bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323386; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Breeding and hibernation of captive meadow jumping mice (Zapus hudsonius) 2 3 4 Ethan A. Brem1, Alyssa D. McNulty1#, William J. Israelsen1* 5 6 7 1 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, 8 United States of America 9 10 #Current Address: Department of Epidemiology and Biostatistics, School of Public Health, Texas 11 A&M University, College Station, Texas, United States of America 12 13 14 *Corresponding author 15 E-Mail: [email protected] (WJI) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323386; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 17 Abstract 18 Hibernating mammals exhibit unique metabolic and physiological phenotypes that have 19 potential applications in medicine or spaceflight, yet our understanding of the genetic basis and 20 molecular mechanisms of hibernation is limited. The meadow jumping mouse, a small North 21 American hibernator, exhibits traits – including a short generation time – that would facilitate 22 genetic approaches to hibernation research. Here we report the collection, captive breeding, and 23 laboratory hibernation of meadow jumping mice. Captive breeders in our colony produced a 24 statistically significant excess of male offspring and a large number of all-male and all-female 25 litters. We confirmed that short photoperiod induced pre-hibernation fattening, and cold ambient 26 temperature facilitated entry into hibernation. During pre-hibernation fattening, food 27 consumption exhibited non-linear dependence on both body mass and temperature, such that 28 food consumption was greatest in the heaviest animals at the coldest temperatures. Meadow 29 jumping mice exhibited a strong circadian rhythm of nightly activity that was disrupted during 30 the hibernation interval. We quantified the length and timing of torpor bouts and arousals 31 obtained from an uninterrupted recording of a hibernating female. Over a 90.6 day hibernation 32 interval, torpor bouts ranged from 2.1 to 12.8 days (mean 7.7 days), and arousal length was 33 relatively constant with a mean length of 9.6 hours. We conclude that it is possible to study 34 hibernation phenotypes using captive-bred meadow jumping mice in a laboratory setting. 35 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323386; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 36 Introduction 37 Daily torpor and hibernation form a continuum of phenotypes that is widespread 38 throughout many orders of mammals [1, 2], suggesting that heterothermy is an ancestral trait that 39 was maintained during the evolution of endothermy [3]. Mammalian hibernators include species 40 of bears, bats, rodents, marsupials, and even primates [4]. Although the state of torpor employed 41 by hibernating animals – profoundly reduced body temperature and significantly slowed 42 metabolic rate – has attracted attention for its potential therapeutic applications in human 43 medicine and spaceflight [5, 6], little is known about its underlying molecular mechanisms or 44 genetics [1]. Hibernation research has historically been closely aligned with ecology and 45 ecological physiology, with the organisms used in hibernation research selected from wild 46 populations locally available to investigators. Traditional species of interest – including bears, 47 bats, ground squirrels, and dormice – exhibit a prolonged hibernation interval fueled by 48 accumulated fat stores, but such species have long generation times and are therefore not readily 49 amenable for genetic studies of hibernation phenotypes. The difficulty in investigating the 50 genetic basis of hibernation is illustrated by the fact that, to our knowledge, only one such study 51 has been published, which identified candidate genes affecting the timing of hibernation onset in 52 the thirteen lined ground squirrel [7]. The golden (or Syrian) hamster (Mesocricetus auratus) is 53 more amenable to genetic approaches [8, 9], but this and other hamster species hoard food and 54 hibernate in a lean state with relatively shorter bouts of torpor [10-14], which would not allow us 55 to study our phenotypes of interest. 56 As part of a long-term goal to accelerate genetic and molecular aspects of hibernation 57 research, we selected the meadow jumping mouse (Zapus hudsonius) as a model organism. The 58 meadow jumping mouse is a small hibernator native to North America; it exhibits pre- 59 hibernation fat storage and a deep hibernation phenotype with long torpor bouts and short 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323386; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 60 interbout arousals [15-18]. The meadow jumping mouse produces multiple litters of offspring 61 per year and is known to reproduce prior to its first hibernation [19, 20], potentially opening the 62 door to genetic experimental approaches that involve breeding and thus require short generation 63 times. Because the meadow jumping mouse uses modifiable environmental cues such as day 64 length and temperature to determine the timing of hibernation [21], this species also enables 65 convenient laboratory study of hibernation phenotypes. Despite the potential utility of the 66 meadow jumping mouse as a model hibernator we were unaware of any reported systematic 67 breeding of this species. Here we report our successful efforts to collect wild meadow jumping 68 mice and establish a captive breeding colony. We investigated the conditions required for 69 induction of fat accumulation and hibernation in the laboratory. Both short photoperiod and low 70 temperature contributed to the weight gain and successful hibernation of meadow jumping mice, 71 which confirmed previous work [21]. We measured food consumption during pre-hibernation 72 fattening and hibernation and identified an interaction effect between animal body mass and 73 housing temperature, in that cold temperature increases pre-hibernation food consumption to the 74 greatest degree in the heaviest mice. We determined the amount and timing of daily locomotor 75 activity before and during hibernation, and quantified the lengths of torpor bouts and the lengths 76 and timing of interbout arousals from the complete hibernation interval of a captive animal. 77 Torpor bout length changed throughout hibernation, but arousal length remained constant and the 78 timing of arousals with respect to photoperiod showed an unexpected degree of regularity. 79 Materials and Methods 80 Animal collection and health testing 81 Animal work described in this manuscript has been approved and conducted under the 82 oversight of the UT Southwestern Institutional Animal Care and Use Committee (protocol 2014- 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323386; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 83 152 & 2015–101240) and the Massachusetts Institute of Technology Committee for Animal Care 84 (protocol 0514-048-17 & 0718-064-21). Animal collection was approved by the Massachusetts 85 Division of Fisheries and Wildlife and conducted according to the Guidelines of the American 86 Society of Mammalogists for the use of wild mammals in research [22]. Wild meadow jumping 87 mice were collected on the Bolton Flats Wildlife Management Area, near Bolton, MA, USA. 88 Live traps (LFA Folding Trap, HB Sherman, Tallahassee, FL, USA) were baited with a mixture 89 of peanut butter and rolled oats and left open overnight. Traps were checked near dawn and left 90 closed during the day. Species other than meadow jumping mice were recorded and released. To 91 mitigate risk of zoonotic illness, masks were worn in the field while working with occupied traps 92 and while handling captive animals of unknown disease status and their soiled bedding, as 93 informed by established guidelines [23]. Occupied traps were washed using a bleach solution 94 prior before being returned to the field; this was a precaution against zoonotic disease and 95 allowed collection of Zapus feces from clean traps for pathogen testing. Following capture, 96 health screening was performed for each animal by MIT veterinary staff. Fresh feces from the 97 trap were used for parasite screening (via fecal float test) and commercial pathogen screening, 98 which included the Charles River Laboratory Mouse FELASA Complete PRIA panel with 99 additional PCR tests for mites, Leptospira, and New World Hantavirus (Charles River 100 Laboratories, Wilmington, MA, USA). In the first year, fecal samples were PCR tested by a 101 second laboratory for Hantavirus (Assay #S0135, Zoologix, Chatsworth, CA, USA). Additional 102 endo- and ectoparasite screening was performed via fur pluck and anal tape exam. All animals 103 were treated with ivermectin for 4 weeks upon entry into the colony. Topical pyrethrin was used 104 as an ectoparasiticide the first year of collection, and animals positive for Giardia parasite were 105 successfully treated with a course of metronidazole in the third year of collection.