Genetics: Early Online, published on June 25, 2018 as 10.1534/genetics.118.301038
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1 A crucial caste regulation gene detected by comparing termites and sister group
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4 Yudai Masuoka1, 2, Kouhei Toga3, Christine A. Nalepa4, Kiyoto Maekawa2*
5 1. Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555,
6 Japan
7 2. Institute of Agrobiological Sciences, National Agriculture and Food Research
8 Organization, Tsukuba, Ibaraki 305-8634, Japan
9 3. Department of Integrated Science in Physics and Biology, Nihon University, Tokyo
10 156-8550, Japan
11 4. Department of Entomology, North Carolina State University, Raleigh, NC 27695-7613,
12 USA
13
14 *Corresponding author (email address: [email protected])
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16 Running head (about 35 characters inc. spaces): Caste regulation gene in termites
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18 Keywords: termites, Cryptocercus, soldier differentiation, juvenile hormone,
19 20-hydroxyecdysone
20
21 Author contributions
22 YM and KM designed experiments; YM, KT and CN collected samples and performed
23 application analysis with JHA; YM performed molecular experiments and analyzed data; YM,
Copyright 2018.
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24 CN and KM wrote the manuscript; KM conceived of the study, designed the study,
25 coordinated the study; All authors read and gave final approval for publication.
26
27 Abstract
28 Sterile castes are a defining criterion of eusociality; investigating their evolutionary origins
29 can critically advance theory. In termites, the soldier caste is regarded as the first acquired
30 permanently sterile caste. Previous studies showed that juvenile hormone (JH) is the primary
31 factor inducing soldier differentiation, and treatment of workers with artificial JH can
32 generate presoldier differentiation. It follows that a shift from a typical hemimetabolous JH
33 response might be required for soldier formation during the course of termite evolution
34 within the cockroach clade. To address this possibility, analysis of the role of JH and its
35 signaling pathway was performed in the termite Zootermopsis nevadensis and compared with
36 the woodroach Cryptocercus punctulatus, a member of the sister group of termites. Treatment
37 with a JH analog (JHA) induced a nymphal molt in C. punctulatus. RNA interference (RNAi)
38 of JH receptor Methoprene tolerant (Met) was then performed, and it inhibited the presoldier
39 molt in Z. nevadensis and the nymphal molt in C. punctulatus. Knockdown of Met in both
40 species inhibited expression of 20-hydroxyecdysone (20E; the active form of ecdysone)
41 synthesis genes. However, in Z. nevadensis, several 20E signaling genes were specifically
42 inhibited by Met RNAi. Consequently, RNAi of these genes were performed in JHA-treated
43 termite individuals. Knockdown of 20E signaling and nuclear receptor gene, Hormone
44 receptor 39 (HR39/FTZ-F1β) resulted in newly-molted individuals with normal worker
45 phenotypes. This is the first report of the JH-Met signaling feature in termites and
46 Cryptocercus. JH-dependent molting activation is shared by both taxa, and mediation
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47 between JH receptor and 20E signalings for soldier morphogenesis is specific to termites.
48
49 Introduction
50 The complex eusocial society of one-piece termites (those utilizing a single log as food and
51 nest) consists of a reproductive caste (queen and king) and temporarily or permanently sterile
52 castes (workers, also known as helpers, pseudergates or alloparents, and soldiers,
53 respectively). Termites are a monophyletic group within cockroaches (Lo et al. 2000; Inward
54 et al. 2007; Bourguignon et al. 2017), and the soldier caste is regarded as the first acquired
55 permanently sterile caste (Nalepa 2011). The molecular basis of termite soldier evolution,
56 however, is still far from fully understood. Increasing juvenile hormone (JH) titers triggers
57 soldier differentiation in workers via an intermediate presoldier stage (Noirot 1985; Roisin
58 1996), and can be induced in many termite species by treating workers with JH or JH analogs
59 (JHA) (Watanabe et al. 2014; Scharf 2015). This is in contrast to other insects, in which JH
60 maintains larval traits and has an inhibitory function on molting via suppression of PTTH
61 (prothoracicotropic hormone) release (Gilbert 2012). It is also known that treatment with
62 JHA can inhibit or delay 20-hydroxyecdysone (20E; the active form of ecdysone) synthesis
63 and suppress expression of the 20E signaling genes (Berger et al. 1992; Zufelato et al. 2000;
64 Aribi et al. 2006). In the German cockroach, Blattella germanica, JHA treatment of young
65 instars inhibited 20E synthesis and resulted in developmental arrest in the nymphal stage
66 (Hangartner and Masner 1973; Masner et al. 1975). Furthermore, JH inhibits expression
67 levels of the 20E induced heat shock protein gene in Drosophila melanogaster (Berger et al.
68 1992), but in D. melanogaster and Manduca sexta, JH activates expression level of the
69 20E-inducible nuclear receptor gene, E75 (Dubrovskaya et al. 2004). There is therefore a
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70 possibility that one or more unidentified JH signaling pathways related to the involvement of
71 20E in both molting (from worker to presoldier) and morphological modification (formation
72 of weapons such as enlarged mandibles) were acquired during the course of termite evolution.
73 To clarify this hypothesis, it is necessary to analyze the role of JH in nymphal development in
74 additional cockroaches, particularly those of the sister group of termites, cockroaches in the
75 family Cryptocercidae (woodroaches; Cryptocercus spp.).
76
77 Recently, the presence of JH signaling genes has been established in some model insect
78 species (Jindra et al. 2015). In both hemimetabolous (without pupal stage, including termites
79 and cockroaches) and holometabolous (with pupal stage) insects, a JH receptor, methoprene
80 tolerant (Met) and a steroid receptor coactivator (SRC; taiman; FISC) induce the expression
81 of Krüppel homolog 1 (Kr-h1), which is necessary for JH to function in maintaining
82 developmental status quo (Jindra et al. 2015; Riddiford 2013). Met and Kr-h1 knockdown
83 inhibited molts in the penultimate instar and induced precocious metamorphosis in Tribolium
84 castaneum (Konopova and Jindra 2007; Minakuchi et al. 2009) and B. germanica (Lozano
85 and Belles 2011, 2014). On the other hand, although Met is generally involved in insect
86 ovarian development, Kr-h1 function differed somewhat among species (Konopova et al.
87 2011; Song et al. 2014). Specifically, Kr-h1 was not required for ovarian development in the
88 linden bug, Pyrrhocoris apterus (Smykal et al. 2014). In termites, a previous study
89 demonstrated that RNA interference (RNAi) of Met suppressed soldier-specific
90 morphogenesis in Zootermopsis nevadensis (Masuoka et al. 2015). Roles of other JH
91 signaling genes, including Kr-h1, for termite soldier differentiation, however, have not been
92 clarified. Moreover, in Cryptocercus cockroaches no studies have focused on the function of
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93 JH signaling genes during molting.
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95 To determine potential differences in the role of JH during molting in C. punctulatus and
96 termites, JHA treatment of young nymphs was performed in C. punctulatus. To further
97 clarify the function of JH signaling genes in these taxa, RNAi knockdown of Met and Kr-h1
98 was conducted in both Z. nevadensis and C. punctulatus. Furthermore, expression and
99 functional analysis of 20E signaling genes was performed during JHA-induced soldier
100 differentiation. Based on the results, we discuss how the termite specific JH pathway is
101 related to soldier development, which involves notable morphological changes during the
102 molting processes.
103
104 Materials & Methods
105 Insects
106 Seventh instars of Z. nevadensis were sampled from three mature colonies, which were
107 collected at Hyogo Prefecture, Japan, in May 2015 and 2016 and kept at approximately 25 °C
108 in constant darkness until the following experiments were performed. Young instar nymphs
109 (head width = 1.31-1.57 mm, Class 1 (3rd or 4th instars); and head width = 1.91-2.12 mm,
110 Class 2 (probably 5th instars); Nalepa 1984, 1990) of C. punctulatus were collected at
111 Mountain Lake Biological Station, Giles County, Virginia, USA, in April 2015-2017. These
112 individuals were kept at 15 °C in constant darkness until use.
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114 JHA treatment
115 In Z. nevadensis, according to the methods of Saiki et al. 2014, filter paper was treated with 0
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116 (for control) or 10 μg JHA (pyriproxyfen; Wako, Osaka, Japan) dissolved in 400 μL acetone
117 and placed in a 90 mm petri dish with 10 individual 7th instars. In C. punctulatus, filter paper
118 and 200 mg cellulose powder (Wako) was treated with 0 (for control) or 100 μg pyriproxyfen
119 dissolved in 200 μL acetone and placed in a 60 mm petri dish with 10 Class 1 or 2 nymphs.
120 All petri dishes were kept in an incubator at 25 °C (Z. nevadensis) or 15 °C (C. punctulatus)
121 in constant darkness for 30 days. Dishes were checked for dead and newly molted individuals
122 every 24 hours. Molting rates in each species were compared between JHA and acetone
123 control treatments. Fisher's exact test was performed using Mac Statistical Analysis ver. 2.0
124 (Esumi, Tokyo, Japan).
125
126 RNA interference (RNAi) experiment
127 Each double-strand RNA (dsRNA) was generated by the partial cDNA sequences amplified
128 by the gene-specific primers (Table S1) using T7 RNA polymerase with a MEGA script T7
129 transcription kit (Ambion, Austin, TX, USA). As in previous studies (Masuoka et al. 2015,
130 2018; Masuoka and Maekawa 2016a, b), GFP was selected as a control gene, and dsRNA
131 was generated using GFP vector pQBI-polII (Wako, Osaka, Japan). Specific primers of the
132 following genes of Z. nevadensis, ZnMet (Gene ID: Znev_09571; Terrapon et al. 2014),
133 ZnSRC (Znev_05083), ZnKr-h1 (Znev_04171), ZnShr (Znev_16529), ZnSpo (Znev_04417),
134 ZnEcR (Znev_13925), ZnE74 (Znev_00833), ZnE75 (Znev_11406), ZnHR3 (Znev_14707),
135 and ZnHR39 (Znev_00332) were designed from genome sequence data using Primer3 plus
136 software (Untergasser et al. 2007). Specific primers of the following genes of C. punctulatus,
137 CpMet (EST ID: Cp_TR6397) and CpKr-h1 (Cp_TR7552), were designed from
138 transcriptome sequence data (Hayashi et al. 2017; DDBJ Sequence Read Archive database
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139 accession number: DRA004598) using Primer3 plus. Each dsRNA (500 ng in 136 nL (Z.
140 nevadensis), 4 μg in 272 nL (C. punctulatus)) was injected into the side of the thorax of
141 individuals using a Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL,
142 USA). Within 24 hours of the injection, all individuals were placed in a petri dish with a filter
143 paper (and also cellulose powder for C. punctulatus) treated with pyriproxyfen or acetone,
144 and the dish was kept in an incubator as in the previous section. Molting rate was compared
145 between treatments, and Fisher's exact test was performed for the statistical analysis using
146 statistical software R v. 3.1.2 (Ihaka and Gentleman 1996). To evaluate the effects of ZnMet
147 dsRNA injection timing, dsRNA was injected every 24 hours after JHA treatment (until 120
148 hours, day 0-5).
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150 Gene expression analysis
151 Three individuals were collected three days after the dsRNA injection. Total RNA was
152 extracted from the whole body of each individual using ISOGEN (NipponGene, Tokyo,
153 Japan). The extracted RNA was purified with DNase treatment and used for cDNA synthesis
154 using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Specific
155 primers of 20E related genes of Z. nevadensis and C. punctulatus (Nvd: Znev_04416 and
156 Cp_TR25860; Shr: Znev_16529 and Cp_TR25505; Spo: Znev_04417 and Cp_TR54771;
157 Phm: Znev_00957; Dib: Znev_08701 and Cp_TR16740; Sad: Znev_14659; Shd:
158 Znev_02808; EcR: Cp_TR4152; USP: Znev_11534; Br-C: Znev_09723; E63: Znev_06687
159 and Cp_TR16589; E74: Znev_00833 and Cp_TR3685; E75: Cp_TR8108; E93: Znev_02008;
160 HR3: Znev_14707 and Cp_TR38613; HR4: Znev_17691; HR38: Znev_16131; HR39:
161 Znev_00332 and Cp_TR1259; HR78: Znev_03071; HR96: Znev_06284 and Cp_TR49824;
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162 FTZ-F1: Znev_18259) were newly designed as shown in the previous section (Table S1). JH
163 signaling genes of C. punctulatus (CpMet: Cp_TR6397 and CpKr-h1: Cp_TR7552) were also
164 newly designed as shown in the previous section. Primers of JH signaling genes (ZnMet,
165 ZnSRC and ZnKr-h1) and 20E signaling genes of Z. nevadensis (ZnEcR, ZnBr-C, ZnHR4 and
166 ZnE75) were previously described (Masuoka et al. 2015, Masuoka and Maekawa 2016a). The
167 expression level of each gene was quantified using a THUNDERBIRD SYBR qPCR Mix
168 (TOYOBO, Osaka, Japan) and MiniOpticon Real-Time System detection system (Bio-Rad,
169 Hercules, CA, USA). An endogenous control gene was selected from the following three
170 genes, EF1-alpha (Zn: Accession No. AB915828, Cp: Accession No. AFK49795), beta-actin
171 (Zn: No. AB915826, Cp: Cp_TR19468) and NADH-dh (Zn: No. AB936819, Cp:
172 Cp_TR49774), using GeNorm (Vandesomple et al. 2002) and NormFinder (Andersen et al.
173 2004). EF1-alpha was selected in all real-time qPCR analyses performed in this study (Table
174 S2). Real-time qPCR analysis was performed in biological triplicates. Statistical analysis was
175 performed using Mann−Whitney's U test for comparison between a target gene and GFP
176 RNAi treatment using statistical software Mac Statistical Analysis ver. 2.0 (Esumi, Tokyo,
177 Japan). For Z. nevadensis, prior to the use of ANOVA, we performed the Browne-Forsythe
178 test on the variance equality using statistical software R v. 3.1.2 (Ihaka and Gentleman 1996).
179
180 Data availability
181 The authors have uploaded supplementary materials to figshare. The authors affirm that all
182 data necessary for confirming the conclusion of the article are present within the article and
183 supplementary material.
184
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185 Results
186 JHA treatment in C. punctulatus and Z. nevadensis
187 In the Class 1 nymphs (3rd or 4th instars) of C. punctulatus, the rates of nymphal molts
188 within 30 days were significantly higher in the JHA treated individuals than in the acetone
189 controls (76.7% and 10.0%, respectively, p < 0.01; Fig. 1). Additionally, JHA treatments in
190 the Class 2 nymphs (5th instars) resulted in the similar tendencies (JHA: 66.7%, acetone:
191 20%, p < 0.01; Fig. 1). In Z. nevadensis, most JHA treated individuals (85.0%) molted into
192 presoldiers within 30 days, whereas no molted presoldiers were observed in the control
193 treatment (Fig. 1). These results are consistent with previous reports (Miura et al. 2003; Itano
194 and Maekawa 2008; Saiki et al. 2014).
195
196 RNAi of JH signaling genes under the JHA treatment in C. punctulatus and Z.
197 nevadensis
198 RNA interference (RNAi) of JH signaling genes was performed in the JHA-treated
199 individuals of Z. nevadensis and C. punctulatus. First, in Z. nevadensis, significant RNAi
200 knockdown effects were observed in ZnMet, ZnSRC and ZnKr-h1, compared to the GFP
201 control (25.80, 52.62 and 39.00%, respectively; Fig. S1). Knockdown of ZnMet strongly
202 inhibited the presoldier molts, and only one tenth of individuals molted into presoldier-like
203 individuals with smaller head capsules and shorter mandibles, compared to the control (Fig.
204 2). Knockdown of ZnSRC showed similar results, and only one in ten termites molted into a
205 presoldier-like individual (Fig. 2). However, ZnKr-h1 RNAi did not have a significant effect
206 on the molts, and seven of ten individuals molted into presoldiers with normal morphological
207 characters (Fig. 2). JHA-induced molting rates under ZnMet RNAi were significantly higher
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208 when dsRNA was injected 3-5 days after the JHA treatment (day 3-5), compared to those just
209 before the treatment (day 0) (Fig. S2). The former molted individuals possessed the enlarged
210 mandibles of normal presoldiers (Fig. S2).
211
212 In C. punctulatus, significant RNAi knockdown effects were observed in CpMet and
213 CpKr-h1 compared to the GFP control (41.99 and 51.31%, respectively; Fig. S1). CpMet
214 RNAi strongly inhibited the nymphal molts, and only one in ten individuals molted into the
215 next instar. CpKr-h1 RNAi, however, did not have a significant effect on the nymphal molts,
216 and 60% of individuals molted into a subsequent instar (Fig. S3).
217
218 Expression of 20E synthesis and signaling genes under the Met RNAi
219 Changes in expression levels of 20E related genes in the JHA-treated individuals were
220 observed under the Met RNAi both in Z. nevadensis and C. punctulatus. In Z. nevadensis,
221 ZnMet knockdown significantly inhibited the expression levels of two 20E synthesis genes
222 (ZnShr and ZnSpo) and seven signaling genes including 20E receptor gene (ZnEcR, ZnE63,
223 ZnE74, ZnE75, ZnHR3, ZnHR39 and ZnHR96) (Fig. 3). On the other hand, in C. punctulatus,
224 expression levels of different 20E synthesis genes (CpNvd and CpDib) were decreased by
225 CpMet RNAi treatment (Fig. 4). Although expression of some 20E signaling genes (CpE63,
226 CpHR3 and CpHR96) were negatively affected by the CpMet RNAi as shown in Z.
227 nevadensis, expression levels of CpEcR, CpE74, CpE75 and CpHR39 were not significantly
228 decreased by the RNAi treatment (Fig. 4).
229
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230 RNAi of 20E synthesis and signaling genes during JHA-induced presoldier
231 differentiation
232 RNAi of 20E synthesis (ZnShr and ZnSpo) and signaling genes (ZnEcR, ZnHR3, ZnE74,
233 ZnE75 and ZnHR39) was performed during artificial presoldier differentiation (Fig. 5).
234 Expression levels of each of these genes except HR3 were negatively affected by Met RNAi
235 in Z. nevadensis, but not in C. punctulatus. Consequently, these expression changes might
236 have crucial roles in presoldier-specific molting events. Expression levels of HR3 were
237 significantly decreased by Met RNAi in both species, and thus HR3 might have a similar role
238 in their molting processes. The expression levels of ZnSpo, ZnE75, ZnHR3 and ZnHR39 were
239 also significantly repressed by ZnSRC RNAi treatment, however ZnKr-h1 RNAi did not
240 affect expression levels of any gene examined (Fig. S4). RNAi treatment of ZnShr and ZnE74
241 did not affect JHA-induced presoldier differentiation, similar to those of GFP controls.
242 ZnSpo and ZnE75 RNAi significantly inhibited the molting process, but were nevertheless
243 treated with JHA. Although knockdown of ZnEcR and ZnHR3 did not affect the rate of gut
244 purged individuals (those that eliminate their gut contents before molt), all injected
245 individuals failed to shed old cuticles (0% molting rate). Interestingly, ZnHR39 RNAi did not
246 inhibit the molting process, but the molted individuals possessed worker-like phenotypes
247 with shorter mandibles and smaller head capsules.
248
249 Discussion
250 Termites and Cryptocercus possess a similar JH dependent molting system
251 Molting events were caused by the JHA treatments not only in Z. nevadensis (presoldier
252 differentiation) but also in C. punctulatus (nymphal molts), suggesting that in these taxa, JH
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253 has a role in activating the molting process. Generally, JH has an inhibitory role in molting
254 via the repression of PTTH secretion and subsequent 20E synthesis (Gilbert 2012). In the
255 german cockroach B. germanica, JHA treatment delays nymphal molt via inhibition of 20E
256 synthesis (Hangartner and Masner 1973; Masner et al. 1975). In some lepidopteran species,
257 however, JH can activate the prothoracic gland during pupation (Hiruma et al. 1978;
258 Cymborowski and Stolaz 1979). Moreover, in the damp-wood termite Hodotermopsis
259 sjostedti, JHA induced growth in the prothoracic gland of pseudergates (Cornette et al. 2008).
260 Recent phylogenetic analyses strongly supported a monophyly of termites within the
261 cockroach clade and sister-group relationships between termites and Cryptocercus
262 cockroaches (Bourguignon et al. 2017). Although further JH treatment assay on some
263 cockroach species are needed, there is a possibility that a role for JH in the activation of the
264 molting process is possessed by both termites and Cryptocercus cockroaches.
265
266 Role of JH signaling genes in termites and Cryptocercus
267 In both Z. nevadensis and C. punctulatus, knockdown of JH receptor, Met, inhibited the
268 molting event instigated by JHA treatment of non-adult individuals. In addition,
269 presoldier-specific morphogenesis (e.g. elongation of mandibles) was also inhibited by Met
270 RNAi in Z. nevadensis. These phenotypic effects were similar to those when RNAi of insulin
271 receptor gene was performed in H. sjostedti (Hattori et al. 2013). Surprisingly, however,
272 knockdown of the Met target gene, Kr-h1, had no influence on the JHA-induced molting rates
273 in both termites and woodroaches, nor morphogenetic changes in termites. These results
274 suggest that the JHA-inducible process of molting (and also specific morphogenesis in
275 termites) is activated via a JH receptor non-Kr-h1 signaling pathway. During metamorphosis
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276 in holometabolous insects, JH acts to maintain “developmental status quo” in the larval stage
277 via Kr-h1 pathway (Minakuchi et al. 2009). Kr-h1 works as an important early transcription
278 factor within the JH signaling pathway, and is known to be involved in other JH-triggered
279 phenomena such as ovarian development in T. castaneum and Locusta migratoria (Minakuchi
280 et al. 2009; Konopova et al. 2011; Kayukawa et al. 2012). However, in the linden bug P.
281 apterus, Kr-h1 had little influence on ovarian development (Song et al. 2014). Further
282 investigations are needed to determine whether there is a non-Kr-h1 signaling pathway for
283 the JH-inducible process of molting in termites and woodroaches, and in the specific
284 morphogenesis found in termites.
285
286 Met regulates expression of 20E synthesis and signaling genes in both species
287 Met knockdown repressed expression levels of some 20E related genes under JHA
288 application both in Z. nevadensis and C. punctulatus. The expressions of the different 20E
289 synthesis genes were inhibited by Met knockdown in Z. nevadensis (ZnShr and ZnSpo) and C.
290 punctulatus (CpNvd and CpDib). There is a possibility that Met is involved in 20E synthesis
291 activity via expression changes of different synthesis genes in the prothoracic glands of
292 termites and Cryptocercus (Fig. 6). A notable difference was also observed between the two
293 species, when the expression levels of 20E related genes were examined after Met RNAi.
294 Expression levels of ZnEcR, ZnE74, ZnE75, and ZnHR39 were significantly repressed after
295 ZnMet RNAi in Z. nevadensis but no significant decreased levels were observed after CpMet
296 RNAi in C. punctulatus. One possibility is that such differences in 20E-related gene
297 expression changes via JH action may be related to soldier-specific morphogenesis in
298 termites. RNAi-mediated function analysis was performed in this study to clarify this
14
299 possibility.
300
301 The function of 20E related genes in termites
302 Expression levels of both ZnHR3 and CpHR3 were significantly decreased by Met RNAi in
303 the JHA-treated individuals. RNAi of ZnHR3 resulted in the failure of ecdysis, and all
304 molting individuals died before the completion of ecdysis as shown in other insects
305 (Tribolium castaneum, Tan and Palli 2008a; Locusta migratoria, Zhao et al. 2018), including
306 cockroaches (B. germanica, Cruz et al. 2007). These results suggest that an ecdysis related
307 function of HR3 is conserved among insects and its expression occurs under JH signaling
308 both in Z. nevadensis and C. punctulatus. To clarify the specific role of JH receptor signaling
309 for 20E-related gene expression changes in termites, functional analyses of genes with
310 different expression patterns after ZnMet and CpMet RNAi were performed. ZnShr and
311 ZnE74 knockdown treatments did not have any significant effects on presoldier
312 differentiation, and resulted in phenotypes similar to those found in the GFP control. These
313 genes may not have an important role for the molting event accompanied with morphological
314 changes. ZnSpo and ZnE75 RNAi resulted in the inhibition of molting, although the
315 individuals were treated with enough JHA to induce the presoldier molt. In Bombyx mori,
316 E75 was involved in the activation of expression of 20E synthesis genes including Spo (Li et
317 al. 2016). In the early process of termite presoldier molting, Spo may have a critical role in
318 20E synthesis under JH signaling via E75 expression (Fig. 6). ZnEcR RNAi resulted in a
319 failure of the shedding of old cuticle, although a newly formed cuticle was generated under
320 the old cuticle, as shown in the presoldier-soldier molt in Z. nevadensis (Masuoka and
321 Maekawa 2016a), the imaginal molt in B. germanica (Cruz et al. 2006) and the larval molt in
15
322 T. castaneum (Tan and Palli 2008b). On the other hand, ZnHR39 RNAi produced a unique
323 effect and the newly molted worker-like individuals had no presoldier-specific
324 morphogenesis. In holometabolous species, orphan nuclear receptor gene, HR39 (FTZ-F1β),
325 had multiple functions in metamorphosis including neuronal remodeling and muscle
326 generation (Tan and Palli 2008a; Boulanger et al. 2011; Zirin et al. 2013). Present results
327 strongly suggest that termite HR39 is necessary for the drastic morphological changes that
328 occur during soldier differentiation (Fig. 6). Note that these changes in termites can be
329 produced under the high levels of JH that result from artificial JHA treatment, whereas a
330 metamorphosis in holometabolous insects is initiated by a reduction of larval JH titer. An
331 important future topic will be to determine the differences in the JH-HR39 regulatory
332 mechanism between termites (soldier differentiation) and holometabola (metamorphosis).
333
334 Conclusion
335 In this study, a comparative analysis of the role of the JH signaling pathway during molting
336 was done in termites (Z. nevadensis) and sister group woodroaches (C. punctulatus). The
337 results showed that JH-inducible molting via receptor (Met) occurred in both termites
338 (presoldier differentiation) and woodroaches (nymphal molt). Further, termite 20E signaling
339 gene HR39 is expressed under JH signaling via Met, and has a crucial function in presoldier
340 morphogenesis. The present study provides important insights into the proximate
341 mechanisms of soldier evolution in termites. Namely, two crucial changes might be necessary
342 for the evolution of termite soldiers: 1) the acquisition of a molting activation mechanism
343 induced by high levels of JH (a feature shared by termites and woodroaches), and 2) a novel
344 mediation between JH receptor and 20E signalings for specific morphogenesis (only in
16
345 termites). Although some caution should be exercised when using the German cockroach B.
346 germanica as a baseline for comparisons with termites, recent in-depth transcriptome analysis
347 showed consistent expression patterns of 20E related genes among B. germanica and termites
348 (Harrison et al. 2018). Furthermore, we recently clarified that TGFβ signaling is involved in
349 the mediation between JH and 20E pathways during soldier differentiation (Masuoka et al.
350 2018). These insights also support that a novel 20E signaling role might trigger a soldier
351 evolution within the cockroach clade.
352
353 Acknowledgement
354 We are grateful to the director and staff of Mountain Lake Biological Station for permission
355 to collect Cryptocercus punctulatus on the grounds. Thanks are also due to Takumi
356 Kayukawa and Tetsuro Shinoda for productive discussions. This study was supported in part
357 by Grants-in-Aid for JSPS Fellows (Nos. JP15J10817 and JP17J06352 to YM) and Scientific
358 Research (Nos. JP25128705 and JP16K07511 to KM) from the Japan Society for the
359 Promotion of Science.
360
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559 Figure legends
560 Fig. 1. Results of JH analog (JHA; pyriproxyfen) and acetone (control) treatment in C.
561 punctulatus and Z. nevadensis. Molting rates indicate the ratio of nymphal (C. punctulatus)
562 and presoldier (Z. nevadensis) molt. Asterisks indicate a significant difference (Fisher’s exact
563 test, **P < 0.01).
564
565 Fig. 2. Phenotype of newly molted individual, and molting rate after the dsRNA
566 injection of JH signaling genes under JHA treatment in Z. nevadensis. The fraction on
567 each column indicates number of molted individuals (numerator) and number of treated
568 individuals (denominator). Asterisks indicate significant differences when compared to the
569 control (GFP) (Fisher’s exact test, *P < 0.05, **P < 0.01, n.s. not significant). GP refers to
570 gut purged. External morphologies of the molted individuals are shown in the upper panels.
571 These individuals were photographed 7 days after the molt.
572
573 Fig. 3. Expression levels (mean ± S.D., biological triplicates) of 20E synthesis and
26
574 signaling genes in 0-5 days after JHA treatment under Met RNAi in Z. nevadensis.
575 Expression levels were normalized by EF1a expression. Relative expression levels were
576 calibrated using the mean expression level of individuals just before the JHA treatment (d0)
577 as 1.0. The statistical results of two-way ANOVA are described in each box (*P < 0.05, **P <
578 0.01). The data is consistent with the use of parametric statistics by the Browne-Forsythe test
579 (ZnMet: P = 7.91E-01 (GFP), 5.90E-01 (ZnMet RNAi); ZnNvd: P = 7.88E-01 (GFP),
580 7.91E-01(ZnMet RNAi); ZnShr: P = 5.37E-01 (GFP), 5.89E-01 (ZnMet RNAi); ZnSpo: P =
581 7.77E-01 (GFP), 4.93E-01 (ZnMet RNAi); ZnPhm: P = 4.43E-01 (GFP), 2.89E-01 (ZnMet
582 RNAi); ZnDib: P = 5.24E-01 (GFP), 6.81E-01 (ZnMet RNAi); ZnSad: P = 7.50E-01 (GFP),
583 7.52E-01 (ZnMet RNAi); ZnShd: P = 8.53E-01 (GFP), 9.60E-01 (ZnMet RNAi); ZnEcR: P =
584 9.47E-01 (GFP), 8.75E-01 (ZnMet RNAi); ZnUSP: P = 9.08E-01 (GFP), 4.35E-01 (ZnMet
585 RNAi); ZnBr-C: P = 5.31E-01 (GFP), 2.30E-01 (ZnMet RNAi); ZnE63: P = 8.46E-01 (GFP),
586 6.73E-01 (ZnMet RNAi); ZnE74: P = 8.57E-01 (GFP), 9.93E-01 (ZnMet RNAi); ZnE75: P =
587 9.17E-01 (GFP), 3.02E-01 (ZnMet RNAi); ZnE93: P = 9.99E-01 (GFP), 5.34E-01 (ZnMet
588 RNAi); ZnHR3: P = 2.61E-01 (GFP), 9.48E-01 (ZnMet RNAi); ZnHR4: P = 6.39E-01 (GFP),
589 6.81E-01 (ZnMet RNAi); ZnHR38: P = 6.44E-01 (GFP), 3.78E-01 (ZnMet RNAi); ZnHR39:
590 P = 3.45E-01 (GFP), 4.08E-01 (ZnMet RNAi); ZnHR78: P = 7.95E-01 (GFP), 9.23E-01
591 (ZnMet RNAi); ZnHR96: P = 9.34E-01 (GFP), 6.09E-01 (ZnMet RNAi); ZnFTZ-F1: P =
592 9.88E-01 (GFP), 6.62E-01 (ZnMet RNAi)) prior to the use of the ANOVA. Gene names with
593 significant different expression levels between injected dsRNAs are shown in bold.
594
595 Fig. 4. Expression levels (mean ± S.D., biological triplicates) of 20E synthesis and
596 signaling genes under Met RNAi in C. punctulatus. Expression levels were normalized by
27
597 EF1a expression. Relative expression levels were calibrated using the mean expression level
598 of GFP dsRNA-injected individuals as 1.0. Asterisks denote significant differences
599 (Mann−Whitney's U test, *P < 0.05, **P < 0.01, n.s. not significant).
600
601 Fig. 5. Phenotype of newly molted individual, and molting and gut-purging rate after
602 the dsRNA injection of 20E synthesis and signaling genes under JHA treatment in Z.
603 nevadensis. The fraction on each column indicates number of molted or gut purged
604 individuals (numerator) and number of treated individuals (denominator). Asterisks indicate
605 significant differences when compared to the control (GFP) (Fisher’s exact test, *P < 0.05,
606 **P < 0.01, n.s. not significant). External morphologies of the molted individuals are shown
607 in the upper panels. These individuals were photographed 7 days after the molt. No molting
608 individuals were obtained by ZnEcR and ZnHR3 RNAi, but all gut purged individuals died
609 just before the molt, because of a failure of the shedding of old cuticle, as shown in the upper
610 panel.
611
612 Fig. 6. Hypothetical pathway of JH signaling in woodroaches and termites. A common
613 pathway involved in 20E synthesis may control a downstream molting process via the JH
614 receptor. In termites, soldier-specific morphogenesis may be regulated by a specific JH
615 receptor pathway, probably involved in the 20E signaling genes including HR39.
616
617 SUPPLEMENTAL FIGURES/TABLES
618
619 Figure S1. Expression levels (mean ± S.D., biological triplicates) of target genes after
28
620 RNAi treatments in Z. nevadensis (A) and C. punctulatus (B). Expression levels were
621 normalized by EF1a expression. Relative expression levels were calibrated using the mean
622 expression level of GFP dsRNA-injected individuals as 1.0. Asterisks denote significant
623 differences (Mann−Whitney's U test, *P < 0.05).
624
625 Figure S2. Phenotype of newly molted individual and molting rate after the ZnMet
626 dsRNA injection 1-5 days after JHA treatment in Z. nevadensis. The fraction on each
627 column indicates number of molted individuals (numerator) and number of treated
628 individuals (denominator). Different letters above the bars denote significant differences
629 (Tukey’s test, P < 0.05). External morphologies of the molted individuals are shown in the
630 upper panels. These individuals were photographed 7 days after the molt.
631
632 Figure S3. Molting rate of Class 1 (3rd or 4th instar) nymphs after the dsRNA injection
633 under JHA treatment in C. punctulatus. The fraction on each column indicates number of
634 molted individuals (numerator) and number of treated individuals (denominator). Asterisks
635 indicate significant differences when compared to the control (GFP) (Fisher’s exact test, **P
636 < 0.01, n.s. not significant).
637
638 Figure S4. Expression levels (mean ± S.D., biological triplicates) of 20E synthesis and
639 signaling genes in 0-5 days after JHA treatment under SRC and Kr-h1 RNAi in Z.
640 nevadensis. Expression levels were normalized by EF1a expression. Relative expression
641 levels were calibrated using the mean expression level of individuals just before the JHA
642 treatment (d0) as 1.0. The statistical results of two-way ANOVA are described in each box
29
643 (*P < 0.05, **P < 0.01). The data is consistent with the use of parametric statistics by the
644 Browne-Forsythe test (ZnShr: P = 5.37E-01 (GFP), 7.15E-01 (ZnSRC RNAi), 4.76E-01
645 (ZnKr-h1 RNAi); ZnSpo: P = 7.77E-01 (GFP), 5.90E-01 (ZnSRC RNAi), 7.34E-01 (ZnKr-h1
646 RNAi); ZnEcR: P = 9.47E-01 (GFP), 9.80E-01 (ZnSRC RNAi), 6.65E-01 (ZnKr-h1 RNAi);
647 ZnE74: P = 8.57E-01 (GFP), 7.45E-01 (ZnSRC RNAi), 3.72E-01 (ZnKr-h1 RNAi); ZnE75: P
648 = 9.17E-01 (GFP), 7.78E-01 (ZnSRC RNAi), 6.68E-01 (ZnKr-h1 RNAi); ZnHR3: P =
649 2.61E-01 (GFP), 7.26E-01 (ZnSRC RNAi), 5.81E-01 (ZnKr-h1 RNAi); ZnHR39: P =
650 3.45E-01 (GFP), 4.77E-01 (ZnSRC RNAi), 6.93E-01 (ZnKr-h1 RNAi)) prior to the use of the
651 ANOVA.
652
653 Table S1
654 Sequences of primers used in this study.
655
656 Table S2
657 Ranking and stability values of reference genes using GeNorm and NormFinder.
658 659 Z. nevadensis 100 **
50 molting rate (%) 0/20 17/20 0 control JHA
C. punctulatus control (acetone) 100 JHA (pyriproxyfen) ** **
50 molting rate (%)
3/30 23/30 2/10 20/30 0 Class 1 Class 2 (3rd of 4th instar) (5 instar)
Fig.1 GFP ZnMet RNAi ZnSRCRNAi ZnKr-h1RNAi
80 * n.s. 70 * 60 （％） 50 40 30 20 10
molting rate 0 12/20 2/20 1/10 7/10
GFP ZnMet ZnSRC ZnKr-h1
Fig. 2 ZnMet gene:7.9E-04** day:2.6E-04 ** inter.:2.5E-01
4
2 GFP RNAi ZnMet 0 d0 d1 d2 d3 d4 d5
20E synthesis genes 20E signaling genes
ZnNvd gene:7.4E-01 day:1.3E-05** inter.:2.9E-01 gene:2.0E-04** day:7.2E-03** inter.:2.8E-01 gene:2.4E-02* day:1.8E-01 inter.:4.8E-01 12 6 ZnEcR ZnHR3 16
3 6 8
0 0 0 ZnShr gene:1.5E-02* day:3.2E-01 inter.:5.1E-01 5 ZnUSP gene:2.1E-01 day:1.2E-03** inter.:9.1E-01 ZnHR4 gene:1.2E-01 day:7.8E-03** inter.:2.4E-01 6 12
2.5 3 6
0 0 0 2 ZnSpo gene:3.4E-02* day:3.5E-01 inter.:6.5E-01 ZnBr-C gene:1.8E-01 day:1.4E-01 inter.:9.6E-01 ZnHR38 gene:7.1E-01 day:1.9E-03** inter.:7.0E-01 40 2
1 1 20
0 0 0 gene:1.9E-01 day:3.2E-01 inter.:3.7E-01 gene:8.1E-03** day:7.6E-03** inter.:5.9E-01 ZnPhm ZnE63 gene:8.8E-03** day:1.5E-02* inter.:6.6E-01 ZnHR39 3 2 3
1 1.5 1.5
0 0 0 1.6 ZnDib gene:2.8E-01 day:3.8E-01 inter.:3.4E-01 ZnE74 gene:1.0E-02* day:5.2E-01 inter.:2.7E-01 4 ZnHR78 gene:5.0E-01 day:1.5E-05** inter.:4.1E-01 3
0.8 2 1.5
Relative expression (/ EF1a ) 0 0 0 1.6 ZnSad gene:9.4E-01 day:4.5E-01 inter.:6.3E-01 12 ZnE75 gene:2.2E-04** day:1.0E-05** inter.:4.1E-01 1.6 ZnHR96 gene:2.9E-02* day:6.7E-02 inter.:1.3E-01
0.8 6 0.8
0 0 0 8 ZnShd gene:3.3E-01 day:4.3E-01 inter.:9.8E-01 5 ZnE93 gene:7.7E-01 day:1.8E-03* inter.:4.0E-01 ZnFTZ-F1 gene:6.7E-01 day:9.5E-02* inter.:4.0E-01 4
2.5 4 2
0 0 0 Fig.3 d0 d1 d2 d3 d4 d5 d0 d1 d2 d3 d4 d5 d0 d1 d2 d3 d4 d5 * GFP RNAi CpMet
n.s. n.s. * ** n.s. ** ** * n.s. ** n.s. 1 Relative expression (/ EF1a )
0
CpDib CpMet CpNvd CpShr CpSpo CpEcR CpE63 CpE74 CpE75 CpHR3 CpHR39 CpHR96 synthesis genes signaling genes
Fig.4 GFP ZnShr RNAi ZnEcR RNAi ZnE75 RNAi ZnE74 RNAi ZnHR39 RNAi ZnHR3RNAi ZnSpoRNAi
did not molt
70 n.s. 60
（％） 50 n.s. n.s. 40 30 20 10 ** ** * ** 12/20 4/10 0/10 0/10 6/10 1/10 0/10 8/20 molting rate 0
70 n.s. n.s. n.s. 60 （％） 50 n.s. n.s. 40 30 20 10 ** * 12/20 4/10 0/10 6/10 6/10 1/10 6/10 8/20 0 gut-purging rate
GFP ZnShr ZnSpo ZnEcR ZnE74 ZnE75 ZnHR3 ZnHR39 synthesis genes signaling genes
Fig.5 Woodroaches Termites (C. punctulatus) (Z. nevadensis) High JH High JH
Receptor Receptor Met/(SRC?) Met/SRC
termite Ecdysone Ecdysone specific pathway synthesisKr-h1+ signal synthesis + signal Ecdysone signal (Nvd, Dib?) (E63, HR3, HR96?) (Spo) (EcR, E75, HR3) (HR39)
morphological molt molt modification
Nymphal Soldier molt differentiation
Fig.6