Photoperiod and temperature separately regulate nymphal development through JH and insulin/TOR signaling pathways in an insect Taiki Mikia,1, Tsugumichi Shinoharaa,1, Silvia Chafinoa,b, Sumihare Nojic, and Kenji Tomiokaa,2 aGraduate School of Natural Science and Technology, Okayama University, 700-8530 Okayama, Japan; bInstitute of Evolutionary Biology, Spanish National Research Council (CSIC)–Pompeu Fabra University, E-08003 Barcelona, Spain; and cIndustrial and Startup Laboratories, Tokushima University, 770-8501 Tokushima, Japan Edited by David L. Denlinger, The Ohio State University, Columbus, OH, and approved February 3, 2020 (received for review December 30, 2019) Insects living in the temperate zone enter a physiological state of down or accelerating the developmental rate and changing the arrested or slowed development to overcome an adverse season, number of instars (9, 10). such as winter. Developmental arrest, called diapause, occurs at a The mechanism of nymphal development underlying the sea- species-specific developmental stage, and embryonic and pupal sonal adaptation includes regulation of cellular and tissue growth diapauses have been extensively studied in mostly holometabo- and/or molting (11, 12). The cellular and tissue growth rates are lous insects. Some other insects overwinter in the nymphal stage mainly controlled by the insulin signaling pathway, in which the with slow growth for which the mechanism is poorly understood. insulin-like peptide (ILP) binds to the insulin receptor (InR) to Here, we show that this nymphal period of slow growth is stimulate a series of signal transduction cascades and eventually regulated by temperature and photoperiod through separate promotes protein synthesis, cell growth, and cell division (13). The pathways in the cricket Modicogryllus siamensis. The former reg- target of rapamycin (TOR) pathway shares a number of key reg- ulates the growth rate, at least in part, through the insulin / target ulators and associates with the insulin signaling pathway, sensing of rapamycin (TOR) signaling pathway. Lower temperature down- nutritional states and also promoting cellular and tissue growth ’ regulates the expression of insulin-like peptide (Ms Ilp) and Target (14–16). Therefore, these two pathways are normally referred to of rapamycin (Ms’Tor) genes to slow down the growth rate with- as the insulin/TOR signaling pathway. However, the growth of PHYSIOLOGY out affecting the number of molts. The latter regulates the num- insects is restricted by the sclerotized cuticle. To allow growth and ber of molts independent of temperature. Short days increase the development, the old cuticle must be shed and replaced with a number of molts through activation of the juvenile hormone (JH) new and larger one as the insect grows. This process is called pathway and down-regulation of myoglianin (Ms’myo), a member molting and is controlled by hormonal factors. When body size of the TGFβ family, which induces adult metamorphosis. In con- reaches a certain level, the molting process starts, including se- trast, long days regulate Ms’myo expression to increase during the cretion of a cerebral neuropeptide, prothoracicotropic hormone fifth to sixth instar to initiate adult metamorphosis. When Ms’myo expression is suppressed, juvenile hormone O-methyl transferase (Ms’jhamt) was up-regulated and increased molts to prolong the Significance nymphal period even under long-day conditions. The present find- ings suggested that the photoperiod regulated Ms’myo,andtheJH Many insects change their development and/or physiology to signaling pathway and the temperature-controlled insulin/TOR path- overcome adverse times of the year. Although photoperiod and way cooperated to regulate nymphal development for overwintering temperature are known to regulate this change, the underly- to achieve seasonal adaptation of the life cycle in M. siamensis. ing mechanism largely remains to be elucidated. The crickets Modicogryllus siamensis overwinter as nymphs with a reduced nymphal development | photoperiod | temperature | myo | insulin/TOR growth rate and increased molts. This study showed that their signaling pathway nymphal development is regulated by photoperiod and tem- perature by two separate pathways: the JH signaling pathway is itting life cycles to a seasonally changing environment is a involved in photoperiodic regulation of molts, and the insulin/ Fprerequisite for insects living in the temperate zone (1, 2). target of rapamycin signaling pathway regulates growth rate in Their common strategy is to enter a state of developmental ar- a temperature-dependent manner. Our findings not only deepen our understanding of the molecular mechanisms for seasonal rest (diapause) or reduced growth rate, during which insects adaptation but also shed light on the insects’ evolutionary strat- overcome the adverse period of the year, which is unsuited for egy to invade temperate zones where photoperiod and tem- growth and reproduction. The most widely used and important perature change seasonally. cue for entering this state is day length or night length in a 24 h day (photoperiod). The photoperiodic information is perceived Author contributions: T.M. and K.T. designed research; T.M., T.S., and S.C. performed by specific photoreceptors and transmitted to a photoperiodic clock, research; T.M., T.S., S.N., and K.T. analyzed data; and T.M., S.C., and K.T. wrote the paper. which measures the lengths of the photophase or scotophase to The authors declare no competing interest. determine the given photoperiod to be a long day (LD) or short day This article is a PNAS Direct Submission. (SD) (3). The LD or SD information is then conveyed to the reg- Published under the PNAS license. ulatory system that induces diapause/reduced growth rate or non- Data deposition: The complementary DNA sequences of Ms’myo, Ms’jhamt, Ms’Kr-h1, diapause/faster growth rate responses. There are lines of evidence Ms’Ilp, Ms’Inr,andMs’Tor have been deposited in DNA Data Bank of Japan (DDBJ), http://getentry.ddbj.nig.ac.jp/top-j.html. Their accession numbers are LC457977, suggesting that a circadian clock is involved in the photoperiodic LC458777, LC460294, LC460295, LC458778, and LC460296, respectively. – clock (4 6). Temperature also plays an important role in regulat- 1T.M. and T.S. contributed equally to this work. ing development. It often modifies the degree of the photoperi- 2To whom correspondence may be addressed. Email: [email protected]. odic response (7, 8) and sometimes alters the critical day length This article contains supporting information online at https://www.pnas.org/lookup/suppl/ for switching between LD and SD responses (2). In addition, doi:10.1073/pnas.1922747117/-/DCSupplemental. temperature changes the developmental time course, by slowing www.pnas.org/cgi/doi/10.1073/pnas.1922747117 PNAS Latest Articles | 1of7 Downloaded by guest on September 26, 2021 (PTTH) (17), which stimulates the prothoracic gland to produce a A steroid hormone, ecdysone (18). Ecdysone is then converted to 20 hydroxyecdysone (20E) that promotes molting through the ex- pression of various molting-related genes (19, 20). However, the nature of molting is determined by juvenile hormone (JH) (21, 22). When humoral JH levels are maintained higher, 20E induces nymphal molting. When humoral JH is eliminated or reduced be- low a certain level, the metamorphosis from nymph to adult is initiated. The action of JH is mediated by a JH-inducible gene, Krüppel homolog 1 (Kr-h1) (23, 24). JH is produced in the corpora allata (CA) from Acetyl–CoA through various enzymatic reactions D (25) (SI Appendix,Fig.S1). The final reaction of JH production is BC the conversion from the inactive JH precursor to active JH, and this reaction is controlled by JH acid O-methyltransferase (JHAMT), a rate-limiting enzyme encoded by the gene jhamt (26). Recently, it was reported that a member of the TGF-β family, myoglianin (myo), down-regulates the expression of jhamt, and, consequently, reduces the level of JH and leads to metamorphosis (27). Insects enter diapause at a species-specific stage with developmental arrest through modulating a certain step of the above-mentioned de- velopmental process. For example, the tobacco hornworm Manduca sexta enters diapause at the pupal stage by inactivation of cerebral PTTH secretion (28), the silk moth Bombyx mori as an embryo by Fig. 1. Effects of photoperiod and temperature on nymphal development Drosophila melanogaster in the cricket Modicogryllus siamensis under long-day (LD, 16L:8D) or short- a diapause hormone (29), and the fruit fly day conditions (SD, 12L:12D) at 25 °C and 30 °C. (A and B) Adult emergence and the mosquito Culex pipiens as an adult by a mechanism in- patterns after hatching from eggs (A) and proportions of molt numbers volving the insulin signaling pathway (12). However, for those before adult emergence (B). The number of animals used was 84, 46, 92, and overwintering as nymphs with a reduced developmental rate, the 29 for LD 30 °C, LD 25 °C, SD 30 °C, and SD 25 °C, respectively. Numbers in underlying molecular mechanism is still largely unknown. Hence, different color fractions in B indicate the number of molts. (C) Adult body this study aimed to explore the mechanism controlling the weight (mean ± SEM) measured at 1 d after adult emergence. Values with nymphal developmental rate in a bivoltine insect, the cricket different letters significantly differ from each other (Tukey test, P < 0.05). Modicogryllus siamensis, which occurs twice a year and overwinters Numbers in parenthesis indicate the number of animals used. (D) Photo- as nymphs and shows clear photoperiodic responses in its nymphal graphs of LD and SD adult crickets raised at 30 °C. (Scale bar, 1 cm.) development (30, 31). At 25 °C, most nymphs become adult at ∼60 d after hatching from eggs, undergoing 7 or 8 molts under LD, under SD, adults occurred between 41 and 101 d with a median of 49 whereas under SD, the nymphal period elongates with an in- d, after 9–13 molts (Fig.