Gender difference in unconditioned and conditioned predator fear responses in Smith's zokors (Eospalax smithii)

Accepted Manuscript Gender difference in unconditioned and conditioned predator fear responses in Smith's zokors (Eospalax smithii) Ibrahim M. Hegab, Zhang Qian, Qiangsheng Pu, Zhicheng Wang, Kang yukun, Cai zhiyuan, Guo huailiang, Haifang Wang, Weihong Ji, A.M. Hanafy, Junhu Su PII:

S2351-9894(18)30359-7

DOI:

https://doi.org/10.1016/j.gecco.2018.e00503

Article Number: e00503 Reference:

GECCO 503

To appear in:

Global Ecology and Conservation

Received Date: 3 October 2018 Revised Date:

7 December 2018

Accepted Date: 7 December 2018

Please cite this article as: Hegab, I.M., Qian, Z., Pu, Q., Wang, Z., yukun, K., zhiyuan, C., huailiang, G., Wang, H., Ji, W., Hanafy, A.M., Su, J., Gender difference in unconditioned and conditioned predator fear responses in Smith's zokors (Eospalax smithii), Global Ecology and Conservation (2019), doi: https:// doi.org/10.1016/j.gecco.2018.e00503. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Gender difference in unconditioned and conditioned predator fear

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responses in Smith’s zokors (Eospalax smithii)

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Ibrahim M. Hegab1,2,3, Zhang Qian1, Qiangsheng Pu1, Zhicheng Wang1, Kang yukun1, Cai

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zhiyuan1, Guo huailiang1, Haifang Wang1, Weihong Ji2,4, Hanafy A.M1,2,5, Junhu Su1,2*

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Gansu Agricultural University, Lanzhou 730070, China

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Gansu Agricultural University, Lanzhou 730070, China

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College of Grassland Science, Key Laboratory of Grassland Ecosystem (Ministry of Education),

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Gansu Agricultural University-Massey University Research Centre for Grassland Biodiversity,

Department of Hygiene, Zoonoses and Animal Behaviour and Management, Faculty of

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Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt

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Shore Mail Centre 0632, Auckland, New Zealand

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Egypt

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Institute of Natural and Mathematical Sciences, Massey University, Private Bag 102904 North

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Animal Production Department, Faculty of Agriculture, Suez Canal University, 41522 Ismailia,

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College of Grassland Science, Key Laboratory of Grassland Ecosystem (Ministry of Education),

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Gansu, Agricultural University, Lanzhou 730070, China

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e-mail: [email protected]

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Tel. +86-931-7631213

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Corresponding author: Dr. Junhu Su

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Abstract

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Risk taking is imperative for the survival and fitness of animals since they constantly face

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innumerable threats from various sources. Indeed, the ability of the individual to balance

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between the costs and benefits of various options and adopt a wise decision is critical for the

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well-being of the animal. In the current study, we investigated gender differences in risk taking

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and decision making in male and female Smith’s zokors (Eospalax smithii), a subterranean

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rodent species, in the defensive withdrawal apparatus (DWA) under novel, predator and

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conditioned contexts. Under novel context, males showed more exploratory and less hiding

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behavior than females especially during the first half of the testing session. However, under

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predator and conditioned contexts, non-significant sexually dimorphic behavioral differences

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were observed. Nevertheless, analysis of the performance of each gender separately across the

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three contexts revealed that males showed strong aversion to the predator and conditioned

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contexts. By contrast, females did not significantly vary their behavioral responses when exposed

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to the predator and conditioned cues. Our results suggest that males are more inclined to indulge

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in riskier exploratory activities while females may be regarded as risk-aversive. The current

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findings highlighted the functional significance of the trade-off between risk and exploration in

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the natural environment such that male and female zokors differed in how they gather and

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process information during risky confrontations that ultimately elicit significant variations in

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their decision-making and coping strategies to dangerous events.

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Keyword: conditioning, behavior, sex differences, risk-taking, subterranean rodents

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1. Introduction Rodents are daily jeopardized with a myriad of threats, and as dangerous confrontations

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escalate, they have to constantly trade-off risk-prone behaviors such as feeding with risk-averse

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ones such as vigilance to build up a series of assessments and decisions to adopt a sound strategy

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for surviving with unanticipated events (Blanchard et al. 2005) and can thus have greater fitness

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outcomes. This fact may be true for aboveground dwelling rodents that usually encounter ever-

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changing events by showing adaptive alterations in their foraging, feeding, general activity and

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shelter-seeking behaviors, which ultimately may affect their survival and fitness (Drakeley et al.

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2015). Nevertheless, the underground ecotope represents a unique habitat for nearly 250

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subterranean rodent species in which the underground dwellers inhabit, for most of their lives, a

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complex sealed burrow system separated from the aboveground environment by mounds of soil

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(Shams et al. 2005). This relatively secured habitat structure with a monotonous and stable

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environment offers shelter to the subterranean dwellers against predators and adverse

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environmental conditions (Šumbera et al. 2006). Moreover, this environment is relatively

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constant and predictable, in comparison with the aboveground niche (Burda et al. 2007). Indeed,

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this environmental predictability may exert ecological and evolutionary pressures on the risk

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assessment and state anxiety in subterranean species in such a way that the more predictable

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environment would require more preparations be launched for a possible dangerous encounter

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(Parihar et al. 2009). Likewise, empirical studies demonstrate the effects of different

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environmental sensory stimuli on risk assessment and threat perception (Munoz and Blumstein

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2012; Fischer et al. 2017). For aboveground species, various sensory cues may facilitate the

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acquisition of valid information from the environment in which the visual capacity of the animal

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is sufficient to permit robust scanning of the surroundings concurrently with other sensory

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systems, including hearing and olfaction. By contrast, the unchanging, extraordinary

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underground niche exerts restriction on the sensory perception of the subterranean dwellers,

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compared with that of aboveground taxa, because the underground burrows lack sufficient light

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and ventilation, which eliminate vision and lessen the magnitude of olfactory stimuli by the soil

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(Kimchi and Terkel 2004). Thus, a reasonable assumption is that these constraints on sensory

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perception may hamper the cognitive ability of underground inhabitants to extract rational

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meanings from common sensory inputs, which in turn might affect their risk assessment,

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rationalizing and behavior guidance (Hegab et al. 2018). Bearing all this in mind, we assume

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that the subterranean rodents may show atypical risk-assessment and anxiety-related behaviors

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when challenged with risky and threatening circumstances, inconsistent with those previously

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described in aboveground species (Jin et al. 2018; Sievert and Laska 2016).

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Predator odors are non-intrusive naturalistic stressors of high ethological relevance that

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provoke various defensive responses in prey species (Hegab et al. 2015). The anti-predator

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behavioral responses to a predation threat include reduced locomotor activities and retreating to a

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safer shelter which, on one hand, minimize the probability of a possible fatal prey-predator

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conflicts and, on the other hand, monitor of the predator threat from a strategic location (Stryjek

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et al. 2018). Unequivocally, prior to the initiation and expression of the adaptive defensive

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behaviors, first and foremost to avoid predation risk is to detect and identify the predator and the

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source of threat perception as well. Equally important, remembering the place where a predator

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or its odor had been previously encountered facilitates the avoidance of potential predators thus

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protecting individuals from danger (Ferrari et al. 2010). This association between an

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environmental context and aversive stimuli is known as contextual fear conditioning (Endres

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and Fendt 2007). Contextual fear conditioning paradigm is widely used to study predator odor

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emotional associative learning and memory. The basic protocol of this model is very

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straightforward (Takahashi et al. 2008); first it involves adapting the individual to the test

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apparatus before exposure to the predator stimulus. Then, animals are exposed to the predator

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stimulus within the same context and the unconditioned fear-related behavioral responses are

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measured. Following exposure to the predator odor, the animal is returned to its home-cage and

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tested the next day(s) for retention of contextual fear behavior in the same conditioning apparatus

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with predator odor had been removed. A generous proportion of the literature described the

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behavioral defensive responses in many aboveground rodent species following exposure to

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predator odors, for example, Norway rats (Rattus norvegicus, Yin et al. 2013), Brandt’s voles

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(Lasiopodomys brandtii, Hegab et al. 2014a), Pateau pika (Ochotona curzoniae, Yin et al.

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2017), Wood mice (Apodemus sylvaticus, Navarro-Castilla et al. 2018), Bank voles

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(Clethrionomys Glareolus, Borowski and Owadowska 2001) and Ship Rats (Rattus rattus,

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Bramley and Waas 2001) and to a predator odor fear conditioning paradigm (Takahashi et al.

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2005, Takahashi 2014, Halonen et al. 2016). However, very few studies have investigated the

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effect of predator odors on the underground rodents such as in Talas Tuco-tuco (Ctenomys

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talarum, Brachetta et al. 2016) and to the best of our knowledge, the predator odor contextual

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conditioning paradigm had not been performed previously on a subterranean species.

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Emerging evidences have demonstrated that males and females differ in some forms of

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risky decision-making (Ward et al. 2004; Cavigelli et al. 2011; Orsini et al. 2016). Generally,

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individuals could be classified based on their risk preference as “risk prone”, “risk neutral” or

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“risk aversive” (Blumstein and Bouskila 1996; Platt and Huettel 2008). These sex variations

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in the willingness to take risk are not only explained by the disparities in the morphological

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(Samia et al. 2015), physiological or neural (van den Bos et al. 2013) architectures between

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genders, but extended beyond to the differences in life-history trade-offs that may also divulge

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how males and females process and utilize information, and thus affect their risk taking and

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learning responses. The speed of decision making during exposure to a predation threat may

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seems to be advantageous, for the first instance, for the individuals’ fitness as being faster at

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information collection may help earning more rewards but, by contrast, may hinder an interactive

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and thorough exploration of the risky context and increase the probability of predation encounter

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as well which ultimately putting their life at stake (Jolles et al. 2015). On the other hand,

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although meticulous information gathering and data acquisition may incur in a slower decision

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making which might minimize the likelihood to grasp valuable rewards from the surrounding

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environment, it may be a safer strategy that preserve the animals’ survival and provides more

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detailed information about the nature of risk (Mathot et al. 2012). Therefore, it is reasonable to

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assume that during many real-world decision, the dynamics of decision making are not only

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curtailed on the potential rewards the animals can benefit, but also on the potential negative

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reinforcements and outcomes. While males tend to be risk-inclined by being proactive and

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rapidly performing more risky choices than females (Harris et al. 2010), females may be more

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motivated to collect comprehensive information about their environment and making them more

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flexible learners (Jolles et al. 2015). However, several decision-making models that have been

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commonly used to explore gender differences do not involve the potential for some life-

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threatening consequences that is sometimes encountered during exposure to dangerous events

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such as a predatory encounter. Besides, the vast variations in the species (Bettis and Jacobs

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2009) or even the strains (Rex et al. 1996) used in different risky-decision tests make a general

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speculation on the magnitude and direction of dimorphic sexual behavioral responses to risky

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situations invalid. Smith's zokors (Eospalax smithii) are a typical subterranean rodent species that dwell in

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areas above 2000 m on the Qinghai-Tibetan Plateau with other zokor species such as plateau

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zokors (Eospalax baileyi) and (Eospalax rufescents), which show closely related morphological

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and phylogenetic relationships (Su et al. 2014). Zokors live in very intricate burrow systems

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with closed endings separated from the underground ecotope with mounds of soil (Su et al.,

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2018). Zokors show frequent aboveground trips, but they remain in tunnels most of their lives

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(Zhou and Dou 1990). Their skulls are also found in the pellets of many aerial predators

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revealing that they most likely disperse aboveground (Cui et al. 2003). During the aboveground

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dispersal, zokors would have increased vulnerability to multiple risks including both terrestrial

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and flying predators (Schaller 1998). In the current study, we repeatedly exposed male and

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female Smith's zokors (Eospalax smithii) to a defensive withdrawal apparatus (Hegab et al.

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2014b) which composed of two parts; a testing arena and a hide box, and observed their

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behavioral responses under three conditions based on the classical fear conditioning paradigm

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(Takahashi et al. 2008); (1) when the context is novel at the first exposure to measure the

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responses of both sexes to novelty, (2) when the context comprised a predator odor (Cat odor) to

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gauge the anti-predator behavioral responses in both genders, and finally (3) when the odor of

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the predator was removed, to quantify responses to associatively learned risk. Within the

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breeding season, males zokors are more inclined to indulge in territorial behaviors and

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competing endeavors over access to burrows and mates (Zhang 2007), whereas females may

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invest more time and activities in parental care. Additionally, males and females show a

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significant difference in their home range size in the breeding season, with males exhibiting

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wider home ranges than those of females (Hegab et al. 2018). Initially, we assume that these

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sex-specific disparities in life history strategies and priorities may reflect differences in risk-

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assessment/taking behavioral responses in zokors, and therefore, we hypothesized that male

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zokors, when exposed to critical situations, will perform more risky behaviors whereas females

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will display more risk-aversive actions to ensure their safety. Nevertheless, we may also present

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an alternative hypothesis, contrary to the previous one, as zokors remained most or, even nearly,

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all their entire life locked to a well-defined and a well-structured burrow system like a “train to

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its rails” or a like a “spider to its web” except for few aboveground dispersal events (Zhang

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2007) and , therefore, the limited space in which the zokors can utilize to launch various anti-

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predator behavioral responses and maneuvering the predation threat would be limited and

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ultimately this will abolish any apparent gender dimorphic variations in their threat perception,

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decision making and the displayed behavioral responses to a predation risk.

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2. Materials and Methods

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2.1. Subjects and housing

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Forty-three adult Smith's zokors (Eospalax smithii) [Twenty-two males and twenty-one

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females] were captured alive in specialized tube traps (Baoji Ludixincheng Co., Ltd, Xian, China)

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from the grassland fields of Minxian County in the northwest part of the Qinghai-Tibetan Plateau

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(34.43807° N, 104.03688° S), Gansu Province. Neither trap deaths nor injuries were recorded.

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Experts trapped the zokors during the breeding season (March-April 2018). Animals were then

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transported to a laboratory animal room in which their health status was checked, and they were

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sexed and individually housed in plastic cages (75 × 50 × 40 cm) with soil (20 cm depth) as

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bedding material to mimic their natural habitat. Animals were fed ad libitum with carrots,

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potatoes and sweet potatoes. Soil change and cage cleaning was performed as required. Animals

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were housed in controlled conditions at 23 ± 1 °C and humidity of 55 ± 5% in a remote animal

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room to avoid noises at Gansu Agricultural University.

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2.2. Experimental set-up

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All animals were experimentally naïve and had not undergone any previous behavioral

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experiments. Testing was conducted in a separate room connected to the animal room with a

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swinging door. Animals were always handled and carried from the animal room to the testing

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room by the same experimenter, thereby reducing stress and increasing the validity of the results.

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To investigate the zokors’ behavioral responses to risk, we subjected them repeatedly to one of

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three identical defensive withdrawal apparatus (DWA). The apparatus consisted of an opaque

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glass wall divided the box into two compartments; the ‘testing arena’ consisted of a rectangular

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area (60 × 30 × 30 cm), and the second compartment, termed the ‘hide box,’ was constructed

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from black glass (15 x 30 x 30 cm). A small, square hole (8 × 8 cm) in the front wall of the hide

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box allowed the tested individuals to enter the arena (Hegab et al. 2014b). The front wall of the

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arena contained a double-face adhesive tape above the floor that held a piece of clothes (2×4 cm

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strip), termed the ‘stimulus’. We used either a ‘cat odour stimulus’, created by placing the

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clothes on laboratory cat for 10 consecutive days, or a ‘control stimulus’ that had not been in

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contact with a cat. Both control and cat odor stimuli were kept separately in airtight plastic bags

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at −20°C and were always manipulated with latex gloves. The zokors’ movements were recorded

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using a camera in the center of the top-unit of the apparatus.

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2.3. Experimental procedure

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Zokors were subjected to the DWA set-up for three 20 min sessions on three subsequent

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days. On day 1, the testing arena contained the control stimulus and was new to the zokors

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‘novel context’. On day 2, the testing arena contained the cat odour stimulus ‘predator odour

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context’. On day 3, the testing arena again contained the control stimulus, thus considered as a

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‘conditioned context’. Zokors were placed in the hiding compartment of the apparatus at the

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beginning of each trial. Both control and cat odour cues were replaced using disposable gloves

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before testing a new individual. To avoid the transfer of zokor odours between trials, the DWA

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(both the testing arena and hide box) was thoroughly cleaned with 50% ethanol solution and

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paper towels after each trial. Between test days the arenas were thoroughly cleaned an extra time.

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2.4. Behavioral measures

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All testing sessions were recorded with an infrared video network camera (Model.

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ZKXC.TD136U2RZT, Zhongke Electric Co., Ltd, Shenzhen, China) mounted above the

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apparatus and connected to a monitor and a computer in an adjacent room for recording and

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monitoring animal behavior. Videos were analyzed using Behavioral Observation Research

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Interactive Software (BORIS, v. 2.95, Friard and Gamba 2016). Data were collectively

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categorized as follows: (1) the proportion of times zokors were completely hidden inside the

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hide-box (‘hidden’) including the tail, (2) the individual’s body is in the hide-box with its head

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outside the opening, a typical risk-assessment position (Head-out; Dielenberg McGregor 2001)

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and (3) the individual’s body completely outside the hide-box (‘out’). These measures are

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mutually exclusive and reflect different levels of risk-taking and/or engagement with the

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environment: low (hidden), intermediate (head-out) and high (out). Additionally, the latency

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from placing the individual inside the hide box at the start of the experiment till the whole zokor

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or part of it appear outside the box was also recorded alongside the duration of locomotor

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activities and contact to the neutral or predator stimulus at the relevant contexts (Hegab et al.

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2014c).

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2.5. Data analysis

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The SPSS 22.00 statistical software package (Armonk, NY: IBM Corp.) was used for all

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analyses. In each context, Student’s test was used to examine the differences between males and

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females for different behavioral variables. Repeated-measures analyses of variance (ANOVAs)

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were used within each context to analyze the difference between the first and second halves of

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the testing sessions (10 minutes each) with sex (between-subjects factor) and time (first and

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second halves as within-subjects factor) effects on the different behavioral responses. Also, One-

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Way repeated-measures ANOVAs were run separately for each gender across the different

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contexts. Duncan’s post hoc test was used to compare different behavioral responses if

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necessarily. Effect size [partial-eta squared (η2) values for repeated measures ANOVA were

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calculated. The level of significance at which the null hypothesis was rejected was α= 0.05.

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3. Results

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3.1. Gender differences inside the novel context

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When male and female zokors firstly placed in the novel context with the control stimulus,

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males spent more time outside the hide box (t41= 2.19, p= 0.03; Fig. 1-C) and were less time

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hidden (t41=−2.43, p= 0.02; Fig. 1-A) than females. The mean duration of locomotor activities

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was higher in males (t41=−2.96, p= 0.01; Fig. 1-D), but females showed longer latencies till leave

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the hide box for the first time (t41=−3.84, p< 0.01; Fig. 1-F). Both genders did not display

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significant differences in the time spent neither for head-out (t41= 1.66, p= 0.10; Fig. 1-B) nor

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sniffing the control stimulus (t41= 0.76, p= 0.45; Fig. 1-E). Means of different behavioral activities of male and female Smith’s zokors (Eospalax smithii)

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during the first and second 10 minutes of the testing session under the novel context were

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presented in Supplementary table (1). Analysis of the behavioral variables in both genders

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between the first and second 10 minutes of the observation session using repeated measures

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ANOVA also yielded clear sex differences. Comparison of the hiding levels showed that there

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was no significant time (F1,41= 0.52, p= 0.48, η2= 0.012) effect on the mean duration of hiding

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but there were significant effects of sex (F1,41= 5.50, p= 0.024, η2= 0.12) and Time x Sex

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interaction (F1,41= 5.06, p= 0.03, η2= 0.11) that males hid more than females and Duncan’s post

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hoc test showed that the highest proportion of time spent in the hide box was in the first half of

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the testing trial for females (0.70 ± 0.07) while males spent the lowest proportion of time hiding

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during the first half of the trial (0.41 ± 0.05). A significant effect of time (F1,41= 8.68, p= 0.005,

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η2= 0.18) on head-out behavior was observed that zokors tend to significantly decrease (t84= 2.92,

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p= 0.02) head out in the second half of the testing session (First half; 0.03 ± 0.007, Second half:

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0.02 ± 0.004), but there were neither significant sex (F1,41= 2.71, p= 0.11, η2= 0.06) nor Sex ×

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Time interaction (F1,41= 0.21, p= 0.65, η2= 0.005) effects. Our results also showed significant sex

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(F1,41= 4.77, p= 0.04, η2= 0.104) and Sex × Time interaction (F1,41= 4.98, p= 0.03, η2= 0.108)

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effects that males spent more time out of cover during the testing session than females (Male;

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0.49 ± 0.05, Females; 0.31 ± 0.07) and Duncan’s post hoc test showed that the highest proportion

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of time spent out of cover was recorded during the first half of the testing session for males (0.54

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± 0.05) while females displayed the lowest time (0.27 ± 0.06) out of cover during the first half

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of the testing session, whereas there was no significant effect of the time of the trial (F1,41= 0.01,

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p= 0.76, η2= 0.002). Zokors tended to significantly (Time: F1,41= 8.24, p= 0.006, η2= 0.167) display more

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locomotor activities (sec) during the first half of the trial (83.70 ± 9.34) compared to the second

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one (55.98 ± 8.19) and males significantly (Sex: F1,41= 8.52, p= 0.006, η2= 0.172) showed more

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locomotion than females (178.40 ± 17.63 and 98.18 ± 20.66, respectively), while there was no

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significant Time × Sex interaction (F1,41= 0.81, p= 0.37, η2= 0.019) was observed. Finally,

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neither Time (F1,41= 0.45, p= 0.51, η2= 0.011), Sex (F1,41= 0.58, p= 0.45, η2= 0.014) nor Time ×

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Sex interaction (F1,41= 0.05, p= 0.80, η2= 0.001) showed any effects on the mean duration of

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contact time (sec) to the control stimulus in males (First half; 7.88 ± 2.14, Second half; 9.21 ±

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4.95) and females (First half; 4.71 ± 1.50, Second half; 7.29 ± 2.67).

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3.2. Gender differences inside the predator odor context

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Upon exposure to the field test arena containing the cat odour stimulus, male and female

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zokors showed non-significant differences in their responses to the predator threat. Both genders

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spent nearly the same proportion of hiding time (t41=−1.42, p= 0.16; Fig. 1-A), head out (t41=

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1.19, p= 0.24; Fig. 1-B) and out of cover (t41= 1.36, p= 0.18; Fig. 1-C). Similarly, the mean

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duration of both locomotor activities (t41=0.88, p=0.38; Fig. 1-D) and contact to the odor source

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(t41= 1.47, p= 0.15; Fig. 1-E) did not significantly change between genders. However, females

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tended to spend longer latencies till first emerge from the hide box than males (t41=−2.76, p=

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0.01; Fig. 1-F).

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Similarly, repeated measures analysis of within testing session (10 minutes interval) of the

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behavioral responses of both sexes revealed absence of significance sex differences in the zokors

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responses toward predator odors over time. Neither Sex nor the Time × Sex interaction showed

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any significant effects on the proportion of hiding time (Sex: F1,41= 2.21, p= 0.15, η2= 0.05/

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Time × Sex: F1,41= 0.12, p= 0.73, η2= 0.003), head out (Sex: F1,41= 1.08, p= 0.31, η2= 0.03/

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Time × Sex: F1,41= 0.36, p= 0.55, η2= 0.009), out of cover (Sex: F1,41= 2.05, p= 0.16, η2= 0.05/

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Time × Sex: F1,41= 0.17, p= 0.68, η2= 0.004), the mean duration of locomotion (Sex: F1,41= 0.92,

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p= 0.34, η2= 0.02/ Time × Sex: F1,41= 1.42, p= 0.24, η2= 0.03) and contact to the predator

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stimulus (Sex: F1,41= 2.13, p= 0.15, η2= 0.05/ Time × Sex: F1,41= 0.96, p= 0.33, η2= 0.02).

294

Means of different behavioral activities of male and female Smith’s zokors (Eospalax smithii)

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during the first and second 10 minutes of the testing session under the predator odor context were

296

presented in Supplementary table (1)

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However, zokors significantly decrease their head out (F1,41= 9.65, p= 0.003, η2= 0.190)

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during the second half of the testing session (First half: 0.03 ± 0.004, Second half: 0.02 ± 0.004),

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and similarly showed reduced locomotion (F1,41= 11.29, p= 0.002, η2= 0.22) activities over time

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during the second half of the testing session (First half: 72.39 ± 8.87, Second half: 47.35 ± 7.05).

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zokors neither significantly vary their proportion of hiding time (F1,41= 0.33, p= 0.57, η2= 0.008)

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and time spent out of cover (F1,41= 0.07, p= 0.80, η2= 0.002) nor the mean contact time to the

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odor source (F1,41= 3.23, p= 0.08, η2= 0.07) between the first and second halves of the testing

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session.

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3.3. Gender differences to the conditioned context

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Similar to the results of the predator context, testing zokor in the conditioned context

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disclosed absence of gender differences to the conditioned context regarding the proportion of

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hiding time (t41=−0.07, p= 0.94; Fig. 1-A), head out (t41= 1.07, p= 0.29; Fig. 1-B), out of cover

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(t41=−0.06, p= 0.95; Fig. 1-C) and the mean duration of both locomotor activities (t41= 0.28, p= 14

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0.78; Fig. 1-D) and contact to the neutral stimulus as well (t41= 0.21, p= 0.83; Fig. 1-E) did not

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significantly change between genders. However, females tended to spend longer latencies till

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first emerge from the hide box than males (t41=−2.62, p= 0.01; Fig. 1-F).

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Repeated measures analysis of within testing session of the behavioral responses of both

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sexes revealed absence of significance sex differences in the zokors responses toward the

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conditioned context over time. Neither Sex nor the Time × Sex interaction showed any

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significant effects on the proportion of hiding time (Sex: F1,41= 0.005, p= 0.94, η2= 0.0001/ Time

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× Sex: F1,41= 3.50, p= 0.07, η2= 0.08), head out (Sex: F1,41= 1.15, p= 0.29, η2= 0.03/ Time × Sex:

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F1,41= 1.33, p= 0.26, η2= 0.03), out of cover (Sex: F1,41= 0.003, p= 0.96, η2= 0.00008/ Time ×

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Sex: F1,41= 2.49, p= 0.12, η2= 0.06), the mean duration of locomotion (Sex: F1,41= 0.08, p= 0.78,

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η2= 0.002/ Time × Sex: F1,41= 0.10, p= 0.75, η2= 0.002) and contact to the predator stimulus

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(Sex: F1,41= 0.05, p= 0.83, η2= 0.001/ Time × Sex: F1,41= 0.09, p= 0.76, η2= 0.002). Zokors

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significantly hid more (F1,41= 5.20, p= 0.03, η2= 0.11) during the second half of the testing

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session (First half: 0.67 ± 0.04, Second half: 0.76 ± 0.05). Nevertheless, both gender

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significantly showed less head out (F1,41= 8.03, p= 0.007, η2= 0.16) behavior during the second

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half of the testing trial (First half: 0.04 ± 0.01, Second half: 0.01 ± 0.002) and significantly

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lowered (F1,41= 11.63, p= 0.001, η2= 0.22) their locomotion as well over time (First half: 61.40 ±

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8.63, Second half: 35.49 ± 7.05). Finally, zokors neither alter their time out of cover (F1,41= 2.34,

328

p= 0.13, η2= 0.05) nor contact time to the odor source (F1,41= 0.19, p= 0.66, η2= 0.005) between

329

the first and second parts of the testing trial. Means of different behavioral activities of male and

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female Smith’s zokors (Eospalax smithii) during the first and second 10 minutes of the testing

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session under the conditioned context were presented in Supplementary table (1)

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3.4. Comparison of gender behavioral responses between the different contexts

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We also conducted separate repeated measures analysis (ANOVA) on the different

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behavioral responses to gauge the different reactions of each gender separately between the

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novel, predator and conditioned contexts. Male zokors significantly (F2,42= 8.62, p= 0.001, η2=

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0.29) hid more in the conditioned context (Fig. 1- A) than they did in the predator and novel ones,

337

while females did not show significant changes in the proportion of hiding time between the

338

different contexts (F2,42= 0.22, p= 0.80, η2= 0.01). Conversely, males significantly (F2,41= 8.22,

339

p= 0.001, η2= 0.28) spent less time out of cover in the conditioned context than they did in the

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predator and novel ones (Fig. 1-C) while females did not show significant changes in the

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proportion of time outside the hide box between different contexts. Neither males (F2,42= 0.12,

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p= 0.79, η2= 0.006) nor females (F2,42= 0.14, p= 0.86, η2= 0.007) showed any significant changes

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in the proportion time for head-out between the different contexts (Fig. 1-B). Males tended to

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significantly modify their locomotion activities (F2,42= 12.14, p< 0.0001, η2= 0.37) between

345

different contexts that they showed the lowest locomotor activities during exposure to the

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conditioned context while females did not significantly alter their locomotor activities (F2,42=

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0.23, p= 0.80, η2= 0.01) between different contexts (Fig. 1-D). Both males (F2,42= 4.41, p= 0.02,

348

η2= 0.17) and females (F2,42= 3.70, p= 0.03, η2= 0.16) displayed the lowest durations of time to

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contact the source of the stimulus when exposed to the conditioned context than they did in the

350

predator or novel ones (Fig. 1-E). Finally, the latency to leave the hide box for the first time

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significantly vary between different contexts in males (F2,42= 2.29, p= 0.14, η2= 0.01) that they

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showed the higher latency when exposed to the conditioned context (Fig. 1-F) while females did

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not significantly (F2,42= 1.08, p= 0.34, η2= 0.05) change the latency when exposed to different

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contexts.

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4. Discussion

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To investigate sex differences in Smith's zokors (Eospalax smithii), a subterranean rodent

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species, under risky environment, we used the predator-odor fear conditioning paradigm to

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measure the behavioral responses when the context is novel (Low risk), when a predator odors

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was presented (High risk) and finally the association between the predator odor context and the

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conditioned context (Fear-Conditioning) when the predator odor was removed in the next day.

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Our findings that males displayed more exploration, spend more time in locomotion and hid less

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than females during exposure to the novel context support the previous claims that males showed

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more “boldness” and were more risk-prone than females. Our results are consistent with those

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reported by (King et al. 2013; Anchan et al. 2014) using a range of species; however, other

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studies show that males when tested in a novel context are more-risk aversive and less

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exploratory than females (Johnston and File 1991; Kokras and Dalla 2014). Several aspects

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might be regarded to explain these contrasting findings and interpretation of such studies. Firstly,

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the nature of the testing apparatus might reflect different states of emotionality and risk

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perception between the different testing conditions. Rodents have a natural response to risk by

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hiding or escaping (Kavaliers and Choleris 2001), however, previous studies using the novel

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contexts without offering a hiding place are more likely to measure the “Compulsory

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exploration”, while in paradigms (Similar to our study) the motivational state of exploration

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would be termed “Voluntary exploration” where the animal would have the opportunity to either

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hide or explore the testing arena. Furthermore, gender differences are not always detected in a

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consistent direction when testing subjects even within the same behavioral paradigm of anxiety

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(Meng and Drugan 1993). For example, female Lister hooded rats showed less social

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interaction than males, which denotes higher levels of anxiety, and in the same test, displayed a

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higher level of exploration and ambulation that reflects a low anxiety level (Johnston and File

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1991). Finally, these sexually dimorphic behavioral patterns in during exposure to the novel

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context might suggest variability in life-history strategies between male and female zokors.

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Zokors are solitary subterranean rodents, and males during the breeding season are more active

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and maintain larger home ranges than those of females to seek a mate. In nature, zokors are free-

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roaming individuals who occupy a large home range of 1790 ± 720 m2 for males and 260 ± 112

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m2 for females (Zhang, 2007). Therefore, taken into considerations that animals had been

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captured during the breeding season, these huge differences in home-range sizes may elicit more

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exploration and less hiding in males that emulate gender difference in the motivational states

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between males and females when they explored novel areas. Also, close inspection and analysis

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of the zokors’ activity patterns during the novel context exposure session showed clear gender

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differences in the behavioral responses between the first and second parts of the trial. Males

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tended to display more exploratory behaviors and hid less than females during the first half of the

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trial which support our previous assumption that during exposure to risk, males tended to be

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more risk-prone while females show more risk-aversive reactions (Anchan et al. 2014).

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Based on the finding that males took more risk in the predator odor and conditioned

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contexts, we can clearly imply that female zokors did not follow the classical anti-predator

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defensive responses (Decreasing time out of cover and shelter-seeking behaviors) as commonly

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observed not only in the above-ground rodents (Bramley and Waas 2001; Staples 2010;

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Ferrero et al. 2011; Pérez-Gómez et al. 2015), but also compared to their male conspecifics

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which had been tested under the same testing conditions. This finding suggests that males zokors

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have clearer anti-predatory and stronger fear conditioning avoidance behavioral responses than

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females. Several aspects of the life-history strategies would be discussed to explain our results.

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The nature of zokors as solitary subterranean rodent that live in an intricate underground

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burrow system mandates dispersal of individuals to new locations rather than staying in their

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natal home range. Male-biased dispersal had been documented in zokors that males maximize

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reproductive success by mating with multiple females and should disperse for both social

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(competition for mates from dominant older males) and genetic (inbreeding avoidance) reasons.

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However, the pattern of dispersal, if occur underground, would result in more energetically

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highly demanding ambulatory and digging activities that would eventually incur a heavy energy

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expenditure (Vleck 1979); thus, males choose the aboveground route for dispersal, because as

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previously reported in (Zhang 2007), “The energetic costs of a potential dispersal through the

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soil appear to be impossibly high”. Animals can learn about dangerous stimuli when they

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directly interact with them. In the wild, young zokors spend the first weeks of life (50 days,

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Zhang et al. 1995) in an enclosed nesting chamber. When they emerge from their natal burrows

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for dispersal, they are exposed to predators that pose significant threats or at least exposed to

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various predatory cues. As mentioned earlier, males might be the more vulnerable sex to the

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predatory risk due to their predominant above-ground dispersion pattern. Therefore, this former

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experience, particularly in young or immature individuals, due to the above-ground trips and the

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earlier acquisition of information regarding the predators’ cues may systematically influence

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male zokors through consolidating of a strong recognition memory especially during the young

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ages which help them recalling their previous dangerous confrontations when they exposed to

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the same threatening stimulus repeatedly during their lifetime even during their adulthood

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(Wiedenmayer 2009), learning about properties of the situation and adjusting its behavior in

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successive encounters. This experience in early ontogeny might shape the expression of anti-

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predator defensive responses in male zokors to mimic those previously reported in the above-

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ground taxa, while females did not encounter the same experience due to the fewer or non-

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aboveground trips compared to males. We hereby cannot assume that female zokors did not

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recognize and identify the predator odor as the anti-predator responses are innate even the prey

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had no prior experience to predation cues (Apfelbach et al. 2005), but the risk of exploring a

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novel area, due to lack of past experience, might provoke an equal perception of threat for

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females as a predator encounter might do. Therefore, females may prefer to launch another tactic

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across the different contexts that rely on being more stationary and less reactive to the changing

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contexts. This is clear in the behavioral response of females in the conditioned context to the

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odor source where they showed stronger aversion and less contact time to the clean towel than

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they did in the novel and predator contexts before.

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Several Studies have demonstrated sex differences in responses to predation risk

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(Blanchard et al. 1990; Harris et al. 2010) and learning performance (Jonasson 2005; van den

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Bos et al. 2013) separately, nevertheless the potential of the predator odor fear-conditioning

437

paradigm involves the use of a natural unconditioned stimulus. Our results clearly revealed that

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male zokors are more risk-prone and depending more on their former experiences, while females

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being more sensitive to any environmental changes. The evolution of gender differences in both

440

parental and reproductive investments in zokors may explain the differences in the anti-predator

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and learning responses. Within the breeding season, males are more inclined to indulge in

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territorial activities and competing endeavors over access to burrows and females (Zhang 2007),

443

whereas females may invest more time and activities in reproductive and parental care make

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them valuable resource which ensures the survival of their offspring. Our results are parallel with

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the asset protection model that predicts that an individual’s past successes at accruing fitness will

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shape its response to current predation threats (Katwaroo-Andersen et al. 2016). Consequently,

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the higher costs and possibly increased predation risk that comes with the higher reproductive

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and maternal investments of females, especially during the breeding period, may make hiding

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and decreased exploratory responses more appropriate to female zokors ensure their safety. Thus,

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prey may modify their behavioral decisions based on both the perceived intensity of acute risk

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and/or the resulting fitness expectations. Similar results were also found in other species in

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which females have a higher reproductive and maternal investment. In Spanish terrapins

453

(Mauremys leprosa), gravid females took longer to emerge from their shells following mimicked

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predator risk, and females with larger clutches took longer to emerge than those with smaller

455

clutches (Ibáñez et al. 2015). Conversely, in Pipefish (Roelke and Sogard 1993), when females

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invest less in parental care and reproductive fitness, they showed strong risk-taking behavioral

457

responses than males which invest more time in broodiness than females.

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Typical odor sources are skin, fur, urine, droppings and secretions of the anal glands are

459

commonly used in different studies (Tidhar et al. 2007; Hacquemand et al. 2010; Sievert and

460

Laska 2016; Yin et al. 2017), and fur/skin source is considered the most potent stimulus

461

providing a strong indication that a predator is nearby now and able to perform a successful

462

strike (Hegab et al. 2015). Therefore, we cannot assume that the odor source was not effective to

463

induce fear-related behavioral responses in zokors since males showed strong anti-predator

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behavioral changes when tested in the predator context and successfully formed an effective risk-

465

conditioned association when the predator odor has been removed. However, our experimental

466

set-up may have contributed to the absence of behavioral response in female zokors. females live

467

in deeper burrows (2–2.5 m depth) than males (0.8–1.5 m) (Shao et al., 2015), and never or

468

rarely leave their nests neither for dispersal nor for foraging as they forge completely

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underground (Zhang 2007). Accordingly, it can be argued that the hide box in the defensive

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withdrawal apparatus may well have presented the same perception of shelter as do the tunnel

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system for females in the wild. Therefore, the hide box, which was found fit testing multiple

472

species (Dielenberg and McGregor 1999; Eilam et al. 1999, Hegab et al. 2014 a,b,c), may be

473

inappropriate for when investigating the risk-perception and decision making under predation

474

threat in female zokors, although it was appropriate when testing males. Furthermore, in an

475

elegant study conducted by Eilam et al. (1999), common spiny mouse (Acomys cahirinus) did

476

not display show any behavioral changes after exposure to owl calls despite increased cortisol

477

levels which are indicative of stress. Indeed, predator odors may elicit various anti-predator

478

responses including behavioral, hormonal and molecular changes in defensive-related brain

479

regions (Apfelbach et al. 2005; Kondoh et al. 2016). The lack of the behavioral changes in

480

female zokors between the different context may be systematically accompanied by hormonal

481

and/or gene expression as previously reported in Eilam et al. (1999). Further studies with a

482

different apparatus and experimental procedure concurrently with analysis of the hormonal

483

profiles might be required to unravel the lack of behavioral response in female zokors.

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Although we used a behavioral testing paradigm that has been extensively used in studies

485

on rodents’ defensive behavior, the results obtained in the current study were somewhat different

486

from those obtained either from common laboratory (Ferrero et al. 2011; Sievert and Laska

487

2016)] or laboratory-bred wild species (Yin et al. 2011; Hegab et al. 2014 a,b,c; Yin et al.

488

2017). First, the levels of head-out behavior did not significantly differ between the different

489

contexts. Head-out is behavioral response aims to collect information regarding the surrounding

490

environment before departing the safe shelter (Dielenberg and McGregor 2001). However, it

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may be considered more risky strategy for zokors than staying concealed completely, especially

492

in the high-risk context of predator odor, which suggests that a predator may be nearby (Jolles et

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al. 2015). Second, zokors did not show aversion to the predator odor source, although both

494

genders showed strong aversion to the neutral odor source in the conditioned context. Similar

495

results were also found in a recent study conducted on wild Norway rats (Rattus norvegicus),

496

where wild rats did not avoid boxes scented with predator odor (Stryjek et al. 2018). This might

497

denote that laboratory-bred individuals in comparable studies may not display similar defensive

498

behavioral responses under risky situations as their wild counterparts will do.

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In conclusion, male and female zokors adopt distinct behavioral strategies when

500

confronted with novel, predator and conditioned contexts where males showed different

501

behavioral responses across contexts, but females did not vary their responses between different

502

degrees of risk. These variations in risk-taking behaviors may arise from gender differences in

503

information gathering and decision-making processes due to the selective pressure of the

504

underground ecotope and life-history traits. A further investigation of the link between

505

behavioral and neuro-endocrinological responses toward predation risk is needed for better

506

illustration of the evolution of defensive anti-predatory tactics in male and female Smith’s zokors.

507

Acknowledgements

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Funding for this work was supported by the Special funds for discipline construction

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of Gansu Agricultural University (GAU-XKJS-2018-003); National Natural Science Foundation

510

of China (Nos. 31460566 and 31760706); Gansu Provincial Natural Science Foundation for

511

Distinguished Young Scholars of China (No. 1606RJDA314); “Fuxi Talent” Plan of Gansu

512

Agricultural University (Gaufx-02J03), Longyuan Youth Innovation and Entrepreneurship

513

Talent Project, the Project of the Innovation Team on Gansu Grassland and Animal Husbandry

514

Sustainable Development (2017C-11) and Talented Young Scientists fellowship (TYSP) of the

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Ministry of Science. The authors would like to thank Professor Shangli Shi (Dean of School of

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Grassland Sciences, Gansu Agricultural University) and the Department of Hygiene, Zoonoses

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and Animal Behavior and Management, Faculty of Veterinary Medicine, Suez Canal University

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for their moral and career support. The authors declare they have no competing interests.

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Supplementary table (1) Means ± SE of different behavioral activities of male and female

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Figure (1) The proportion of time spent in the hide box (A), proportion of time spent in head out

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(B), proportion of time spent out of cover (C), the mean duration of locomotion (D), mean

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exposure of male (n=22; triangles, dashed line) and female (n=21; circle, solid line) Smith’s

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zokors to the novel, predator and conditioned contexts. * Represents gender differences within

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context at p ≤ 0.05. Means with different superscripts for each gender are statistically significant

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between contexts at p ≤ 0.05.

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Highlights • Male and female Smith’s zokors showed different risk-taking responses under different contexts.

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• Males trended to explore the contexts and collect information more than females.

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• Males are more risk-prone while females are risk-aversive.