Monarch Butterflies Use an Environmentally Sensitive, Internal

1 Article type: Original Article 2 3 Monarch butterflies use an environmentally sensitive, internal timer to control overwintering 4 dynamics 5 6 Running head: “Monarch butterfly internal timekeeping” 7 8 Delbert A. Green II1,2 and Marcus R. Kronforst1 9 10 1Department of Ecology and Evolution 11 University of Chicago. Chicago, IL 60637 USA 12 2Current Address: Department of Ecology and Evolutionary Biology 13 University of Michigan. Ann Arbor, MI 48109 USA 14 15 16 Corresponding Author: 17 Delbert A. Green II 18 University of Michigan (Ann Arbor) 19 1105 N. University Ave. 20 Rm. 4014 21 Ann Arbor, MI 48109 22 734-647-5483 23 [email protected] 24 25 26 27 Abstract 28 The monarch butterfly (Danaus plexippus) complements its iconic migration with diapause, a 29 hormonally controlled developmental program that contributes to winter survival at Author Manuscript 30 overwintering sites. Although timing is a critical adaptive feature of diapause, how This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mec.15178 This article is protected by copyright. All rights reserved 31 environmental cues are integrated with genetically-determined physiological mechanisms to time 32 diapause development, particularly termination, is not well understood. In a design that subjected 33 western North American monarchs to different environmental chamber conditions over time, we 34 modularized constituent components of an environmentally-controlled, internal diapause 35 termination timer. Using comparative transcriptomics, we identified molecular controllers of 36 these specific diapause termination components. Calcium signaling mediated environmental 37 sensitivity of the diapause timer, and we speculate that it is a key integrator of environmental 38 condition (cold temperature) with downstream hormonal control of diapause. Juvenile hormone 39 (JH) signaling changed spontaneously in diapause-inducing conditions, capacitating response to 40 future environmental condition. Although JH is a major target of the internal timer, it is not itself 41 the timer. Epigenetic mechanisms are implicated to be the proximate timing mechanism. 42 Ecdysteroid, JH, and insulin/insulin-like peptide (IIS) signaling are major targets of the diapause 43 program used to control response to permissive environmental conditions. Understanding the 44 environmental and physiological mechanisms of diapause termination sheds light on 45 fundamental properties of biological timing, and also helps inform expectations for how monarch 46 populations may respond to future climate change. 47 48 Keywords: monarch butterfly, diapause, ecdysone, juvenile hormone, insulin signaling 49 Introduction 50 Organisms subject to seasonally variable environments have evolved myriad adaptations to 51 translate environmental condition into accurately timed behavioral and physiological responses. 52 The monarch butterfly (Danaus plexippus) has evolved migration and diapause to survive 53 inhospitable winters across its temperate North American range. Each fall, millions of monarchs 54 across the US and southern Canada migrate to specific locations in central Mexico if in the 55 eastern North American population, or along the Pacific Coast if in the western North American 56 population, where they overwinter in reproductive diapause. In spring, they mate, remigrate 57 northwards, and repopulate their breeding grounds over three to four successive generations. Author Manuscript 58 Previous work has revealed environmental cues and sensory modalities required to accurately 59 interpret environmental condition for both migration and diapause initiation (Goehring & This article is protected by copyright. All rights reserved 60 Oberhauser 2002; Goehring et al. 2004; Zhu et al. 2008; Gegear et al. 2008; Merlin et al. 2009; 61 Guerra & Reppert 2013; Guerra et al. 2014), as well as hormonal mechanisms that control 62 monarch diapause development (Barker & Herman 1973; Herman 1975; Dallmann & Herman 63 1978; Herman & Lessman 1981). The missing link, however, is how monarchs, and other 64 diapausers, integrate external cues with internal, genetically-controlled responses to achieve 65 specifically timed seasonal responses (Hand et al. 2016). Here, we use monarch diapause 66 termination as a model to understand molecular control of environmental response and seasonal 67 timing. 68 Diapause development proceeds through stereotypic eco-physiological phases: initiation, 69 maintenance, and termination (Andrewartha 1952; Koštál 2006). Together, initiation and 70 termination define the specific time interval of diapause maintenance, the environmentally 71 insensitive refractory period during which organisms experience suppressed metabolic rate, 72 bolstered stress resistance, and halted reproductive development (Herman 1973). Upon 73 termination, individuals either immediately resume non-diapause development if prevailing 74 conditions are permissive (e.g. warm), or as is more often the case, enter post-diapause 75 quiescence in adverse conditions (e.g. cold), an environmentally sensitive dormancy. Monarch 76 diapause, like that of many other temperate species, is primarily induced by low and decreasing 77 photoperiod, and is also enhanced by low temperature and host plant quality (Goehring & 78 Oberhauser 2002). Termination, on the other hand, is much more variable among insects and is 79 rarely under photoperiodic control in hibernal diapauses (Hodek 2002; Koštál 2006; Liedvogel & 80 Lundberg 2014; Tauber 1986). Eastern and western North American monarch populations 81 terminate diapause before the winter solstice (Herman 1981; Herman et al. 1989), proving that 82 increasing photoperiod is not a relevant termination cue, although not excluding any 83 photoperiodic involvement. Counterintuitively, cold temperature often hastens spontaneous 84 diapause termination in overwintering diapausers (reviewed in Tauber et al. 1986). How cold 85 temperature controls diapause timing in insects is unknown. More generally, how non- 86 photoperiodically controlled developmental timing occurs is a little-explored and open problem. Author Manuscript 87 Hormonal signaling plays critical roles in insect diapauses. In pre-adult diapauses, 20- 88 hydroxyecdysone (20-HE) is most often recognized as the diapause terminator (reviewed in This article is protected by copyright. All rights reserved 89 (Denlinger et al. 2011). The role of 20-HE in adult reproductive diapause is more variable. 20HE 90 plays opposing roles in different organisms, promoting diapause termination and reproduction in 91 fruit flies (D. melanogaster), locusts (L. migratoria), and European firebugs (P. apterus), while 92 being associated with diapause maintenance in Colorado potato beetles (L. decimlineata) 93 (reviewed in Denlinger et al. 2011). Insulin/insulin-like peptide signaling (IIS) has been shown 94 to influence adult reproductive diapause phenotypes in fruit flies and mosquitoes (C. pipens) 95 (Williams et al. 2006; Sim & Denlinger 2008). The consistent controller of adult reproductive 96 diapause across insects is juvenile hormone (JH) (reviewed in Tauber et al. 1986). Increased JH 97 titer is associated with reproductively active monarchs in spring and summer (Lessman et al. 98 1989). JH signaling is required for reproductive development in non-migrating monarchs, and 99 exogenous JH analog application is sufficient to terminate diapause and induce reproductive 100 development in normally non-reproductive migrant monarchs in summer-like conditions (Barker 101 & Herman 1973; Herman 1975). While these studies consistently associate JH signaling with 102 reproductive state, they leave open the specific mechanism by which JH controls diapause 103 because they do not follow JH signaling dynamics with sufficient temporal resolution across 104 diapause development. Despite knowing that these hormonal pathways are associated with 105 particular diapause states, it is not clear how these pathways actually function in diapause 106 development. Are they involved in transducing the environmental signal? Do they engender 107 maintenance phenotypes? Do they specify termination? Moreover, JH, ecdysteroid, and IIS 108 pathways interact to achieve coherent responses in different contexts. For example, in 109 Drosophila, JH does not control metamorphosis timing as it does in Manduca sexta (Nijhout & 110 Williams 1974; Riddiford et al. 2010). Rather, JH indirectly regulates larval growth rate through 111 controlling ecdysone concentration, versus its timing, whereby influencing IIS pathway activity, 112 which is a well-known controller of larval growth rate (Mirth et al. 2014). How do these 113 interactions change in different contexts, and what role, if any, does diapause play in influencing 114 interactions between JH, ecdysteroid, and IIS? 115 Here we examine diapause termination in the western North American monarch Author Manuscript 116 population in order to understand 1) the relative influence of external environmental cues versus 117 internal physiological mechanisms for terminating diapause; 2) what molecular mechanisms This article is protected by copyright. All rights reserved 118 mediate environmental sensitivity during diapause development; and 3) what controls diapause 119 timing. We take a comparative transcriptomics

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