversification of beetle ‘horns’. Heredity 97, 179−191.

483

candidate

pathways

for

the

origin

and

evolutionary

ACCEPTED MANUSCRIPT

Futahashi, R., Fujiwara, H., 2007. Regulation of 20-hydroxyecdysone on the

485

larval pigmentation and the expression of melanin synthesis enzymes and

486

yellow gene of the swallowtail butterfly, Papilio xuthus. Insect Biochem. Mol.

487

Biol. 37, 855−864.

RI PT

484

488

Hattori, A., Sugime, Y., Sasa, C., Miyakawa, H., Ishikawa, Y., Miyazaki, S.,

490

Okada, Y., Cornette, R., Lavine, L.C., Emlen, D.J., Koshikawa, S., Miura, T.,

491

2013. Soldier morphogenesis in the damp-wood termite is regulated by the

492

insulin signaling pathway. J. Exp. Zool. B 320, 295−306.

M AN U

SC

489

493

Heath, H., 1927. Caste formation in the termite genus Termopsis. J. Morphol.

495

Physiol. 43, 387−425.

TE D

494

496

Hiruma, K., Riddiford, L.M., 2009. The molecular mechanisms of cuticular

498

melanization:

499

expression in Manduca sexta. Insect Biochem. Mol. Biol. 39, 245−253.

The

ecdysone

cascade

leading

to

dopa

decarboxylase

AC C

500

EP

497

501

Howard, J.K., Thorne, L.B., 2011. Eusocial evolution in termites and

502

hymenoptera. In: Bignell, D. E., Roisin, Y., Lo, N. (Eds), Biology of Termites: A

503

Modern Synthesis, 2nd edition. Springer, New York, pp. 97−132.

504

ACCEPTED MANUSCRIPT

505

Ishikawa, Y., Aonuma, H., Miura, T., 2008 Soldier-specific modification of the

506

mandibular motor neurons in termites. PLoS ONE 3, e2617.

RI PT

507 Itano, H., Maekawa, K., 2008. Soldier differentiation and larval juvenile hormone

509

sensitivity in an incipient colony of the damp-wood termite Zootermopsis

510

nevadensis (Isoptera, Termopsidae). Sociobiology 51, 151−162.

SC

508

511

Jindra, M., Palli, R.S., Riddiford, M.L., 2013. The juvenile hormone signaling

513

pathway in insect development. Ann. Rev. Entomol. 58, 181−204.

M AN U

512

514

Jindra, M., Uhlirova, M., Charles, J.P., Smykal, V., Hill, R.J., 2015. Genetic

516

evidence for function of the bHLH-PAS protein Gce/Met as a juvenile hormone

517

receptor. PloS Genet. 11, e1005394.

TE D

515

EP

518 Konopova,

520

Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium

521

castaneum. Proc. Natl. Acad. Sci. U.S.A. 104, 10488−10493.

522

B.,

Jindra,

M.,

2007.

Juvenile

hormone

resistance

gene

AC C

519

523

Konopova, B., Smykal, V., Jindra, M., 2011. Common and distinct roles of

524

juvenile hormone signaling genes in metamorphosis of holometabolous and

525

hemimetabolous insects. PloS One 6, e28728.

526

ACCEPTED MANUSCRIPT

Korb, J., 2015. Juvenile hormone: A central regulator of termites caste

528

polyphenism. In: A. Zayed and C. F. Kent. (Eds), Advances in Insect

529

Physiology, Vol. 48; Genomics, Physiology and Behaviour of Social Insects.

530

Elsevier, London, pp. 131−161.

RI PT

527

531

Koshikawa, S., Matsumoto, T., Miura, T., 2002. Morphometric changes during

533

soldier differentiation of the damp-wood termite Hodotermopsis japonica

534

(Isoptera: Termopsidae). Insectes Sociaux 49, 245−250.

M AN U

SC

532

535

Lozano, J., Belles, X., 2014. Role of Methoprene-tolerant (Met) in adult

537

morphogenesis and in adult ecdysis of Blattella germanica. PloS One 9,

538

e103614.

539

TE D

536

Maekawa, K., Nakamura, S., Watanabe, D., 2012. Relationships between

541

frontal-gland formation and mandibular modification during JH III-induced

542

presoldier differentiation in the termite Reticulitermes speratus (Isoptera:

543

Rhinotermitidae). Entomol. Sci. 15, 56−62.

AC C

544

EP

540

545

Maekawa, K., Hayashi, Y., Lee, T., Lo, N., 2014. Presoldier differentiation of

546

Australian termite species induced by juvenile hormone analogs. Austral

547

Entomol. 53, 138−143.

548

ACCEPTED MANUSCRIPT

Marchal, E., Hult, F.E., Huang, J., Pang, Z., Stay, B., Tobe S. S., 2014.

550

Methoprene-tolerant (Met) knockdown in the adult female cockroach, Diploptera

551

punctata completely inhibits ovarian development. PloS One 9, e106737.

RI PT

549

552

Masuoka, Y., Miyazaki, S., Saiki, R., Tsuchida, T., Maekawa, K. 2013. High

554

Laccase2 expression is likely involved in the formation of specific cuticular

555

structures during soldier differentiation of the termite Reticulitermes speratus.

556

Arthropod Struct. Dev. 42, 469−475.

M AN U

SC

553

557

Minakuchi, C., Namiki, T., Shinoda, T., 2009. Krüppel homolog 1, an early

559

juvenile hormone-response gene downstream of Methoprene-tolerant, mediates

560

its anti-metamorphic action in the red flour beetle Tribolium castaneum. Dev.

561

Biol. 325, 341–350.

EP

562

TE D

558

Miura, T., Matsumoto, T., 2000. Soldier morphogenesis in a nasute termite:

564

discovery of a disc-like structure forming a soldier nasus. Proc. R. Soc. Lond. B.

565

Biol. Sci. 267, 1185–1189.

566

AC C

563

567

Miura, T., 2001. Morphogenesis and gene expression in the soldier-caste

568

differentiation of termites. Insectes Sociaux 48, 216−223.

569

ACCEPTED MANUSCRIPT

Miura, T., Scharf, M.E., 2011. Molecular basis underlying caste differentiation in

571

termites. In: Bignell, D.E., Roisin, Y., Lo, N. (Eds), Biology of Termites: A

572

Modern Synthesis, 2nd edition. Springer, New York, pp. 211−253.

573

RI PT

570

Moczek, P.A., Nijhout, H.F., 2002. Developmental mechanisms of threshold

575

evolution in a polyphonic beetle. Evol. Dev. 4, 252−264.

SC

574

576

Naito, Y., Yoshimura, J., Morishita, S., Ui-Tei, K., 2009. siDirect 2.0: updated

578

software for designing functional siRNA with reduced seed-dependent off-target

579

effect. BMC Bioinformatics 10, 392.

M AN U

577

580

Nalepa, A.C., 2011. Altricial development in wood-feeding cockroaches: the key

582

antecedent of termite eusociality. In: Bignell, D.E., Roisin, Y., Lo, N. (Eds),

583

Biology of Termites: A Modern Synthesis, 2nd edition. Springer, New York, pp.

584

69−96.

586 587

EP

AC C

585

TE D

581

Nijhout, H.F., 1994. Insect hormones. Princeton Univ. Press, New Jersey.

588

Noirot, C., 1985. The caste system in higher termite. In: Watson, J. A. L., Okot-

589

Kotber, B. M., Noirot, C., (Eds), Caste differentiation in social insects,

590

Pergamon Press, Oxford, pp. 75−86.

591

ACCEPTED MANUSCRIPT

Parthasarathy, R., Tan, A., Palli, R.S., 2008. bHLH-PAS family transcription

593

factor methoprene-tolerant plays a key role in JH action in preventing the

594

premature development of adult structures during larval-pupal metamorphosis.

595

Mech. Dev. 125, 601−616.

RI PT

592

596

Riddiford, M.L., 2008. Juvenile hormone action: A 2007 perspective. J. Insect

598

Physiol. 54, 895−901.

M AN U

599

SC

597

600

Roisin, Y., 1996. Castes in humivorous and litter-dwelling neotropical nasute

601

termites (Isoptera: termitidae). Insectes Sociaux 43, 375−389.

602

Saiki, R., Gotoh, H., Toga, K., Miura, T., Maekawa, K., 2015. High juvenile

604

hormone titer and abdominal activation of the JH signaling may induce

605

reproduction of termite neotenics. Insect Mol. Biol., in press.

EP

TE D

603

606

Saiki, R., Yaguchi, H., Hashimoto, Y., Kawamura, S., Maekawa, K., 2014

608

Reproductive soldier-like individuals induced by juvenile hormone analog

609

treatment in Zootermopsis nevadensis (Isoptera, Archotermopsidae). Zool. Sci.

610

31, 573−581.

AC C

607

611 612

Scharf, M.E., Ratliff, C.R., Hoteling, J.T., Pittendrigh, B.R., Bennette, G.W.,

613

2003. Caste differentiation responses of two sympatric Reticulitermes termite

ACCEPTED MANUSCRIPT

614

species to juvenile hormone homologs and synthetic juvenoids in two laboratory

615

assays. Insectes Sociaux 50, 346−354.

RI PT

616 Smykal, V., Bajgar, A., Provaznik, J., Fexova, S., Buricova, M., Takaki, K.,

618

Hodkova, M., Jindra, M., 2014. Juvenile hormone signaling during reproduction

619

and development of the linden bug, Pyrrhocoris apterus. Insect Biochem. Mol.

620

Biol. 45, 69−76.

SC

617

M AN U

621

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6:

623

Molecular Evolutionary Genetics Analysis Version 6.0. Biol. Evol. 30, 2725-

624

2729.

625

TE D

622

Terrapon, N., Li, C., Robertson, H.M., Ji, L., Meng, X., Booth, W., Chen, Z.,

627

Childers, C.P., Glastad, K.M., Gokhale, K., Gowin, J., Gronenberg, W.,

628

Hermansen, R.A., Hu, H., Hunt, B.G., Huylmans, A.K., Khalil, S.M., Mitchell,

629

R.D., Munoz-Torres, M.C., Mustard, J.A., Pan, H., Reese, J.T., Scharf, M.E.,

630

Sun, F., Vogel, H., Xiao, J., Yang, W., Yang, Z., Yang, Z., Zhou, J., Zhu, J.,

631

Brent, C.S., Elsik, C.G., Goodisman, M.A., Liberles, D.A., Roe, R.M., Vargo,

632

E.L., Vilcinskas, A., Wang, J., Bornberg-Bauer, E., Korb, J., Zhang, G., Liebig,

633

J., 2014. Molecular traces of alternative social organization in a termite genome.

634

Nat. Commun. 5, 3636.

635

AC C

EP

626

ACCEPTED MANUSCRIPT

Toga, K., Hojo, M., Miura, T., Maekawa, K., 2009. Presoldier induction by

637

juvenile hormone analog in the nasute termite Nasutitermes takasagoensis

638

(Isoptera: Termitidae). Zool. Sci. 26, 382−388.

RI PT

636

639

Toga, K., Hojo, M., Miura, T., Maekawa, K., 2012. Expression and function of a

641

limb-patterning gene Distal-less in the soldier-specific morphogenesis in the

642

nasute termite Nasutitermes takasagoensis. Evol. Dev. 14, 286−295.

643

M AN U

SC

640

644

Tsuchida, T., Koga, R., Horikawa, M., Tsunoda, T., Maoka, T., Matsumoto, S.,

645

Simon, J.C., Fukatsu, T., 2010. Symbiotic bacterium modifies aphid body color.

646

Science 330, 1102−1104.

TE D

647

Tsuchiya, M., Watanabe, D., Maekawa, K., 2008. Effect on mandibular length of

649

juvenile hormones and regulation of soldier differentiation in the termite

650

Reticulitermes sports (Isoptera: Rhinotermitidae). Appl. Entomol. Zool. 43,

651

307−314.

AC C

652

EP

648

653

Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Greurts, R., Leunissen,

654

J.A.M., 2007. Primer 3 Plus, an enhanced web interface to Primer 3. Nucleic

655

Acids Res. 35, 71−74.

656

ACCEPTED MANUSCRIPT

Vandesompele, J., Preter, K.D., Pattyn, F., Poppe, B., Roy, N.V., Paepe, A.D.,

658

Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR

659

data by genometric averaging of multiple internal control genes. Genome Biol.

660

3: 0034:1−0034:11.

RI PT

657

661

Watanabe, D., Gotoh, H., Miura, T., Maekawa, K., 2014. Social interactions

663

affecting caste development through physiological actions in termites. Front.

664

Physiol. 5, 127.

M AN U

SC

662

665

Zhou, X., Riddiford, L.M., 2002. Broad specifies pupal development and

667

mediates the ‘status quo’ action of juvenile hormone on the pupal-adult

668

transformation in Drosophila and Manduca. Development 129, 2259-2269.

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Figure legends

671

Fig. 1

672

ZnMet (upper), ZnKr-h1 (middle) and ZnBr-C (lower) expression changes in the

673

heads of Z. nevadensis during soldier differentiation. Each expression levels

674

were normalized by RPL13a expression. Relative expression levels (mean ±

675

S.D., technical triplicates) were calibrated using the mean expression level of

676

workers (3 days before the presoldier molt) as 1.0. Three real-time quantitative

677

PCR analyses were performed using the same cDNA sample (four different

678

individuals were used; see the section 2.3.) of each time point in the molting

679

process. Different letters above the bars denote significant differences (Tukey’s

680

test, P < 0.05).

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Fig. 2

683

Head and mandibles of soldiers 7 days after the soldier molt. GFP or ZnMet

684

dsRNA was injected each day in the presoldier period (days 0−9). The fraction

685

(4−15/4−15) shows the number of individuals with similar phenotypes shown in

686

each photo (numerator) and the numbers examined (denominator). Scale bar

687

indicates 1 mm. Relative change in ZnMet expression during presoldier period

688

(Fig. 1) is shown in the upper graph.

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Soldier head morphology affected by the dsRNA injection just before the

692

presoldier molt. Both soldiers were photographed 7 days after the soldier molt.

693

GFP or ZnMet dsRNA was injected before the presoldier molt. The fraction

694

(10/10) shows the number of individuals with similar phenotypes shown in each

695

photo (numerator) and the numbers examined (denominator). Scale bar

696

indicates 1 mm.

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697 Fig. 4

699

Plots of the principal component analysis based on measurements of external

700

morphology in each dsRNA-injected individual. A contribution ratio of each

701

principal component is indicated in parentheses. Based on each Eigen-vector,

702

first and second principal components mainly indicated a difference in body size

703

and in weapon size, respectively. Black and white plots indicate ZnMet and

704

GFP dsRNA-injected individuals, respectively. Squares indicate molted soldiers

705

after dsRNA injection during the presoldier period (days 0−9; Fig. 2). Triangles

706

indicate molted soldiers after dsRNA injection before the presoldier molt (bpm;

707

Fig. 3). Circles indicate molted presoldiers after dsRNA injection before the

708

presoldier molt.

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Fig. 5

711

Head capsule (A) and left mandible (B) lengths (mean ± S.D., n = 4) of molted

712

soldiers after dsRNA injection at each time point before the presoldier molt

ACCEPTED MANUSCRIPT

(bpm) and at days 0−9 during the presoldier period. Note that day 7 to 9 during

714

the presoldier period correspond to day 3 to 1 before the soldier molt (bsm).

715

Black and gray columns indicate ZnMet and GFP dsRNA-injected individuals,

716

respectively. Asterisks indicate a significant difference (two-way ANOVA

717

followed by Scheffe’s F test, p < 0.05).

SC

718

RI PT

713

Fig. S1

720

Developmental pathway of Z. nevadensis (modified from Heath, 1927; Castle,

721

1936; Saiki, et al., 2014) (A) and three focal molts shown as the boxed area in

722

A (B). Soldiers differentiate from 5−7th instar larvae (L5−7) via the presoldier

723

stage in the mature colonies. In incipient colonies, however, presoldiers

724

differentiate from 3rd instar larva (L3) (Maekawa et al. 2012). L: larva, N:

725

nymph, Neo: neotenic, A: alate, PS: presoldier, S: soldier. See the text for

726

details.

EP

727

TE D

M AN U

719

Fig. S2

729

Partial cDNA sequences and deduced amino acid (aa) sequences of ZnMet (A),

730

ZnKr-h1 (B) and an alignment of ZnMet aa sequence with those of some taxa

731

(Reticulitermes speratus, LC017912; Diploptera punctata, AIM47235; Blattela

732

germanica, CDO33887; Tribolium castaneum, NP_001092812; Bombyx mori,

733

ACJ04052; Daphnia pulex, BAM83853) (C). The nucleotides in white letters

734

indicate conserved domains; basic-helix-loop-helix (bHLH) DNA-binding domain

AC C

728

ACCEPTED MANUSCRIPT

and two Per-Arnt-Sim (PAS) domains (Ashok et al., 1998) in ZnMet (A), and

736

four C2H2 Zn-finger domains (Minakuchi et al., 2009) in ZnKr-h1 (B). The boxed

737

area and solid underline show the sequences for dsRNA synthesis and primers

738

for qPCR analysis, respectively.

RI PT

735

739 Fig. S3

741

ZnMet (upper), ZnKr-h1 (middle) and ZnBr-C (lower) expression changes in

742

heads during the worker molt. Each expression levels were normalized by

743

RPL13a expression. Relative expression levels (mean ± S.D., technical

744

triplicates) were calibrated using the mean expression level of workers (3 days

745

before the worker molt) as 1.0. Three real-time quantitative PCR analyses were

746

performed using the same cDNA sample (four different individuals were used;

747

see the section 2.3.) of each time point in the molting process. Different letters

748

above the bars denote significant differences (Tukey’s test, P < 0.05).

749 Fig. S4

AC C

750

EP

TE D

M AN U

SC

740

ACCEPTED MANUSCRIPT

Expression levels (mean ± S.D., biological quadruplicates) of ZnMet in the

752

heads of presoldiers at 3 days after dsRNA injection during the presoldier

753

period (injected on day 1). Each expression levels were normalized by RPL13a

754

expression. Relative expression levels were calibrated using the mean

755

expression level of GFP dsRNA-injected individuals as 1.0. Asterisks denote

756

significant differences (Mann-Whitney’s U test, P < 0.05).

SC

RI PT

751

757 Fig. S5

759

Brightness of the molted soldier cuticles in the head capsule (mean ± S.D., n =

760

4) after dsRNA injection before the presoldier molt. N.S. indicates no significant

761

difference (Mann-Whitney’s U test, P < 0.05). See the text for the detail

762

methods to measure the values.

TE D

763

M AN U

758

Fig. S6

765

Expression levels (mean ± S.D., biological quadruplicate) of ZnMet (A) and

766

ZnKr-h1 (B) in the heads of presoldiers just after molting. GFP or ZnMet dsRNA

767

was injected before the presoldier molt. Each expression levels were

768

normalized by RPL13a expression. Relative expression levels were calibrated

769

using the mean expression level of GFP dsRNA-injected individuals as 1.0.

770

Asterisks denote significant differences (Mann-Whitney’s U test, P < 0.05).

AC C

EP

764

771 772

Fig. S7

ACCEPTED MANUSCRIPT

Presoldiers photographed 3 days after the presoldier molt. GFP or ZnMet

774

dsRNA was injected before the presoldier molt. The fraction (10/10) shows the

775

number of individuals with similar phenotypes shown in each photo (numerator)

776

and the number examined (denominator). Scale bar indicates 1 mm.

RI PT

773

777 Fig. S8

779

Head capsule (A) and left mandible (B) lengths (mean ± S.D., n = 4) of molted

780

presoldiers after dsRNA injection before the presoldier molt. N.S. indicates no

781

significant difference (Mann-Whitney’s U test, P < 0.05).

M AN U

SC

778

782 Table S1

784

Primer sequences used in this study and the amplified efficiencies of each

785

primer pairs.

TE D

783

EP

786 Table S2

788

Ranking of reference genes to normalize target gene expressions. Values in the

789

parentheses indicate the stability values (M), which were calculated by the

790

GeNorm and NormFinder. Both softwares recommended RPL13a for the

791

reference gene.

792

AC C

787

ACCEPTED MANUSCRIPT a bcd bcd bcd

def

de ef

de f

ab

0

-3 -2 -1 worker (3rd lavae)

cde

fgh

gh

gh

cde

f

a

efg

cd

ef

a

ab

def

0 +1 +3 +5 +7 +8 +9 0 +1 +3 +5 +7 -3 -2 -1

EP

day

cd

TE D

h

ef

M AN U

cd gh

cd

ef

ZnBr-C fgh

de

ef

SC

c

de

bc

1.5

ab

b

0.6

0 3

a

ZnKr-h1

bcd

de

f

0 1.2

cde

ab

abc

RI PT

1

ZnMet

AC C

Relative expression level (/RPL13a)

2

presoldier

soldier

ACCEPTED MANUSCRIPT

1

3

5

4/4

10/10

15/15

11/11

5/5

11/11

4/4

5/5

7 -3 8/8

8 -2 8/8

AC C

EP

TE D

M AN U

GFP

soldier

9 -1 5/5

RI PT

0

ZnMet

RNAi

injection day

presoldier

SC

worker

ACCEPTED MANUSCRIPT

10/10

AC C

EP

TE D

M AN U

SC

RI PT

10/10

GFP

ZnMet

RNAi

ACCEPTED MANUSCRIPT

2.5

injection timing

2.0

presoldier day 0~9

1.5

bpm

ZnMet

RNAi

GFP

soldier soldier presoldier

1.0 0.5

RI PT

0 -0.5 -1.0

-2.0 -8.0

-6.0

-4.0

-2.0

SC

-1.5 0

EP

TE D

M AN U

first principal component (82.84%)

AC C

second principal component (4.37%)

3.0

2.0

4.0

6.0

ACCEPTED MANUSCRIPT

A

ZnMet RNAi GFP

head capsule

RI PT

*

SC

2000

M AN U

length (μm)

4000

0

injection timing

bpm day0 day1 day2 day3 day4 day5 day6 day7 day8 day9 bsm3 bsm2 bsm1

B

*

EP

mandible

ZnMet RNAi GFP

AC C

length (μm)

3000

TE D

presoldier period

1500

0

injection timing

bpm day0 day1 day2 day3 day4 day5 day6 day7 day8 day9 bsm3 bsm2 bsm1

presoldier period

ACCEPTED MANUSCRIPT Highlights

・ Role of the Methoprene-tolerant (Met) gene in termite soldier differentiation was examined.

・ RNAi knockdown of Met prior to presoldier molt suppressed morphogenesis of

RI PT

soldier-specific characters.

AC C

EP

TE D

M AN U

SC

・ JH acts through its receptor Met to direct social cast polyphenism in termites.

ACCEPTED MANUSCRIPT

Forward

Reverse

Met (RACE PCR)

TGGAGCTGAGGAGGCTTCTG

TCGAAGCTGTTCATCTGGTG

Met (dsRNA synthesis)

GAGGATGATCAGGGTGGAGA

Met (qPCR)

ATCACGATGTGCTGGACAAG

GTGATGATGAACTGGAGGTGTC

117.4

Kr-h1 (qPCR)

CCGCTGCTGTCGTAGAATC

ATGAACACTCGTATGCCTTGTC

106.6

Br-C (qPCR)

TCTGCTGCTCCACACATCAG

TGTAGCTTCAGGTGATGGCG

80.1

b-actin (qPCR)

AGCGGGAAATCGTCCGTGAC

CAATGGTGATGACCTGCCCAT

107

EF-1a (qPCR)

GCATGCACTGTTGGCTTTTA

TTCCTCAAATCGGGTTTCAG

108.3

NADH-dh (qPCR)

CGGCAAGGAAGCAAATAAAG

TTGGGTTGGGGGTATCAGC

123.4

RS49 (qPCR)

CATGCTTCCTACTGGCTTCC

AATTTCGGCACAGAATTTGC

103

RPS18 (qPCR)

CTCCGTGAAGACCTGGAGAG

CGTCTTCGTGTGTTGTCCAC

110.9

RPL13a (qPCR)

CACTTCAGAGCACCAAGCAA

ACGTTTCAATGCTGCCTTTC

103.4

SC

M AN U TE D EP AC C

Efficiencies (%)

RI PT

gene name

ACCEPTED MANUSCRIPT

Normfinder

RPL13a (0.622) RPS18 (0.633) RS49 (0.670) EF-1a (0.684) NADH-dh (1.155) b-actin (1.330)

RPL13a (0.128) RPS18 (0.215) RS49 (0.224) EF-1a (0.227) NADH-dh (0.687) b-actin (0.851)

AC C

EP

TE D

M AN U

SC

1 2 3 4 5 6

geNorm

RI PT

Software

ACCEPTED MANUSCRIPT

A

S

S

PS L

L

2

3

L

4

L

5

L L

gut-purge 3.1 ± 0.51 days

No.1

TE D

3.4 ± 0.57 days

EP

AC C

worker (L3)

7

N

1

worker (L4)

N

2

A

Neo

gut-purge

6.7 ± 0.75 days

presoldier

No.2

6

RI PT

1

SC

B

L

M AN U

Egg

PS

3.5 ± 0.66 days

soldier

A βHLH

ACCEPTED MANUSCRIPT

aaa1aTTACAGTCGTCAGAATCGCAACATGATGGAGAAACACCGACGCGACAAGATGAACGCGCA aaaaaaaYaaSaaRaaQaaNaaRaaNaaMaaMaaEaaKaaHaaRaaRaaDaaKaaMaaNaaAaaH aa611CATCAGCAACCTCGCGCTGCTGGTGCCCACTGTAGCAAATTCTCCAAAGAAAATGGACAA aaaaaaaIaaSaaNaaLaaAaaLaaLaaVaaPaaTaaVaaAaaNaaSaaPaaKaaKaaMaaDaaK 1121aGACAAGCATTTTGAGATTAACTGCTGCTTTCCTTCGACTGCATAAGTTTCTGACTACGAA aaaaaaaTaaSaaIaaLaaRaaLaaTaaAaaAaaFaaLaaRaaLaaHaaKaaFaaLaaTaaTaaK a181aATCCGAGAGGATTAAGAGATTAGGGCTCCCGCACTACTTGAAGACCTACAACCTGTCCCA aaaaaaaSaaEaaRaaIaaKaaRaaLaaGaaLaaPaaHaaYaaLaaKaaTaaYaaNaaLaaSaaQ PAS A a

M AN U

SC

RI PT

a241aAGCTCTGATGGAGGTGCTGGATGGCTTTATGATCATTGTCTCATCCACGGGAAAGATACT aaaaaaaAaaLaaMaaEaaVaaLaaDaaGaaFaaMaaIaaIaaVaaSaaSaaTaaGaaKaaIaaL a301aGTTCGTCACTCATACCGTGGAGACTTTACTCGGCTATCCTCAGAGTTACTTGATGGGGCA aaaaaaaFaaVaaTaaHaaTaaVaaEaaTaaLaaLaaGaaYaaPaaQaaSaaYaaLaaMaaGaaQ a361aGTCAATGCATTCCATCATCTGCCGTGAAGATCACGATGTGCTGGACAAGAACTTAACACC aaaaaaaSaaMaaHaaSaaIaaIaaCaaRaaEaaDaaHaaDaaVaaLaaDaaKaaNaaLaaTaaP a421aTGACTTGGATCTCATGGCAGCCGAACATGGTGTTTCTGGTCAATTGTCACTTGGTGAGGA aaaaaaaDaaLaaDaaLaaMaaAaaAaaEaaHaaGaaVaaSaaGaaQaaLaaSaaLaaGaaEaaD a481aCAGCAGCAGTTCAGGGGACACCTCCAGTTCATCATCACAGAGTGCTATGTCACCCCAACA aaaaaaaSaaSaaSaaSaaGaaDaaTaaSaaSaaSaaSaaSaaQaaSaaAaaMaaSaaPaaQaaQ a541aGCAGCCCGTGTCATCTGCCCCCTCGTTGCAAGACATCACAGGCCCCATTCCGTCTTTGTA aaaaaaaQaaPaaVaaSaaSaaAaaPaaSaaLaaQaaDaaIaaTaaGaaPaaIaaPaaSaaLaaY a601aTCAGCAGCGCCGGTCCTTCTATCTGCGGATGATTCAGAAAGCTGTTTCTCGTAGTGAGCT aaaaaaaQaaQaaRaaRaaSaaFaaYaaLaaRaaMaaIaaQaaKaaAaaVaaSaaRaaSaaEaaL a661aCACCCAGTATGAGTTGGTCCATGTGCTGGGGTTCCTCAGCGTACCACAGCGACCGACACC aaaaaaaTaaQaaYaaEaaLaaVaaHaaVaaLaaGaaFaaLaaSaaVaaPaaQaaRaaPaaTaaP a721aCGGGCCTTCCACACCCTCTCGCACTCGCAACCGTTCGCGTGATGGCAGCAATGCAGGATC aaaaaaaGaaPaaSaaTaaPaaSaaRaaTaaRaaNaaRaaSaaRaaDaaGaaSaaNaaAaaGaaS a781aGGGTAATGACATCGATACTGTGCTGATCGCAGTCGTTCGCATGATGCGTGATAACAGTGT aaaaaaaGaaNaaDaaIaaDaaTaaVaaLaaIaaAaaVaaVaaRaaMaaMaaRaaDaaNaaSaaV

PAS B

AC C

EP

TE D

a841aTGCTCAGCGCTCACTACTTGAGCCAAGTAAAGATGAATATGTGACTCGGCATCTCATTGA aaaaaaaAaaQaaRaaSaaLaaLaaEaaPaaSaaKaaDaaEaaYaaVaaTaaRaaHaaLaaIaaD a901aCGGCCGCATTATTTACAGTGACCACAGGATCTCGGTGGTGGCAGGCTACATGGCCGAGGA aaaaaaaGaaRaaIaaIaaYaaSaaDaaHaaRaaIaaSaaVaaVaaAaaGaaYaaMaaAaaEaaE a961aGATTACAGGAGAGTCTGCCTTCAAGTTTATGCATAAGGATGATGTACGCTTTACAATTGT aaaaaaaIaaTaaGaaEaaSaaAaaFaaKaaFaaMaaHaaKaaDaaDaaVaaRaaFaaTaaIaaV 1021aAGCTCTACGACAGATGTATGATCGTGGTAAAGGGAGTTATGGCAGCAGTTGCTACAGGCT aaaaaaaAaaLaaRaaQaaMaaYaaDaaRaaGaaKaaGaaSaaYaaGaaSaaSaaCaaYaaRaaL 1081aTCTGTGCAAGACCGGCCAGTATATTTACTTGCGTACTCATGGCTACCTTGAGTATGACAA aaaaaaaLaaCaaKaaTaaGaaQaaYaaIaaYaaLaaRaaTaaHaaGaaYaaLaaEaaYaaDaaK 1141aGGACAGCCAGAAGATAGTTTCCTTCATATGCATCAATACCCTTGTGTCGGAGGAAGAAGG aaaaaaaDaaSaaQaaKaaIaaVaaSaaFaaIaaCaaIaaNaaTaaLaaVaaSaaEaaEaaEaaG 1201aTGTGCAACTTGTGCGAGAGATGAAGGCTCGGTTCTCTGCCAACATCTTGACAGCATCTAG aaaaaaaVaaQaaLaaVaaRaaEaaMaaKaaAaaRaaFaaSaaAaaNaaIaaLaaTaaAaaSaaS 1261aCCAGCAGCCAGCTCCACTTCCTCCCGCTTCCATCCCGTCCACTTCCACTGTCACGTCTGG aaaaaaaQaaQaaPaaAaaPaaLaaPaaPaaAaaSaaIaaPaaSaaTaaSaaTaaVaaTaaSaaG 1321aCCCCCTGGAATCAGACCTGTTGGAGGTAGCTATTTCACAACTTATTTCCAACATCCCTGC aaaaaaaPaaLaaEaaSaaDaaLaaLaaEaaVaaAaaIaaSaaQaaLaaIaaSaaNaaIaaPaaA 1381aAACAGAGGATGATCAGGGTGGAGAAGAGGAGAGGCGCCCTGAGGTATCAGATACCCAGTA aaaaaaaTaaEaaDaaDaaQaaGaaGaaEaaEaaEaaRaaRaaPaaEaaVaaSaaDaaTaaQaaY 1441aTATGAAGGTTGTACACTATTCGAAGGCTCTTCCTCCTGCAACAATACAGGCAGCAAAAGT aaaaaaaMaaKaaVaaVaaHaaYaaSaaKaaAaaLaaPaaPaaAaaTaaIaaQaaAaaAaaKaaV 1501aGGGCCTTGACCCACTTATGTCATTGTCTCATGTGGGGCCTACCTCACCTACAAGACCACT aaaaaaaGaaLaaDaaPaaLaaMaaSaaLaaSaaHaaVaaGaaPaaTaaSaaPaaTaaRaaPaaL 1561aTACTGTGACCATCCCTGGAACACCTTCAAGGGTACAGACAGATTCTGCTTGGTCTATGGT aaaaaaaTaaVaaTaaIaaPaaGaaTaaPaaSaaRaaVaaQaaTaaDaaSaaAaaWaaSaaMaaV

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

1621aGAAACGTGAATCTGTTATAACATCTTTAGTGTCTTCTGTTAAGACAGAGCATGTTCCTCG aaaaaaaKaaRaaEaaSaaVaaIaaTaaSaaLaaVaaSaaSaaVaaKaaTaaEaaHaaVaaPaaR 1681aACCTCCACCACCAGTGCCACCTCGAAGGCAAGAGACTGTTGTGGTTAGTGTACAGGCAAA aaaaaaaPaaPaaPaaPaaVaaPaaPaaRaaRaaQaaEaaTaaVaaVaaVaaSaaVaaQaaAaaN 1741aTCAGGCTGGAAATATGCCAGAGTGCATGACAGGGTTGAGAACACAAGCAAATCCAGTGTT aaaaaaaQaaAaaGaaNaaMaaPaaEaaCaaMaaTaaGaaLaaRaaTaaQaaAaaNaaPaaVaaL 1801aGAAGCGGACAAGTAACATTGCAGACTGTGAGGTACAGAGTAGTTCTAAGCGTCAGCATGT aaaaaaaKaaRaaTaaSaaNaaIaaAaaDaaCaaEaaVaaQaaSaaSaaSaaKaaRaaQaaHaaV 1861aGAGCTATGCTCGGCGAAGGGAAAGACGCCCCACTGAACCTTCAGCCCAACTTCATCTTGA aaaaaaaSaaYaaAaaRaaRaaRaaEaaRaaRaaPaaTaaEaaPaaSaaAaaQaaLaaHaaLaaE 1921aGAGTCCCACAATGGATGAACCTAGGAGGACACCAGATGAACAGCTTCGACTTATCATGAT aaaaaaaSaaPaaTaaMaaDaaEaaPaaRaaRaaTaaPaaDaaEaaQaaLaaRaaLaaIaaMaaM 1981aGCGAGACTCTCTTGTCACGAGTCCTCCTGCTAATTCCCTCTTACACTGTGTTGCAAGGGG aaaaaaaRaaDaaSaaLaaVaaTaaSaaPaaPaaAaaNaaSaaLaaLaaHaaCaaVaaAaaRaaG 2041aATTCTCAGAACCCAGTGCACCGAGTCCTGTTTCTCCAGCAGTTGGTGATTTTGTTACTC aaaaaaaFaaSaaEaaPaaSaaAaaPaaSaaPaaVaaSaaPaaAaaVaaGaaDaaFaaVaaT

B

ACCEPTED MANUSCRIPT aa1aTGAGGACGGCTCAGTTCACGGAAGCGCGGAGTGTGCTGGTCCCATGTTGCCAGAGCAACG aaaaaaEaaDaaGaaSaaVaaHaaGaaSaaAaaEaaCaaAaaGaaPaaMaaLaaPaaEaaQaaR a61aCCCAGAACAGCAAGAAGCCCCCGTCAAGCGGCTTGTGTGCAGCCCGGATTTACCCGTGTT aaaaaaPaaEaaQaaQaaEaaAaaPaaVaaKaaRaaLaaVaaCaaSaaPaaDaaLaaPaaVaaF 121aCTCGCTCACTCACGCTTTCGAGGAAGCCGCTGCCGCTGCTGTCGTAGAATCGTCTTCACC aaaaaaSaaLaaTaaHaaAaaFaaEaaEaaAaaAaaAaaAaaAaaVaaVaaEaaSaaSaaSaaP 181aTCCGGCCTCCGCCCCAGCGCCCGAAGACAAGGCATACGAGTGTTCATTTTGCCACAAGAC aaaaaaPaaAaaSaaAaaPaaAaaPaaEaaDaaKaaAaaYaaEaaCaaSaaFaaCaaHaaKaaT 241aTTTCCCTCAGAAGAACACGTACCAGAACCACCTGCGTTCGCACGGAAAGGAGGGCGAAGA aaaaaaFaaPaaQaaKaaNaaTaaYaaQaaNaaHaaLaaRaaSaaHaaGaaKaaEaaGaaEaaD

C2H2 zf

RI PT

301aTCCATACCAGTGCAACATCTGTGGCAAAACATTCGCAGTGCCCGCCCGCCTGACGCGACA aaaaaaPaaYaaQaaCaaNaaIaaCaaGaaKaaTaaFaaAaaVaaPaaAaaRaaLaaTaaRaaH 361aCTACCGCACGCACACGGGCGAGAAGCCCTATCAGTGCGAATACTGCAGCAAGTCCTTTTC aaaaaaYaaRaaTaaHaaTaaGaaEaaKaaPaaYaaQaaCaaEaaYaaCaaSaaKaaSaaFaaS

C2H2 zf

421aCGTGAAGGAGAACCTCAGTGTGCATAGGCGCATTCACACGAAGGAGCGACCCTACAAGTG aaaaaaVaaKaaEaaNaaLaaSaaVaaHaaRaaRaaIaaHaaTaaKaaEaaRaaPaaYaaKaaC

C2H2 zf

SC

481aCGACATATGCGCTAGGGCATTCGAACACAGTGGCAAACTGCATCGCCACATGCGGATCCA aaaaaaDaaIaaCaaAaaRaaAaaFaaEaaHaaSaaGaaKaaLaaHaaRaaHaaMaaRaaIaaH 541aCACGGGCGAGCGACCACACAAGTGCGGTGTCTGCGCCAAGACGTTCATCCAGAGTGGGCA aaaaaaTaaGaaEaaRaaPaaHaaKaaCaaGaaVaaCaaAaaKaaTaaFaaIaaQaaSaaGaaQ

C2H2 zf

M AN U

601aGCTCGTCATCCACATGCGGACTCACACTGGCGAGAAACCCTACGTGTGTGCGGCGTGCGG aaaaaaLaaVaaIaaHaaMaaRaaTaaHaaTaaGaaEaaKaaPaaYaaVaaCaaAaaAaaCaaG

C2H2 zf

AC C

EP

TE D

661aTAAGGGCTTTACCTGCTCCAAACAACTGAAGGTACACACAAGGACCCACACAGGAGAGAA aaaaaaKaaGaaFaaTaaCaaSaaKaaQaaLaaKaaVaaHaaTaaRaaTaaHaaTaaGaaEaaK 721aACCCTACTCTTGCGACATTTGCGGCAAGGCGTTCGGCTACAACCACGTGCTGAAGCTTCA aaaaaaPaaYaaSaaCaaDaaIaaCaaGaaKaaAaaFaaGaaYaaNaaHaaVaaLaaKaaLaaH 781aCCAAGTGGCGCACTACGGGGAGAAAGTGTACAAATGCACGATCTGCAGCCAAACCTTTAC aaaaaaQaaVaaAaaHaaYaaGaaEaaKaaVaaYaaKaaCaaTaaIaaCaaSaaQaaTaaFaaT 841aTTCGAAGAAGACTATGGAGGTTCATATCAAGAGCCACTCGGAGCCCTCGAACACGGCACG aaaaaaSaaKaaKaaTaaMaaEaaVaaHaaIaaKaaSaaHaaSaaEaaPaaSaaNaaTaaAaaR 901aGAGCCCACAGCCCCCCGCACTCCCCCCGACAGGGCGGCAGAACACCGACCCGGGCGAATC aaaaaaSaaPaaQaaPaaPaaAaaLaaPaaPaaTaaGaaRaaQaaNaaTaaDaaPaaGaaEaaS 961aGTCATGTGCTTCATCCAGTAGCGATAA aaaaaaSaaCaaAaaSaaSaaSaaSaaD

C

211

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

281

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

351

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

421

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

491

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

VANSPKKMDK VANAPKKMDK VANSPKKMDK VANSPKKMDK VARSAKRMDK VARSAKRMDK AAAAPRKLDK * * **

TSILRLTAAF TSILRLTAAY TSILRLTAAF TSILRLTAXF TSILRLAATH TSILRLAATH TSTLRLSANF ** *** *

LRLHKFLTTK LRLHKFLNAD LRLHKFLVTD LRLHKFLVAD LRIYQTLLSG LRIYQTLLSG LRIHQNM--**

S-ERIKRLGL TGGNVTRLEF P-GELNRIDF P-GDPNRIEF K--NHPHIQL K--NHPHIQL ---DLRMKPY

PHYLKTYNLS PRYLKDYDMY PNFLKDCNLS PDFLKDCNLS PKHVDQYLLE PKHVDQYLLE NRW--NALAG

QALME---VL QALME---VM QAFME---VM QAFMELLQVM QLVCE---QL QLVCE---QL HSILE---KL *

DGFMIIVS-S DGFMIMVS-R DGFMIIVS-C DGFLIIVS-S GGFLLILT-P GGFLLILT-P DSFLLVVSCC *

TGKILFVTHT TGNILFVTHT SGNILFVSHT TGNILFVSHT NGKIVFVSHT NGKIVFVSHT SGKIIYVTDR * * *

VETLLGYPQS VETLLGHPQS VETLLGHPQT VETLLGHQQN VEHLLGHLQT VEHLLGHLQT VEKLLGHAQV ** ***

YLMGQSMHSI FLMGQKLHSI SLLGQKLHSI FLLGQKLHSI DLMGQSIFNI DLMGQSIFNI DMMGYQLSCF *

ICREDHDVLD TFHEDHDVLN TCREDHDTLN TCREDHDKLN TSPDDHDRLTSPDDHDRLVHQADQDAIE * *

KNLTPDLDLM KNLTPDSDPS KNLNPDPDPS KNLTPDPDPS ------------------KRLSA-----

AAEHGVSGQL LSDHDGSGQL AGDQENSGQL AGDQESTGQM --------RM --------RM -----FAAQV

SLGEDSSSSG SAGDDNSSSG SVGEDSSSSG SVGEDSSSSG YINTESVLDG YINTESVLDG AANPDASDSA

DTSSSSSQSA DTSSSSSQSA DTSNSSSPSV DASSSSSQSL D--------D--------D--------*

MSPQQQPVSS LSPPQPSSST PSPQQQQQQL PSPQHQQQSA ----------------------------

APSLQDI--FSVPANT--QPSTSQQ--PSSSTSTAVA ----------------------------

---TGPIPSL ---SRNSSPD --QEAPSEPL VPEAAPSEPL ----------------------------

YQQRRSFYLR -HQRRSFYVR -RQRRSFYLR -RQRRSFYLR --WKKCFNIR --WKKCFNIR -GQVYSFECR * *

MIQKAVSRSE LSQRTASRSD LSQRTVSRSE LSQKAVSRSE L-KRAGPRTE L-KRAGPRTE LAGRQLSRGE * *

LTQYELVHVL VTQYELVHVQ ITQYELMHVL ITQYELMHXL SAVYEPVRIM SAVYEPVRIM PTVYERVSVS **

GFLSVPQRPT GFLRVPQHTN GHLRVPQRPS GHLRVPQRPN GVHR-----GVHR-----GTFRGPRRRR *

PGPSTPSRTR PGPATHSRTR QGPSTQSRTR PGPSAQSRTR PGFDNDC--PGFDNDC--DWADLKS---

NRSRDGSNAG SRSRDGNSAG SRQRDGSSTG NRQREGSSTG --NKNTSTSK --NKNTSTSK -SDRSVATVQ

SGND-IDTVL SGND-IDTVL SSVD-SDTVL SSAD-SDTVL EIAL-NNDVL EIAL-NNDVL QHNDYSEPLF

IAVVRMMR-D IAVVRTMR-E IAVVRTVR-E IAVVRTVR-E LFFVKVFR-P LFFVKVFR-P IGLVRILQTP *

NSVAQRSLLE RTVSSRSLFH SSVAERSLLE SSLVERSLLE EPLCER-LFE EPLCER-LFE NTLPPLTIMQ

PSKDEYVTRH ATREEYVTRH PSKDEYVTRH PSKDEYVTRH ASREEYVTRH ASREEYVTRH AVQDEYITQH ** *

LIDGRIIYSD LLDGRIIYCD LIDGRIIYSD LIDGRIIYSD LIDGRIIGCD LIDGRIIGCD TTGGTIIQTD * ** *

HRISVVAGYM YRISLVAGYM HRISVVAGYL HRISVVAGYM QRISFIAGYM QRISFIAGYM HRIAIIAGYL ** ***

AEEITGESAF ASEITGMSAF AEEVAGESAF AEEVAGLSAF TEEVSGLSAF TEEVSGLSAF SGEVTGMSAY * **

KFMHKDDVRF KFMHKDDVLY KFMHRDDVRF KFMHKDDMRF KFMHREDVRW KFMHREDVRW DYVYSEDLEY *

TIVALRQMYD TIVALREMYH TIVALRQMYD TMIALRQMYD VMIALRQMYD VMIALRQMYD TLKAQSLMLT * *

RGKGSYGSSC HGKQSFGNSC RGKDSYGSSC RGKDSYGSSC RG-ESKGSSC RG-ESKGSSC R---SEGMVT *

YRLLCKTGQY YRLLSKTGQF YRLLCKTGQY YRLQSKTGQY YRLLSRNGQF YRLLSRNGQF YRLKTSTGRL *** *

IYLRTHGYLE IYLQTHGYLE IYMRTHGYLE IYLRTHGYLE IYLRTFGFLE IYLRTFGFLE IFLRSRGFIQ * *

YDKDSQKIVS YDRDSKKLVS YDKDTQQIVS YDKDTQQIVS ID-DQGTVES ID-DQGTVES YDENTKEIVS * *

FICINTLVSE FLCVNTLVSE FICINTLVPE FICINTLLTE FVCVNTLVSE FVCVNTLVSE FFCINSLIDE * * * * *

EEGVQLVREM EEGLQLMHEM DEGETLVQEM EEGERRIQEM QEGLQLINEM QEGLQLINEM EQGMKEMQEM * **

KARFSANILT KAQYDSGL-Q KTRYSATVMS KARFSATITS KKRYSALINS KKRYSALINS RAMLDKLNIA

ASSQQPAPLP SYCQKASLMA SRQQ-AALLG SRQQ-----X QSCP-----QSCP-----TVTP-----A

PASIPSTSTV PTPVPSTSTS PVPGPSTSTG AILGPSTSTS -ITSSGSTDS -ITSSGSTDS IASSPTSTIE

TSGPLESD-L TCSPVDTQ-D -SSSVEAD-P -VGNVDND-Q SSQSVEDPQQ SSQSVEDPQQ PVGAASTQEP

LEVAISQLIS LETAVSHLLA LDAAISQLIT LEVAISQLIS VEAAIVHLIA VEAAIVHLIA LSRCVRASVG *

NIPATEDDQG SIPPPDG--NIPEVRDDGK NIPEVRDDNK NLPSPGSDQR NLPSPGSDQR KPPPSFSSNG *

---GEEERRP ---AEEEKCS ---SEPSS-----EPSN----STPSP----STPSP-CHLANGSRVS

EVSDTQYMKV EVSDSQYMKV -LPDTQYVKA -LPDTQYAKA ----RVYGNV ----RVYGNV GLPRSSIMPN

VHYSKALPPA LHYSKLLPPV VRYSKVLPSV VRYSKVLPSA NENQDCSTPT NENQDCSTPT GHNSLCISPS

TIQAAKVGLD SVQAAKAGID TEQAAKVGIK TEQAAKVGIK EN-------S EN-------S SE--------

PLMSLSHVGP PYMSLSGEQP NVIPTTRGGP PVIPNARTGP PTKPYYKLKT PTKPYYKLKT --LQYSPSGS

TSPTRPLTVT ASPTRPLTVT TSPTGPLTVA ISPTGPLTVT ANNKRP---ANNKRP---SSSSFS----

IPGTPSRVQT IPEAPSRIQP IPEGPQKSQN IPEXPQRIHP ----PSTELG ----PSTELG ----EERCQS

DSAWSMVKRE ESGWPRVKRE EANWIHVKRE EATWTSIKRE TNIYTSSKRQ TNIYTSSKRQ STPHSLVSSP

RI PT

PAS domain A

SC

141 Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

ISNLALLVPT INSLAQLVPT ISNLALLVPT ISNLALLVPT IGELATLVPM IGELATLVPM ISELYSLVPS * * ***

M AN U

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

KHRRDKMNAH KHRRDKMNAH KHRRDKMNAH KHRRDKMNAH KMRRDKLNSY KMRRDKLNSY KQRRDKLNAY * **** *

TE D

71

YSRQNRNMME YSRQTRNMME YSRQNRNMME YSRQNRNMME NSREMRNRAE NSREMRNRAE SSREMRNRAE ** ** *

EP

Z.Zn nevadensis R. speratus Rs D. punctata Dp Bg B. germanica T.Tc castaneum Bm B. moli Daphnia D. pulex

ACCEPTED MANUSCRIPT Helix-loop-helix domain

AC C

1

PAS domain B

ACCEPTED MANUSCRIPT VVSVQANQAG LVSAQGNQTG LVSAQSDSSS -------------------------------------

NMPECMTGLR SVPECMAGLR DVSRLRGN--------------------------------------

TQANPVLKRT GRANQAMKRT ---NAVLKRT -------------------------------------

SNIADCEVQS NTLADCDVQS SSVADCEVQT -------------------------------------

SSKRQHVSYA SSKRQHVSYA GSKRQHVSYA ---------------------------------VSYP

RRRERRPTEP RRRERWPKEH RRRERRSTEP ---------------------------FAPIPFPWRP

SAQLHLESPT PAKLKPESST TSQHQPKSPT ---------------------------STDCSPITTT

MDEPRRTPDE LDEPRRIPDE LEEPQRTPEE -------------------------------------

QLRLIMMRDS QLRLIMMRES QLRLIMSRNS -------------------------------------

LVTSPPANSL SVTSPSASSS SVTSPSSNSP -----------------------------------SN

LHCVARGFSE SHCTARGCSE LHCIGRGCSE ---------------------------GHVNTREVSK

PSAPSPVSPA P---SPMFSA PGAPSPGSPT ---------------------------S---------

VGDFVT HGG--HGA-----------------------

M AN U

SC

RI PT

PVPPRRQETV PKPPRRHESV ----RRQETV -------------------------------------

TE D

701

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

KTEHVPRPPP KAEPVPPPPP KVEVV-----------------------------------------

EP

631

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

SVITSLVSSV SVITTLGPSI SVITSLVPAV SVITSLVP-RTSPQLSP-RTSPQLSP-MEIPNLVA--

AC C

561

Z.Zn nevadensis R.Rs speratus D.Dp punctata B.Bg germanica T.Tc castaneum B.Bm moli D.Daphnia pulex

ACCEPTED MANUSCRIPT a

ZnMet

a b

bc

4

d

c d

d

0

2.5

a

a

ZnKr-h1

RI PT

5

a

b

bc c

ZnBr-C d

d

-3

-2

d

0

ab

a

+3

+5

b

b

d

0

TE D

-1

worker (3rd instar lavae)

EP

day

a

c

2.5

M AN U

4.5

SC

0

AC C

Relative expression level (/RPL13a)

8

+1

worker (4th instar lavae)

+7

AC C EP TE D

Relative expression level (ZnMet / RPL13a)

0.8

0.6

RI PT

0.4

SC

0.2

0

GFP

M AN U

ACCEPTED MANUSCRIPT

1.4

*

1.2

1

ZnMet

RNAi

ACCEPTED MANUSCRIPT

70

N.S.

RI PT

50

SC

40 30

M AN U

Brightness (%)

60

20

0

TE D

10

AC C

EP

GFP

ZnMet

RNAi

ACCEPTED MANUSCRIPT

1.2

*

ZnMet

1

RI PT

0.8 0.6

SC

0.4 0.2 0

GFP

0.8

TE D

1

ZnKr-h1

ZnMet

RNAi

*

EP

1.2

ZnMet

RNAi

0.6

AC C

Relative expression level (/RPL13a)

B

M AN U

Relative expression level (/RPL13a)

A

0.4 0.2 0

GFP

ACCEPTED MANUSCRIPT

10/10

AC C

EP

TE D

M AN U

SC

RI PT

10/10

GFP

ZnMet

RNAi

2500

ACCEPTED MANUSCRIPT head capsule N.S.

RI PT

1500

0

M AN U

SC

length (μm)

A

GFP

TE D

ZnMet

RNAi

N.S.

EP

2000

mandible

AC C

length (μm)

B

ZnMet

RNAi

1000

0

GFP