Expression of calcineurin in relation to the embryonic diapause process in the silkworm, Bombyx mori

Accepted Manuscript Expression of calcineurin in relation to the embryonic diapause process in the silkworm, Bombyx mori

Hsiao-Yen Hsieh, Shi-Hong Gu PII: DOI: Reference:

S1095-6433(18)30256-3 doi:10.1016/j.cbpa.2018.10.013 CBA 10391

To appear in:

Comparative Biochemistry and Physiology, Part A

Received date: Accepted date:

12 October 2018 14 October 2018

Please cite this article as: Hsiao-Yen Hsieh, Shi-Hong Gu , Expression of calcineurin in relation to the embryonic diapause process in the silkworm, Bombyx mori. Cba (2018), doi:10.1016/j.cbpa.2018.10.013

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ACCEPTED MANUSCRIPT Expression of calcineurin in relation to the embryonic diapause process in the silkworm, Bombyx mori

Hsiao-Yen Hsieh, Shi-Hong Gu* Department of Biology, National Museum of Natural Science, 1 Kuan-Chien Road, Taichung, Taiwan 404, ROC

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To whom correspondence should be addressed. *

Dr. Shi-Hong Gu

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Tel : 886-42-3226940 ext. 542

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Fax: 886-42-3232146

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E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Calcineurin (CN) is a Ca2+/calmodulin-activated serine/threonine protein phosphatase that is essential for translating Ca2+ signals into changes in cell function and development.

In the present study, we investigated changes in CN expression

during the process of embryonic diapause in the silkworm, Bombyx mori. An immunoblot analysis showed that Bombyx eggs contained a 59-kDa catalytic A subunit (CNA), a 19-kDa regulatory B subunit (CNB), and a 27-kDa calcipressin; the

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CNA, CNB, and calcipressin were found to undergo differential changes between diapause and developing eggs during the diapause process. In developing eggs,

However, CNB protein levels

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gradually decreased with embryonic development.

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protein levels of CNA and calcipressin were high during the first stages and then

showed inverse temporal changes, with increased levels being detected during later

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embryonic stages of developing eggs. In diapause eggs, protein levels of CNA and calcipressin remained at relatively high levels during the first 8 days after oviposition, but CNB levels remained at low levels.

CN enzymatic activity was directly

the first 8 days after oviposition.

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determined and results showed that it remained at low levels in diapause eggs during However, in developing eggs, CN enzymatic

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activity sharply increased during the first several days, reached a peak during middle embryonic development, and then greatly decreased 5 or 6 days before hatching. Examination of temporal changes in mRNA expression levels of CNB also showed

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differences between diapause and HCl-treated eggs. These results demonstrated that protein levels of CNA, CNB, and calcipressin, transcriptional levels of CNB, and CN enzymatic activity between diapause and developing eggs are differentially

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regulated, and these regulated changes are likely related to the embryonic diapause process of B. mori.

Keywords: Bombyx mori; calcineurin; phosphatases; diapause; calcipressin; signaling.

ACCEPTED MANUSCRIPT 1. Introduction Insects have evolved various strategies to withstand unfavorable environmental conditions in their habitats.

One of the main strategies is diapause, a programmed

developmental arrest, which allows active adaption to extreme environmental conditions, such as freezing temperatures in winter and extreme heat and desiccation in summer (De Loof, 2011; Hand et al., 2016).

The silkworm, Bombyx mori,

provides an excellent model system for investigating regulatory mechanisms

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underlying the diapause process (Yamashita and Yaginuma, 1991; Zhao et al., 2014). Diapause occurs in the late gastrula stage of embryogenesis, when the embryonic

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cell cycle becomes arrested in the G2 phase of the cell division cycle (Nakagaki et al.,

a diapause state is maintained.

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1991; Xu et al., 1995; Shiomi et al., 2015). As long as eggs are incubated at 25 °C, When diapause is terminated by chilling to 5 °C for

and Yaginuma, 1991).

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2~3 months, embryonic cell division is resumed after transfer to 25 °C (Yamashita Diapause can be prevented if diapause-destined eggs are When developing eggs (non-diapause eggs, HC-treated

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artificially treated with HCl.

eggs, and chilled eggs) are incubated at 25 °C, eggs hatch on about day 9 or 10. Diapause is usually characterized by a reduced metabolic rate (Hand et al., 2016).

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Several key carbohydrate metabolism-related enzymes during the Bombyx embryonic diapause process have been studied (Yamashita and Yaginuma, 1991).

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Moreover, several protein kinases, including extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3β, a multifunctional protein kinase that plays important roles

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in regulating both glycogen synthesis and protein synthesis, appear to be involved in Bombyx embryonic development (Iwata et al., 2005; Fujiwara and Shiomi, 2006; Fujiwara et al., 2006a, b; Lin et al., 2009).

In addition, we previously demonstrated

that phosphorylation of the translational repressor, 4E-binding protein (4E-BP), which is generally accepted as a marker of target of rapamycin (TOR) activity (Hay and Sonenberg, 2004; Bhaskar and Hay, 2007), is an early signaling event involved in egg development, in which diapause initiation is prevented by HCl treatment (Gu et al., 2011). More recently, we further found that injury-induced phosphorylation and activation of c-Jun N-terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways, are also related to diapause termination in dechorionated eggs (Gu and Chen, 2017). In contrast to the multitude of protein kinases and clear signaling transduction

ACCEPTED MANUSCRIPT pathways involved in insect diapause processes (Iwata et al., 2005; Fujiwara and Shiomi, 2006; Fujiwara et al., 2006a, b; Kidokoro et al., 2006; Fujiwara and Denlinger, 2007; Lin et al., 2009; Hand et al., 2016; Gu and Chen, 2017), relative few protein phosphatases are known, and their regulatory mechanisms are unclear.

In

mammalian cells, it is well known that protein phosphatases do not behave as indiscriminate signal terminators, but can function as both negative and positive regulators of specific signaling pathways.

Protein kinases and phosphatases

responses.

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appear to be partners, and their activities are coordinated in regulating signaling A balance between kinase and phosphatase activities controls the

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phosphorylation status of a substrate protein, the alteration of which is capable of

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affecting almost every aspect of its functions, such as activity, stability, and protein-protein interactions (Tonks, 2006, 2013).

Thus, we hypothesized that similar

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to protein kinases, protein phosphatases may be differentially regulated between diapause and developing eggs and thus are involved in the egg diapause process. It was previously reported that high expression of protein phosphatase 2A (PP2A) Aα

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subunit in Helicoverpa armigera was associated with diapause induction during the photoperiod-sensitive stage (Tian and Xu, 2013). We recently found differential

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regulation of PP2A during the embryonic stage between diapause and developing eggs in B. mori. Our results demonstrated that higher PP2A gene expression and protein levels and dramatically increased PP2A enzymatic activity are likely related to

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the embryonic development of B. mori (Gu et al., 2017). However, it is unclear for other protein phosphatases.

Calcineurin (CN) is a highly conserved Ca2+/calmodulin-dependent protein

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phosphatase, that is known to be involved in a myriad of cellular processes and signal transduction pathways (Klee et al., 1998; Rusnak and Mertz, 2000). It is activated by an influx of intracellular calcium, and represents a key signaling node that conveys environmental stimuli into adaptive responses in multiple tissues and organs (Rusnak and Mertz, 2000). CN is a heterodimeric protein comprising a 59-kDa catalytic A subunit, calcineurin A (CNA), and a 19-kDa regulatory B subunit, calcineurin B (CNB) (Klee et al., 1998).

The large CNA subunit has phosphatase

activity and consists of several domains: a catalytic domain which regulates protein dephosphorylation, a CNB-binding domain, a Ca2+/calmodulin-binding domain, and an autoinhibitory domain (Aramburu et al., 2000; Shibasaki et al., 2002). inactive state, the autoinhibitory domain inhibits the catalytic domain.

In the

Binding of

ACCEPTED MANUSCRIPT Ca2+/calmodulin activates CN by alleviating this autoinhibition.

The biological roles

of CN have been extensively studied in diverse groups of organisms (Klee et al., 1998; Rusnak and Mertz, 2000).

In Drosophila melanogaster, CN appears to

promote induction of innate immune responses and plays a pivotal role in controlling systemic energy and body weight homeostasis (Dijkers and O'Farrell, 2007; Pfluger et al., 2015). A high expression pattern of CN in early Drosophila embryos suggests its potential role in embryonic development (Brown et al., 1994). In Bombyx, CN’s

(Fonagy et al., 1999; Yoshiga et al., 2002).

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involvement in sex pheromone biosynthesis signaling was previously reported It was demonstrated that CN signaling in

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Caenorhabditis elegans regulates lipolysis, development, behavior, and aging (Lee et

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al., 2013; Wang et al., 2017). Although numerous studies have shown specific roles for CN in regulating various physiological functions, no study has been conducted on

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differential expression of CN during the insect diapause process. The involvement of CN in Drosophila egg activation (Takeo et al., 2010) prompted us to investigate temporal changes in CN expression during Bombyx embryonic stages and its

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potential involvement in the egg diapause process. In the present study, we investigated the expression of CN and its regulator,

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calcipressin during the embryonic diapause process of B. mori. Our results showed that the CNA and CNB subunits undergo inverse differential changes in protein levels during the embryonic stage between diapause and developing eggs. Similar

detected.

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changes in calcipressin protein levels as those in CNA subunit levels were also The mRNA expression levels of CNB also showed differences between

diapause and HCl-treated eggs. Directly determining CN enzymatic activity further

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confirmed that its activity is regulated during the embryonic diapause process. These results indicated that regulation of CN signaling at protein and mRNA expression levels, and enzymatic activities are likely related to the embryonic process of B. mori.

2. Materials and methods 2.1. Experimental animals and egg treatment A hybrid strain (Guofu × Nongfong) of the silkworm, B. mori, was used as a diapause-egg producer.

Eggs laid during the first 3 h were pooled and then divided

into three groups for the experiments. °C to maintain a diapause state.

The first group of eggs was incubated at 25

The second group was exposed to 5 °C for 70

ACCEPTED MANUSCRIPT days after 15 days post-oviposition and then transferred to 25 °C to allow embryonic development.

To prevent entry into diapause, the third group was treated with HCl

(with a specific gravity of 1.075 at 15 °C) for 5 min at 46 °C at 20 h after oviposition. In addition, the polyvoltine strain (P1) was used as the non-diapause-egg producer as previously reported (Gu et al., 2017). When non-diapause eggs from the polyvoltine strain were incubated at 25 °C, eggs hatched on about day 9 or 10 after HCl-treated eggs and chilled eggs continued development at 25 °C and

9 days later larvae began to hatch.

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oviposition.

Diapause termination was confirmed by more

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than 95% hatchability of non-diapause eggs, HCl-treated eggs, and chilled eggs. Diapause eggs which were destined to initiate diapause were treated with a HCl

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solution at 20 h after oviposition in order to prevent diapause initiation. Subsequently, treated eggs were washed with running water, air-dried, and

was used instead of HCl.

As a control, water at 46 °C

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2.2. CN enzymatic activity assay

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incubated at 25 °C to allow embryonic development.

CN activity was measured with a fluorescent-based RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit (R-33700, Invitrogen, Carlsbad, CA, USA)

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following the manufacturer’s protocol.

According to the manufacturer’s description,

tyrosine phosphatase activity cannot be detected with this assay kit. CN activity can

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be distinguished from protein phosphatase 1, PP2A, and protein phosphatase 2C by adding different metal ions, such as calmodulin, NiCl2, MnCl2, and Ca2+ to the assay buffer, which enables different phosphatase activities to be determined (Cohen,

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1991). As suggested by the manufacturer’s protocol, NiCl2 and calmodulin were specifically used for optimization of the CN assay.

Each egg was homogenized in

100 μl of low-detergent buffer (1% Nonidet P-40, 10 mM HEPES, 150 mM NaCl, 10% glycerol, 1 mM PMSF, and a complete protease inhibitor cocktail).

In total, 50 μl of

egg lysates was incubated with 1 x CN phosphatase reaction buffer (pH 7.0) for 30 min at 37 °C.

The fluorescence intensity was measured using excitation at 355 nm

and emission at 485 nm.

According to the manufacturer’s recommendations, data

are presented as the percentage of CN enzymatic activity compared to eggs from the respective first day of each treatment. 2.3. Antibodies CN is a ubiquitous and highly conserved enzyme (Klee et al., 1998; Rusnak and Mertz, 2000). High similarities of insect CNA, CNB, and calcipressin with those of

ACCEPTED MANUSCRIPT mammals suggest that commercial antibodies against mammalian CNA, CNB, and calcipressin can successfully be used in insect systems (Fonagy et al., 1999; Takeo et al., 2006; Voss et al., 2010). Anti-mouse heat shock protein (HSP) 70 (ab5439), anti-rabbit CNA (ab90540), and anti-rabbit CNB (ab94535) antibodies were purchased from Abcam (Cambridge, MA, USA).

The anti-mouse calcipressin

antibody (sc-374454) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Horseradish peroxidase-linked goat anti-rabbit and anti-mouse second

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antibodies were purchased from PerkinElmer Life Sciences (Boston, MA, USA). 2.4. Western blot analysis

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Each egg from different stages was homogenized in 20 μl of lysis buffer (10 mM Lysates were

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Tris and 0.1% Triton X-100) at 4 °C (Lin et al., 2009; Gu et al., 2011).

boiled in an equal volume of sodium dodecylsulfate (SDS) sample buffer for 4 min

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followed by centrifugation at 15,800 g for 3 min to remove any particulate matter. Each 10 μl of supernatant (approximately 12 μg of total protein) was loaded onto SDS gels.

Following electrophoresis, proteins were transferred to polyvinylidene

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difluoride (PVDF) membranes using the Mini Trans-Blot Electrophoretic Transfer Cell System (Bio-Rad, CA, USA), and then washed with Tris-buffered saline (TBS) for 10 Blots were blocked at room temperature for 1 h in TBS

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min at room temperature.

containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat powdered dry milk, followed by washing three times for 5 min each with TBST. Blots were incubated overnight at

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4 °C with the primary antibody in TBST with 5% bovine serum albumin (BSA). Blots were then washed three times in TBST for 10 min each and further incubated with the HRP-linked second antibody in TBST with 1% BSA. Following three additional

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washes, the immunoreactivity was visualized by chemiluminescence using Western Lightning Chemiluminescence Reagent Plus from PerkinElmer Life Sciences. Films exposed to the chemiluminescent reaction were scanned and quantified using an AlphaImager Imaging System and AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA). 2.5. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA from eggs (29 mg, about 50 eggs) was extracted using 600 μl of the TRI Reagent (Molecular Research Center, OH, USA) according to the manufacturer’s protocol.

The concentration was determined using a NanoPhotometer Pearl

(Implen GMbH, Munich, Germany).

First-strand complementary DNA synthesis was

performed using an iScript cDNA synthesis kit (Bio-Rad, CA, USA).

ACCEPTED MANUSCRIPT For the qRT-PCR analysis, total RNA was extracted from eggs on different days. A qRT-PCR was carried out in a 20-l reaction volume containing 10 l of SYBR1 Green Realtime PCR Master Mix (Bio-Rad), 2 l of a first-strand cDNA template, and 2 M of each primer.

The iQ5 Real-Time PCR Detection System (Bio-Rad) was

used according to the manufacturer’s instructions.

RT-PCR primers were designed

according to parameters (no primer dimers and a product length of no more than 200

annealing temperature for all reactions was 59.5 °C.

The

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bp) outlined in the manual of SYBR1 Green Realtime PCR Master Mix.

Because a previous study

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showed that B. mori ribosomal protein 49 (rp49) (GenBank accession No. NM_001098282.1) is the most stable gene during embryonic development

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(Kobayashi et al., 2014), it was chosen as a reference gene in the current study. The qRT-PCR was performed using the following primers: CNA (GenBank accession

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No. NM_001043560.1) forward, 5'-AGCTGTGATATTAAGCAATGAT-3’ and reverse, 5'-GAATATAAAGACACGCCCAAG-3’; CNB (GenBank accession No.

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NM_001043561.1) forward, 5'- GCGTGTCTCAGTTTAGTGTGAAA-3’ and reverse, 5'-GTGTCCTTTAGATTGTTGCCTACC-3’; and rp49 forward, 5’-CAGGCGGTTCAAGGGTCAATAC-3’ and reverse,

2.6. Statistical analysis

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5’-TGCTGGGCTCTTTCCACGA-3’.

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Data are shown as the mean ± standard error of the mean (SEM). analysis was done using Student’s t-test for comparison of two groups.

Statistical A one- or

two-way analysis of variance (ANOVA) followed by Tukey’s test was also used where

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appropriate. A p value < 0.05 was considered significant.

In addition, for the gene

expression experiments, we set the significance value at > 2.0 for upregulated genes, and at < 0.5 for downregulated genes.

Genes that exhibited more-significant

perturbations were then identified by ANOVA. 3. Results 3.1.

Expressions of CNA, CNB, and calcipressin proteins in silkworm eggs

In the present study, as the first step to study the involvement of CN in the B. mori embryonic diapause process, a Western blot analysis was used to examine whether differences existed in protein levels of CNA, CNB, and calcipressin between diapause eggs and eggs in which diapause initiation was prevented by HCl treatment. As shown in Fig. 1, protein levels of the CNA subunit were relatively lower in eggs in

ACCEPTED MANUSCRIPT which diapause initiation was prevented by HCl treatment compared to those in diapause eggs (treated with water) on day 4 after treatment.

However, an inverse

change in the CNB subunit protein was detected between diapause eggs and HCl-treated eggs. In addition, we also examined changes in calcipressin protein levels, a highly conserved regulator of CN (Hilioti and Cunningham, 2003).

Results

showed that calcipressin protein level in diapause eggs did not significantly change compared to HCl-treated eggs on day 4 after HCl treatment.

Total protein levels, as

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indicated by the anti-HSP70 antibody, did not significantly change.

This result

indicates the possible involvement of CNA and CNB in the embryonic diapause We then studied temporal changes in each protein level during the

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process.

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embryonic diapause process in more detail.

3.2. Changes in protein levels of CNA, CNB, and calcipressin upon HCl treatment at

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20 h after oviposition and in diapause eggs

To further elucidate whether CN is related to the embryonic development of eggs in which diapause initiation was prevented by HCl treatment, temporal changes in

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protein levels of CNA, CNB, and calcipressin were examined in samples of diapause-destined eggs treated with HCl at 20 h after oviposition to prevent the eggs

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from entering diapause. Results (Fig. 2) showed that levels of the CNA and CNB subunits exhibited inverse changes.

Gradually decreasing levels of the CNA

subunit and increasing levels of the CNB subunit were detected during the first 8

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days of the embryonic stage in HCl-treated eggs. However, in diapause eggs, levels of the CNA subunit remained at relatively high levels during the first 8 days after oviposition, although the levels fluctuated (Fig. 3).

The level of the CNB

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subunit was detectable on day 0, and remained low on days 1~3.

It then decreased

to an almost undetectable level on days 4~8 after oviposition (Fig. 3).

Calcipressin

protein levels remained at relatively high levels during the first 8 days after oviposition in diapause eggs. In contrast, in HCl-treated eggs, calcipressin protein levels were high during the first stages, and then gradually decreased on days 7~8.

3.3. Changes in protein levels of CNA, CNB, and calcipressin in both non-diapause eggs and eggs in which diapause was terminated by chilling Correlations between changes in protein levels of CNA, CNB, and calcipressin and embryonic development prompted us to further study changes in protein levels of CNA, CNB, and calcipressin in non-diapause eggs and eggs in which diapause was

ACCEPTED MANUSCRIPT terminated by chilling during embryonic development.

As shown in Fig. 4, in

non-diapause eggs, increased protein levels of the CNB subunit were detected during the middle and later stages of embryonic development.

Similar to those in

non-diapause eggs, increased protein levels of the CNB subunit were also detected in eggs in which diapause was terminated by chilling during the first 8 days after eggs were transferred to 25 °C (Fig. 5).

For protein levels of the CNA subunit and

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calcipressin, gradually decreasing levels were detected in both non-diapause eggs and eggs in which diapause was terminated by chilling during embryonic These results further confirmed that changes in protein levels of CNA,

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development.

CNB, and calcipressin are likely related to embryonic development.

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3.4. Changes in CN enzymatic activity

If CN is a potential candidate for regulating the B. mori embryonic diapause

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process, it would be expected that CN enzyme activity would be differentially regulated in diapause and developing eggs. To test this hypothesis, CN enzyme As shown in

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activity was determined in extracts of diapause and developing eggs.

Fig. 6, in diapause eggs, CN activity was relatively high on the first day after oviposition.

It then decreased to a low level the next day and was maintained at low

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levels on days 2~8. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by

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chilling, CN activity increased during the first several days, reached a peak during the middle embryonic stage, and then greatly decreased 5 or 6 days before hatching. These results clearly indicate that high CN activity is likely related to B. mori embryonic development.

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3.5. Changes in mRNA expression levels of CNA and CNB A further experiment was conducted to examine changes in mRNA expression levels of CNA and CNB in diapause eggs and diapause-destined eggs treated with HCl at 20 h after oviposition to prevent the eggs from entering diapause.

Results

(Fig. 7) showed that mRNA levels of both subunits were high the first day, and they then sharply decreased the next day and remained at low levels.

No significant

difference in CNA levels was detected between HCl-treated and diapause eggs. Similar changing patterns in mRNA expression levels of CNB as CNA were also detected.

On days 6~9, significantly higher levels of CNB were detected in

HCl-treated eggs compared to diapause eggs.

ACCEPTED MANUSCRIPT 4. Discussion Our recent study demonstrated that differential regulation of PP2A between diapause and developing eggs of B. mori is likely linked to the embryonic diapause process (Gu et al., 2017).

The present study further extends the previous study and

clearly shows that CN, another serine-threonine phosphatase, also exhibits different changing patterns in protein levels of CNA, CNB, and calcipressin, in transcriptional levels of CNB, and in enzymatic activities between diapause and developing eggs These regulated changes clearly imply the possible

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during embryonic stages.

involvement of CN in the B. mori embryonic diapause process.

CN and its

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regulation by calcipressin are highly conserved across multiple species (Rusnak and

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Mertz, 2000; Mehta et al., 2009). Although a role for CN during egg activation was demonstrated in Drosophila and Xenopus (Hilioti and Cunningham, 2003; Mochida

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and Hunt, 2007; Nishiyama et al., 2007; Takeo et al., 2010), to the best of our knowledge, this is the first study to demonstrate differential regulation of CN between

embryonic diapause process.

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diapause and developing eggs and its potential correlation with the B. mori CN consists of two subunits, CNA and CNB, of ∼59 and 19 kDa, respectively.

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CNA includes a catalytic domain in the N-terminal half, a regulatory portion with a CNB-binding domain, and a calmodulin-binding domain, followed by an autoinhibitory domain (Klee et al., 1998; Rusnak and Mertz, 2000).

The CNB subunit contains four

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EF-hand Ca2+-binding motifs and binds tightly to activate CNA.

It was demonstrated

that non-catalytic domains in CNA negatively regulate enzyme activity and act as intramolecular inhibitors, and that in the presence of calmodulin and/or CNB, enzyme

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activities increase with removal of three functional domains (Klee et al., 1998; Rusnak and Mertz, 2000; Liu et al., 2005).

In the present study, protein levels of the

CNA and CNB subunits showed inverse changing patterns in both diapause and developing eggs. In diapause eggs, protein levels of CNA remained at relatively high levels, in contrast to the very low levels of CNB. However, in developing eggs (non-diapause eggs, HCl-treated eggs, and eggs in which diapause had been terminated by chilling), protein levels of CNA gradually decreased during embryonic development, leading to almost undetectable levels near the end of the embryonic stage, in contrast to the gradually increasing protein levels of CNB. Considering that non-catalytic domains of CNA negatively regulate enzyme activity, we speculated that the decrease in protein levels of CNA and the increase in protein levels of CNB

ACCEPTED MANUSCRIPT during embryonic development in developing eggs may both contribute to increased CN enzymatic activity.

Similar results (i.e., increased activity but decreased protein

levels) were previously reported in the rat brain treated with antipsychotics (Rushlow et al., 2005). It was suggested that changes in protein levels may be an attempt to compensate for changes in activity (Rushlow et al., 2005). In daf-2 long-lived mutants of C. elegans, it was reported that a higher abundance of the TAX-6 protein, the mammalian homologue of CNA was detected compared to the controls (Dong et

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al., 2007). Moreover, calcipressin protein levels showed similar changing patterns as those in CNA during the embryonic diapause process.

Calcipressins are highly

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conserved regulators of CN. Overexpression experiments in several organisms It was also suggested that

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revealed that calcipressins inhibit CN activity (Liu, 2003).

calcipressins operate as endogenous feedback inhibitors of CN (Kingsbury and

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Cunningham, 2000). Thus, we hypothesized that high protein levels of calcipressin detected in diapause eggs may also contribute to very low endogenous CN activity. In addition, our results showed that CN enzymatic activity sharply increased

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during the first several days, and reached a peak during the middle embryonic development in non-diapause eggs, eggs whose diapause initiation was prevented These

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by HCl, and eggs in which diapause had been terminated by chilling.

temporal changes in enzymatic activity appeared to differ from changes in protein levels of the CNB subunit, which gradually increased during embryonic development

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in developing eggs. Thus, protein expressions of the CNB subunit are not always a direct reflection of CN enzymatic activity.

It was demonstrated that CN enzymatic

activity is not only Ca2+ dependent but is controlled by two structurally similar but

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functionally different Ca 2+-binding proteins, calmodulin and the CNB subunit (Klee et al., 1998). The increase in intracellular Ca2+ activates CN through Ca2+/calmodulin binding to the CNA subunit (Klee et al., 1998; Rusnak and Mertz, 2000). This binding reversibly changes the structure of CNA, causing the autoinhibitory sequence to dissociate from the catalytic groove (Klee et al., 1998; Shibasaki et al., 2002). So far as we know, no study has been carried out on temporal changes in intracellular Ca2+ concentrations and activities of calmodulin during the embryonic development in silkworms.

However, in Drosophila and vertebrate systems, it was demonstrated

that Ca2+ plays an important role in regulating embryonic development and that Ca2+ transients clearly and more intensely accrue in tissues undergoing rearrangement/migration, indicating its participation in tissue movements, cell shape

ACCEPTED MANUSCRIPT changes, and the reorganization of contractile cytoskeletal elements in developing embryos (Markova and Lenne, 2012). Thus, it was hypothesized that in addition to increased protein levels of CNB, dramatic changes in intracellular Ca2+ concentrations and calmodulin activities during the early and middle embryonic development in developing eggs of silkworms may contribute to sharp increases in CN enzymatic activity. of CN activity.

Future studies are needed to clarify the temporal regulation

In addition, significantly higher levels in mRNA expression of CNB

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(on days 6~9) were detected in HCl-treated eggs compared to diapause eggs. Currently, it is not known whether these differential changes in transcriptional levels

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are involved in CN’s enzyme activity.

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In mammalian systems, CN appears to lie at the intersection of protein phosphorylation and Ca2+ signaling cascades and thus plays important roles in the

2000).

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transduction of Ca2+ signals to regulate various cellular processes (Rusnak and Mertz, In Bombyx, CN was demonstrated to be present in pheromone glands, and it

was found that CN appears to be involved in signaling of a pheromone

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biosynthesis-activating neuropeptide (Fonagy et al., 1999; Yoshiga et al., 2002). A genetic study in C. elegans showed that CN plays a pivotal role in regulating a wide

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variety of cellular processes related to development, fertility, proliferation, behavior, and lifespan regulation (Lee et al., 2013). It was also demonstrated that CN plays a pivotal role in controlling systemic energy and body weight homeostasis.

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Knockdown of CN in D. melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure (Pfluger et al., 2015). Because Bombyx diapause eggs showed suppressed metabolic rates and arrested morphogenesis

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(Yamashita and Hasegawa, 1985), it was speculated that low CN enzymatic activity in silkworm diapause eggs likely contributes to regulation of suppression of multiple cellular processes and metabolism.

It was reported that a reduction in PP2A activity

during ground squirrel hibernation appeared to be linked to inhibition of the elongation phase of protein synthesis and a reduction in protein synthesis (Chen et al., 2001). In contrast, we hypothesized that similar to PP2A, high CNB subunit expression and enzymatic activity in developing eggs may positively affect cell proliferation and differentiation, and enhance metabolism and protein synthesis during embryonic development.

It was suggested that in developing eggs of B. mori,

not only PP2A and CN, but other families of protein phosphatases are also activated during embryonic development, and that activation of protein kinases, coordinated

ACCEPTED MANUSCRIPT together with activation of protein phosphatases, precisely regulates embryonic development. In summary, the present study demonstrates that protein levels of CNA, CNB, and calcipressin, transcriptional levels of CNB, and CN enzymatic activities are differentially regulated between diapause and developing eggs in B. mori. High expression levels of the CNB protein and enzymatic activities in developing eggs suggest a potential role of CN in regulating Bombyx embryonic development.

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Additional studies to clarify the regulation of CN activity and map upstream and downstream signaling would provide new insights into understanding possible

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mechanisms regulating embryonic development in B. mori.

ACCEPTED MANUSCRIPT Acknowledgements The authors would like to thank the Taiwan Ministry of Science and Technology for a grant (MOST 104-2311-B-178-002-MY3) and the National Museum of Natural

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Science, Taichung for their financial support.

ACCEPTED MANUSCRIPT References Aramburu, J., Rao, A., Klee, C.B., 2000. Calcineurin: from structure to function. Curr. Top. Cell. Regul. 36, 237-295. Bhaskar, P.T., Hay, N., 2007. The two TORCs and Akt. Dev. Cell 12, 487-502. Brown, L., Chen, M.X., Cohen, P.T., 1994. Identification of a cDNA encoding a Drosophila calmodulin regulated protein phosphatase, which has its most abundant expression in the early embryo. FEBS Lett. 339, 124-128.

PT

Chen, Y., Matsushita, M., Nairn, A.C., Damuni, Z., Cai, D., Frerichs, K.U., Hallenbeck, J.M., 2001. Mechanisms for increased levels of phosphorylation of

RI

elongation factor-2 during hibernation in ground squirrels. Biochemistry 40,

SC

11565-11570.

Cohen, P., 1991. Classification of protein-serine/threonine phosphatases:

NU

identification and quantitation in cell extracts. Methods Enzymol. 201, 389-398. De Loof, A., 2011. Longevity and aging in insects: Is reproduction costly; cheap; beneficial or irrelevant? A critical evaluation of the "trade-off" concept. J. Insect

MA

Physiol. 5, 1-11.

Dijkers, P.F., O'Farrell, P.H., 2007. Drosophila calcineurin promotes induction of

ED

innate immune responses. Curr. Biol. 17, 2087-2093. Dong, M.Q., Venable, J.D., Au, N., Xu, T., Park, S.K., Cociorva, D., Johnson, J.R., Dillin, A., Yates, J.R.3rd, 2007. Quantitative mass spectrometry identifies insulin

EP T

signaling targets in C. elegans. Science 317, 660-663. Fonagy, A., Yokoyama, N., Ozawa, R., Okano, K., Tatsuki, S., Maeda, S., Matsumoto, S., 1999. Involvement of calcineurin in the signal transduction of PBAN in the

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silkworm, Bombyx mori (Lepidoptera). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 124, 51-60.

Fujiwara, Y., Denlinger, D.L., 2007. High temperature and hexane break pupal diapause in the flesh fly, Sarcophaga crassipalpis, by activating ERK/MAPK. J. Insect Physiol. 53, 1276-1282. Fujiwara, Y., Shindome, C., Takeda, M., Shiomi, K., 2006a. The roles of ERK and P38 MAPK signaling cascades on embryonic diapause initiation and termination of the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 36, 47-53. Fujiwara, Y., Shiomi, K., 2006. Distinct effects of different temperatures on diapause termination, yolk morphology and MAPK phosphorylation in the silkworm, Bombyx mori. J. Insect Physiol. 52, 1194-1201.

ACCEPTED MANUSCRIPT Fujiwara, Y., Tanaka, Y., Iwata, K., Rubio, R.O., Yaginuma, T., Yamashita, O., Shiomi, K., 2006b. ERK/MAPK regulates ecdysteroid and sorbitol metabolism for embryonic diapause termination in the silkworm, Bombyx mori. J. Insect Physiol. 52, 569-575. Gu, S.H., Chen, C.H., 2017. Injury-induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori. Insect Sci. 24, 248-258.

PT

Gu, S.H., Hsieh, H.Y., Lin, P.L., 2017. Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori. J. Insect Physiol. 103,

RI

117-124.

SC

Gu, S.H., Young, S.C., Tsai, W.H., Lin, J.L., Lin, P.L., 2011. Involvement of 4E-BP phosphorylation in embryonic development of the silkworm, Bombyx mori. J. Insect

NU

Physiol. 57, 978-985.

Hand, S.C., D.L. Denlinger, Podrabsky, J.E., Roy, R., 2016. Mechanisms of animal diapause: recent developments from crustaceans, insects, nematodes and

MA

fish. Am. J. Physiol. Regul. Integr. Comp. Physiol. 310, R1193-R1211. Hay, N., Sonenberg, N., 2004. Upstream and downstream of mTOR. Genes Dev. 18,

ED

1926-1945.

Hilioti, Z., Cunningham, K.W., 2003. The RCN family of calcineurin regulators. Biochem. Biophys. Res. Commun. 311, 1089-1093.

EP T

Iwata, K., Shindome, C., Kobayashi, Y., Takeda, M., Yamashita, O., Shiomi, K., Fujiwara, Y., 2005. Temperature-dependent activation of ERK/MAPK in yolk cells and its role in embryonic diapause termination in the silkworm Bombyx mori. J.

AC C

Insect Physiol. 51, 1306-1312. Kidokoro, K., Iwata, K., Takeda, M., Fujiwara, Y., 2006. Involvement of ERK/MAPK in regulation of diapause intensity in the false melon beetle, Atrachya menetriesi. J. Insect Physiol. 52, 1189-1193. Kingsbury, T.J., Cunningham, K.W., 2000. A conserved family of calcineurin regulators. Genes Dev. 14, 1595-1604. Klee, C.B., Ren, H., Wang, X., 1998. Regulation of the calmodulin-stimulated protein phosphatase, calcineurin. J. Biol. Chem. 273, 13367-13370. Kobayashi, N., Takahashi, M., Kihara, S., Niimi, T., Yamashita, O., Yaginuma, T., 2014. Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression

ACCEPTED MANUSCRIPT and function. J. Insect Physiol. 68, 58-68. Lee, J.I., Mukherjee, S., Yoon, K.H., Dwivedi, M., Bandyopadhyay, J., 2013. The multiple faces of calcineurin signaling in Caenorhabditis elegans: development, behaviour and aging. J. Biosci. 38, 417-431. Lin, J.L., Lin, P.L., Gu, S.H., 2009. Phosphorylation of glycogen synthase kinase-3beta in relation to diapause processing in the silkworm, Bombyx mori. J. Insect Physiol. 55, 593-598.

PT

Liu, J.O., 2003. Endogenous protein inhibitors of calcineurin. Biochem . Biophys. Res. Commun. 311, 1103-1109.

RI

Liu, P., Huang, C., Jia, Z., Yi, F., Yu, D.Y., Wei, Q., 2005. Non-catalytic domains of

SC

subunit A negatively regulate the activity of calcineurin. Biochimie. 87, 215-221. Markova, O., Lenne, P.F., 2012. Calcium signaling in developing embryos: focus on

NU

the regulation of cell shape changes and collective movements. Semin. Cell Dev. Biol. 23, 298-307.

Mehta, S., Li, H., Hogan, P.G., Cunningham, K.W., 2009. Domain architecture of the

MA

regulators of calcineurin (RCANs) and identification of a divergent RCAN in yeast. Mol. Cell Biol. 29, 2777-2793.

ED

Mochida, S., Hunt, T., 2007. Calcineurin is required to release Xenopus egg extracts from meiotic M phase. Nature 449, 336-340. Nakagaki, M., Takei, R., Nagashima, E., Yaginuma, T., 1991. Cell cycles in embryos

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of the silkworm, Bombyx mori: G2-arrest at diapause stage. Roux. Arch. Dev. Biol. 200, 223-229.

Nishiyama, T., Yoshizaki, N., Kishimoto, T., Ohsumi, K., 2007. Transient activation of

AC C

calcineurin is essential to initiate embryonic development in Xenopus laevis. Nature 449, 341-345. Pfluger, P.T., Kabra, D.G., Aichler, M., Schriever, S.C., Pfuhlmann, K., Garcia, V.C., Lehti, M., Weber, J., Kutschke, M., Rozman, J., Elrod, J.W., Hevener, A.L., Feuchtinger, A., Hrabe de Angelis, M., Walch, A., Rollmann, S.M., Aronow, B.J., Muller, T.D., Perez-Tilve, D., Jastroch, M., De Luca, M., Molkentin, J.D., Tschop, M.H., 2015. Calcineurin Links Mitochondrial Elongation with Energy Metabolism. Cell Metab. 22, 838-850. Rushlow, W.J., Seah, Y.H., Belliveau, D.J., Rajakumar, N., 2005. Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics. J. Neurochem. 94, 587-596.

ACCEPTED MANUSCRIPT Rusnak, F., Mertz, P., 2000. Calcineurin: form and function. Physiol. Rev. 80, 1483-1521. Shibasaki, F., Hallin, U., Uchino, H., 2002. Calcineurin as a multifunctional regulator. J. Biochem. 131, 1-15. Shiomi, K., Takasu, Y., Kunii, M., Tsuchiya, R., Mukaida, M., Kobayashi, M., Sezutsu, H., Ichida Takahama, M., Mizoguchi, A., 2015. Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling

PT

pathway in Bombyx. Sci. Rep. 5, 15566.

Takeo, S., Hawley, R.S., Aigaki, T., 2010. Calcineurin and its regulation by

RI

Sra/RCAN is required for completion of meiosis in Drosophila. Dev. Biol. 344,

SC

957-967.

Takeo, S., Tsuda, M., Akahori, S., Matsuo, T., Aigaki, T., 2006. The calcineurin

NU

regulator sra plays an essential role in female meiosis in Drosophila. Curr. Biol. 16, 1435-1440.

Tian, K., Xu, W.H., 2013. High expression of PP2A-Aalpha is associated with

MA

diapause induction during the photoperiod-sensitive stage of the cotton bollworm, Helicoverpa armigera. J. Insect Physiol. 59, 588-594.

ED

Tonks, N.K., 2006. Protein tyrosine phosphatases: from genes, to function, to disease. Nat. Rev. Mol. Cell Biol. 7, 833-846. Tonks, N.K., 2013. Protein tyrosine phosphatases--from housekeeping enzymes to

EP T

master regulators of signal transduction. FEBS J. 280, 346-378. Voss, M., Fechner, L., Walz, B., Baumann, O., 2010. Calcineurin activity augments cAMP/PKA-dependent activation of V-ATPase in blowfly salivary glands. Am. J.

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Physiol. Cell. Physiol. 298, C1047-1056. Wang, Y., Xie, C., Diao, Z., Liang, B., 2017. Calcineurin Antagonizes AMPK to Regulate Lipolysis in Caenorhabditis elegans. Molecules 22,1062. Xu, W.H., Sato, Y., Ikeda, M., Yamashita, O., 1995. Molecular characterization of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) of the silkworm, Bombyx mori and its distribution in some insects. Biochim. Biophys. Acta. 1261, 83-89. Yamashita, O., Hasegawa, K., 1985. Embryonic diapause. In: Gilbert, L.I., Kerkut, G.A. (Eds), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Pergamon Press, Oxford, Vol 1, pp 407-434. Yamashita, O., Yaginuma, T., 1991. Silkworm eggs at temperatures: implications for

ACCEPTED MANUSCRIPT sericulture. In: Lee, R.E.J., Denlinger, D.L. (Eds), Insects at Low Temperature, Chapman & Hall, New York, pp 424-445. Yoshiga, T., Yokoyama, N., Imai, N., Ohnishi, A., Moto, K., Matsumoto, S., 2002. cDNA cloning of calcineurin heterosubunits from the pheromone gland of the silkmoth, Bombyx mori. Insect Biochem. Mol. Biol. 32, 477-486. Zhao, L.C., Hou, Y.S., Sima, Y.H., 2014. Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm, Bombyx mori.

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Insect Sci. 21, 39-46.

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Fig. 1. Western blot analysis of CNA, CNB, and calcipressin (CAL) in silkworm eggs. Following SDS-PAGE and immunoblotting, silkworm egg extracts (a quarter of one

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egg for each lane) were probed with anti-CNA, anti-CNB, anti-CAL, and anti-HSP 70 (HSP) antibodies. W, extracts from control diapause eggs (4 days after water treatment); H, extracts from eggs whose diapause was prevented by HCl (4 days

gel.

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after HCl treatment).

Molecular weight markers are shown on the right side of the

Results shown in the upper panel are representative of three independent

experiments.

Data are expressed as fold change over egg lysates treated with

water after being normalized to the total amount of HSP 70 (loading control). Asterisks indicate significant differences compared to controls (treated with water) (by Student’s t-test, ** p < 0.01).

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Fig. 2. Effect of HCl treatment on protein levels of CNA, CNB, and calcipressin during the first 8 days after treatment.

Diapause-destined eggs that had been incubated at

25 °C for 20 h after oviposition were treated with HCl and then incubated at 25 °C. Egg lysates from each stage (a quarter of one egg for each lane) were prepared and subjected to an immunoblot analysis with anti-CNA, anti-CNB, anti-CAL, and anti-HSP 70 (HSP) antibodies. Results shown in the upper panel are representative of three independent experiments (A).

Quantified protein levels relative to HSP

(loading control) were standardized to levels from the first day after HCl treatment (B). Different letters above the bars indicate significant differences (ANOVA followed by Tukey’s test).

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Fig. 3. Western blot analysis of protein levels of CNA, CNB, and calcipressin in diapause eggs.

Egg lysates from each stage after oviposition (a quarter of one egg

for each lane) were prepared and subjected to an immunoblot analysis with anti-CNA, anti-CNB, anti-CAL, and anti-HSP 70 (HSP) antibodies. Results shown in the upper panel are representative of three independent experiments (A).

Quantified protein

levels relative to HSP (loading control) were standardized to levels from the first day after oviposition (B). Different letters above the bars indicate significant differences (ANOVA followed by Tukey’s test).

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Fig. 4. Western blot analysis of protein levels of CNA, CNB, and calcipressin in non-diapause eggs.

Egg lysates from each stage after oviposition (a quarter of one

egg for each lane) were prepared and subjected to an immunoblot analysis with anti-CNA, anti-CNB, anti-CAL, and anti-HSP 70 (HSP) antibodies. Results shown in the upper panel are representative of three independent experiments (A). Quantified protein levels relative to HSP (loading control) were standardized to levels from the first day after oviposition (B).

Different letters above the bars indicate

significant differences (ANOVA followed by Tukey’s test).

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Fig. 5. Western blot analysis of protein levels of CNA, CNB, and calcipressin in eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then transferred to 25 °C. Egg lysates from each stage after being transferred to 25 °C (a quarter of one egg for each lane) were prepared and subjected to an immunoblot analysis with anti-CNA, anti-CNB, anti-CAL, and anti-HSP 70 (HSP) antibodies. Results shown in the upper panel are representative of three independent experiments (A).

Quantified protein levels relative to HSP

(loading control) were standardized to levels from the first day after transfer to 25 °C (B).

Different letters above the bars indicate significant differences (ANOVA

followed by Tukey’s test).

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Fig. 6. Changes in CN enzymatic activity in Bombyx eggs. A, diapause eggs; B,

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non-diapause eggs; C, eggs whose diapause initiation was prevented by HCl; D, eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then transferred to 25 °C.

Egg extracts from each stage were

prepared, and CN enzymatic activity was determined. Each data point indicates the mean ± SEM (n = 3).

Different letters above the bars indicate a significant

difference (ANOVA followed by Tukey’s test).

Results of ANOVA: diapause eggs: F

= 8.24, p < 0.001; non-diapause eggs: F = 181.95, p < 0.001; HCl-treated eggs: F = 157.23, p < 0.001; chilled eggs: F = 198.36, p < 0.001.

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Fig. 7. Changes in the mRNA expression levels of CNA (A) and CNB (B) in diapause eggs and HCl-treated eggs. Egg extracts from each stage were prepared, and mRNA expression levels were determined by a qRT-PCR.

The mRNA expression

levels of CNA and CNB relative to rp49 were standardized to the means of day 0 of diapause eggs.

Significant differences were calculated from ANOVA. * p < 0.05.

Each data point indicates the mean ± SEM (n = 4).

Results of ANOVA: diapause

eggs of CNA levels: F = 134.06, p < 0.001; diapause eggs of CNB levels: F = 112.84, p < 0.001; HCl-treated eggs of CNA levels: F = 5.43, p < 0.001; HCl-treated eggs of CNB levels: F = 89.63, p < 0.001.

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Fig. 8. Our current understanding of the signaling network involved in the Bombyx embryonic diapause process. See text for details.

ACCEPTED MANUSCRIPT Research highlights Bombyx eggs contained a 59-kDa calcineurin catalytic A subunit (CNA), a 19-kDa regulatory B subunit (CNB), and a 27-kDa calcipressin.

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Protein levels of CNA, CNB, and calcipressin were found to undergo differential changes between diapause and developing eggs.

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CN enzymatic activity remained at low levels in diapause eggs.

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An increase in CN activity was detected in developing eggs. CN appears to be related to Bombyx embryonic diapause process.

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