Hastings Natural History Reservation

30 Aug, 02 AM - 19 Dec, 12 AM

Individuals often need to alter allocation of time and energy to different behaviors. In response to changing social conditions this may need to occur rapidly, especially in a biparental species with the competing needs of a mate and offspring versus maintenance of an exclusive territory. We will examine a simple mechanism in a territorial and monogamous species that is expected to alter time and energy allocation within a territory: rapid and transient release of testosterone (T). While T manipulations via long-lasting implants have been used under field conditions in birds and lizards to mimic altered baseline levels, transient increases in T have not. In many species, T increases in response to both male-male and male-female interactions, but it is unknown how transient pulses of T function differently between the two social contexts or whether there is one similar function (Gleason et al. 2009). We suggest that the temporal patterns and context of T release are critical: T has rewarding effects that induce conditioned place preferences (CPPs). The adaptive function of CPPs has not been tested in the field or lab, despite being a well-established paradigm used for studying addiction and reward. Our central hypothesis is that one similar function of T within the different social contexts is to induce a CPP in a territorial location and alter social interactions related to that location. The expectation is that changes in both spacing and social behavior will occur in our model system, the monogamous and territorial California mouse (Peromyscus californicus). On a spatial level, there are competing demands for associating with the mate and the pups in the nest versus associating with intruding males and maintaining an exclusive territory. On a behavioral level, communication via mechanisms such as ultrasonic vocalizations can be altered within the bonded pair versus between males at territorial boundaries. We predict that T-injections administered to males near the nest will increase time at the nest and alter the number and characteristics of USVs to the female whereas T-injections administered near the territorial boundaries with a neighboring male will increase time patrolling the boundaries and the number and characteristics of USVs towards males near these boundaries. Rationale: At our established research site at the Hastings Natural History Reserve (HNHR), single- and multi-syllable ultrasonic vocalizations (USVs) in male-male and male-female interactions have been studied (Kalcounis-Rueppell 2006, 2010, Carney 2009, Briggs 2009, Petric 2010). To test our hypothesis, that one similar function of T within the different social contexts is to induce a CPP in specific areas of the territory and alter social interactions related to that location, we will trap resident males and administer T or sham injections. When the T pulse occurs near the nest, we expect the pair bond to be stronger because males with higher T spend more time in the nest with the female and maintain a closer proximity to the female in the lab (Gleason & Marler unpub. data). Moreover, T levels and paternal behavior are positively associated (Trainor & Marler 2001, 2002). Our collaboration also reveals increased use of USVs in response to T manipulations by both sexes and possibly duetting (Pultorak, Kalcounis-Rueppell & Marler, unpub. data). We expect that a stronger pair bond will be expressed by increased USVs by the pair and closer spatial proximity. Males also release T in response to encounters with other males (i.e. territorial boundary). We expect that a male experiencing T pulses at the territory edges will spend more time patrolling and calling at the boundaries and less with the female and pups. The altered male focus could have significant negative repercussions for both females and pups. 3 Why EAGER funding? We view this proposal as high risk, but exciting. It is multi-faceted with respect to techniques and approaches: telemetry allows individual and interaction identification, infrared techniques allow observation of social interactions, and recording arrays allow measurements of USVs (used by Kalcounis-Rueppell). The risk arises from integration of this remote sensing approach with transient increase in T administered to males trapped in different locations. These measurements/manipulations will be conducted and coordinated in the field. The risk and creativity also lies in the integration of a naturalistic approach by the Kalcounis-Rueppell lab and an experimental approach by the Marler lab. This unique collaboration will move the field forward by revealing new concepts about hormone-behavior interactions, their relation to CPPs, as well as the balance and coordination of behaviors within a bonded pair in the wild. We will create a new niche for integrating different research approaches to extend to questions of sexual selection, ecology of space use, exposure to predators, behavioral plasticity in space use, alterations of territorial size, and observations of the dynamics and compatibility of pair-bonded individuals. We expect to make major advances in behavioral field studies of small, nocturnal mammals and identify long term functions of transient increases in T on male allocation of time and energy towards the different functions of raising young and defending a territory. This in turn is predicted to influence individual spacing under field conditions. Such results will positively impact our understanding of the physiological mechanisms contributing to enduring social patterns within a species. Specific Aim 1: In the field, transient increases in T influence USVs and increase site specificity based on the sexual and aggressive contexts associated with that location. Prediction 1A: T-injections administered to the male when he is adjacent to the nest will increase time spent associated with the nest and change the number and characteristics of USVs to the female. These males are expected to spend less time patrolling their territories. Prediction 1B: T-injections administered near the territorial boundaries with a neighboring male will increase patrolling near these boundaries and the number and characteristics of USVs towards individuals near his territory boundary. These males are expected to spend less time associated with their mates and pups. Expected Significance: In order to secure resources and respond appropriately to a mate and/or offspring, individuals need to balance time and energy allocation. Rapid and transient changes in T are expected to contribute to the ability to respond quickly to these competing and changing demands. This contrasts with the use of silastic implants in the field to produce long-term changes in hormones to mimic altered baseline hormone levels (e.g. Marler & Moore 1988; Wingfield et al. 1990; Ketterson et al. 1992). Rapid and transient changes in T have not been used under field conditions, despite many studies measuring rapid changes in T in response to competition (Hirschenhauser et al. 2003). Our approach provides a new perspective on mechanisms influencing re-allocation of male effort in response to changing social conditions and the effect on the family unit. The integration of techniques and the resulting outcomes are expected to fundamentally change how we assess rapid changes in behavior. 4 Functions of T increases in male-male and male-female interactions across species: Male-Male: Even though it is widely documented that male encounters can induce T elevations in response to challenges from other males (e.g. Hirschenhauser et al. 2003; Wingfield et al. 1990; Challenge Hypothesis), the function of these increases remain unclear (Gleason et al. 2009). Numerous rodent studies show that T has rewarding properties (Dimeo & Wood 2006) and elicits a conditioned place preference (CPP) (Alexander et al. 1994; Wood et al. 2004). Moreover, androgen-induced CPPs are blocked by dopamine receptor agonists (Packard et al. 1998), suggesting that T activates dopamine receptors to create a reward and induce a CPP. Individuals can form CPPs for locations in which they have previously won fights (Farrell & Wilczynski 2006; Martinez et al. 1995; Meisel & Joppa 1994). This research indicates that T pulses following a fight might induce a CPP for the encounter location (Marler et al. 2005; Gleason et al. 2009) and influence territoriality by adjusting site preferences. This would also provide interesting insight into the adaptive function of the ?Challenge Effect?. Male-Female: Questioning the function of T pulses across male-male and male-female contexts arose because of the striking similarity between the T response after male?male encounters, such as P. californicus, and male?female encounters in Mus musculus (Gleason et al. 2009), but the functions of these T surges are not entirely understood. In contrast to the study of male?male encounters, T pulses in sexual contexts have been primarily examined in house mice and rats. Males respond to female stimuli with a rise in plasma T (Clancy et al. 1988; Coquelin & Bronson 1980; Macrides et al. 1975; Marunick & Bronson 1976). The T reflex does not rely on previous sexual experience (Clancy et al. 1988), but can be triggered by a female?s scent (Marunick & Bronson 1976). This phenomenon is observed across species, and likely has an adaptive function (Nyby 2008). Sexually-stimulated T release occurs in naive males, but is also shaped by experience; while female urine elicits LH response in sexually na?ve male mice, the response is more robust when animals are sexually experienced (Clancy et al 1988) as in rams (Borg et al. 1992). Classical conditioning also impacts rapid T release in response to pairing with sexually receptive females and the scent of wintergreen oil (Graham & Desjardins 1980). One gap is the integration of T response to female stimuli and location preference; male rodents permitted access to an estrous female consistently show a preference for the chamber in which mating occurred (Camacho et al. 2004; Hughes et al. 1990; Mehara & Baum 1990; Miller & Baum 1987). Likewise, peripheral injections of T also induce CPPs (Alexander et al. 1994; Rosselini et al. 2001), leading to our hypothesis that it is the T release that induces this location preference. Within sexual contexts, relatively low circulating T maintains sexual behavior, again calling into question why rapid T increases are commonly observed if not required for mating, especially when T pulses in response to females vary depending on mouse strain without an effect on ability to mate (James et al. 2006). The rapid rise in T may be involved in courtship interactions that may be more apparent in a monogamous species in which the assessment period prior to mating is more prolonged. There are, however, intriguing hints that the ability of a male to mount a rapid rise in T is not simply a response to a social stimulus, but also a reflection of male quality (McGlothin et al. 2008). Additional work needs to tease apart the functions of T pulses at a behavioral, physiological and neural level, but the similarity of rapid T response in differing social contexts presents an exciting new research direction. 5 Function of USVs and T: USVs are an important component of multimodal communication in rodents (Costantini & D'Amato 2006; Geyer & Barfield 1979; Hahn & Thornton 2005; Portfors 2007; Sales 1999). Rodent USVs are structured signals that impart meaning and cause predictable behavioral responses in recipients (Brudzynski 2005). For mice, in particular, USVs are associated with social interactions (Costantini & D'Amato 2006; Portfors 2007; Musolf et al. 2010; Hammerschmidt & Fisher 2009). Depending on the context, USVs in same-sex encounters mediate aggression, establish social relations and dominance hierarchies (Gourbal et al. 2004; Nyby et al. 1976; Sales 1972a; Wysocki et al. 1982), and facilitate sex and individual recognition (D'Amato 1997; D'Amato & Moles 2001; Wysocki et al. 1982, Musolf et al 2010). Males produce USVs during courtship and mating interactions (Barthelemy et al. 2004; Holy & Guo 2005; Liu et al. 2003; Nunez et al. 1985; Nyby 1983; Pomerantz & Clemens 1981; Pomerantz et al. 1983; Sales 1972b; Sales 1999; Musolf et al. 2010; Hammerschmidt & Fisher 2009). Our preliminary data show that both sexes of P. californicus produce USVs during male-female encounters. Furthermore, there is individual variation in USVs (Mus: (Holy & Guo 2005; Musolf et al 2010); Peromyscus: (Briggs 2009, Petric 2010) that may reflect individual quality. Besides social context, USVs are also influenced by T. In house mice, deer mice (Pomerantz et al. 1983) and male Alston?s singing mice (Pasch et al. 2011), T increases the frequency of USV songs. In house mice, T release after exposure to a novel female is accompanied by USVs (Sales 1972b; Nyby 1983, 2001; James 2005). In P. californicus, T increases the frequency of USVs in males with a similar pattern in the responding females (Pultorak, Kalcounis Rueppell & Marler, unpub data). The monogamous California mouse: P. californicus, a strictly monogamous species, displays high levels of territoriality and paternal care (e.g. Dewsbury 1983; Gubernick & Alberts 1987; Ribble & Salvioni 1990; Gubernick & Nordby 1993; Bester-Meredith et al. 1999; Bester-Meredith & Marler 2001, 2003a,b, 2007; Trainor & Marler 2001, 2002). Males maintain exclusive territories with no extra-pair fertilizations (Ribble & Salvioni 1990; Ribble 1990, 1991). When presented with the opportunity to mate with a novel individual, the majority of animals do not (Gubernick & Nordby 1993). In the field and lab, male care enhances offspring survival (Gubernick & Teferi 2000, Cantoni & Brown 1997; Gubernick et al. 1993). Both sexes produce USVs and USVs are a major component of the behavioral repertoire at the nest and within the territory (Kalcounis-R?ppell et al. 2010, In Prep, Briggs 2009). Field Site: HNHR is a primary location for field studies with P. californicus (Ribble & Salvioni 1990; Ribble 1991, 1992; Gubernick & Teferi 2000; Kalcounis-R?ppell & Millar 2002; Kalcounis-R?ppell et al. 2006, 2010) in the foothills of the Santa Lucia mountains (CA) where they breed from November-April (McCabe & Blanchard, 1950). There are 3 long-term live-trapping grids for P. californicus extensively used by Kalcounis-R?ppell (Kalcounis-R?ppell & Millar 2002; Kalcounis-R?ppell et al. 2006; Briggs 2009). Mice have been live-trapped and radio-collared since the 1980s; this is not expected to induce stress compared to unmanipulated mice as they do not differ from other populations with respect to foraging bout rate, timing of reproduction, litter number/size, or dispersal behavior. Field metabolic rate (FMR) was measured using injections of doubly-labeled water on our study grid and there was a high tolerance for injection, as FMR was as predicted based on allometric relationships (Kalcounis-R?ppell 2000; Kalcounis-R?ppell & Ribble 2007; Kalcounis-R?ppell 2007). Recapture rates within 48hrs for injected mice were similar to unmanipulated mice (Kalcounis-R?ppell unpub.). 6 We have a high success rate of recapturing mice to retrieve transmitters, and residents wear transmitters several times without moving (Kalcounis-R?ppell 2000; Briggs 2009; Petric 2010). In addition, carrying a mouse-style transmitter does not influence FMR in rodents (Berteaux et al. 1996). Lastly, we have little movement of resident male P. californicus, regardless of manipulations. Overall this is an ideal species and location for our studies. Specific Aim 1: In the field, transient increases in T influence USVs and increase site specificity based on the sexual and aggressive contexts associated with that location. Introduction: We will manipulate transient T levels of males in different territorial locations to test if we can alter a male?s spatial movements through CPPs induced by T. We predict (1A) that T-injections administered to a male when he is near the nest will increase time spent associated with the nest and change the number and characteristics of USVs to the female as well as decrease the amount of time patrolling his territory. In contrast, we predict (1B) that T-injections administered near the territorial boundaries with a neighboring male will increase patrolling by the boundaries and the number of USVs towards individuals near his territory boundary. These males are expected to spend less time with their mates and pups. We will use well-established research sites (HNHR) with marked and well-known resident males. We will administer T or sham injections, release individuals, and record behaviors. We will determine locations in which males will be injected and observe responses to injections using radio-telemetry, territory mapping, live-trapping, acoustic recording, and where possible thermal imaging. We expect to identify alterations in a male?s spatial patterns and acoustic behavior such that the release in T conditions a male to focus on the location/social context in which he received the manipulation. We expect to achieve this through the experiment detailed below. Experimental Design: For T manipulations we will use grids historically used to study P. californicus (See Stromberg HNHR Letter). During Sept-Oct 2011 a live trapping program will be initiated with 1 Sherman trap at each station on the grids (10X10m array). Traps are baited with rolled oats and set 2X weekly to collect demographic data using standard mark/recapture techniques. All mice are ear tagged, sexed, aged (juvenile, sub-adult, adult), weighed, and examined for reproductive condition. By continuously live-trapping established geo-referenced grids, we determine phenology, demography, and residency. Residents are mice captured more than 3Xs with at least 7-d between the last 2 captures (Kalcounis-Rueppell & Millar 2002, Kalcounis-Rueppell et al. 1010). Trapping data is entered into a spatially explicit database and mapped daily into Arcview GIS. Kernel and minimum convex polygon territory size estimates are calculated using Animal Movement SE v 2.04. Grids are trapped 9Xs to determine residency. When >95% of captures are tagged (after ~ 9 trappings), we select focal pairs and fit them with custom built 0.55g M1450 Peromyscus mouse collar transmitters from Advanced Telemetry Systems (ATS). We use hand-held telemetry to find the nest site in the day and map out territory boundaries at night (bi-angulating fixes of individuals from the grid at least 3 Xs/ night for 7 nights). Geo-referenced telemetry fixes are added to establish territory boundaries from trapping data. We will then initiate the experiments and remote sensing for behaviors. Remote Sensing Observations in Territories: Our remote sensing method integrates sound recording, thermal imagery, and radio-telemetry in space and time to determine which individuals produce USVs and their location. For a given pair, we will measure the following after an injection (T or sham). We set up a microphone array (6-12 mics based on territory 7 shape) to record and localize full spectrum sound (sonic, ultrasonic, harmonics; 10-250kHz; Avisoft Ultrasound GateSystem). The array records all sounds produced within the array and localizes USVs using time-delay-of-arrival differences of the same sound at different microphones. This records USVs and location of production (nest or boundary). Members of the pair are outfitted with radio transmitters to determine who produced the USV. To remotely detect the location of radio-collared mice, we attach 4 small antennae (Sigflex 15 cm omni-directional) to a central receiver (4 MHz R4000), antenna switch box, and data logger (DSU D50410; all from ATS) to search continuously for both frequencies of the pair. 3 antennae will be positioned at the edge of a territory and one at the nest. If a frequency is detected at any antennae, the receiver will record signal strength at all 4 antennae and move to the next frequency. To determine transmitter position based on signal strength, we make an array within the receiver space to produce a reference validation grid upon which to compare signal strength data from radio-collared mice, and identify the caller. To visualize social interactions, we use a thermal imaging camera (Photon 320 14.25 mm; Flir/Core By Indigo) where possible (limited by number of camera relative to number of pairs of mice and canopy structure). The camera is suspended ~10 m above the focal area using a pulley system set in the canopy. The camera feeds images directly (via A/V cabling) to a ground-based 30GB hard disk digital video recorder (JVC Everio DVR) that records the entire night in real time. All equipment (cameras, telemetry components, microphone array, computer) is powered by inverters and 12 V 33AMP batteries. We will follow 2 pairs at a time. We successfully used this method to record USVs from known mice at HNHR (see methods in Kalcounis- R?ppell 2010). T-manipulation: Nest sites and boundaries will be saturated with traps 5m around the nest (4 traps in a circular orientation) and 5 m on either side of the territorial boundary as defined in the program Animal Movement (traps every 5m on the boundary on either side). At least 10 resident males will be identified and receive 3 injections over 6-d with at least 1-d between injections. Males will be randomly assigned to receive (1) 3 T-injections within 5m of the nest (Nest Group), (2) 3 T-injections within 5m of a boundary with a neighboring male (Boundary Group) or (3) 3 vehicle injections (Control Group). Random assignment will be constrained so that no T-injected males are in neighboring territories. Males receiving T-injections every other day display cumulative changes in ability to win (Trainor et al. 2004; Gleason et al. 2009; Fuxjager et al. 2009, 2010a,b; Fuxjager & Marler 2010). Traps will be checked 1x/hr when a male is assigned to be injected. Behaviors and USVs will be recorded for 3-d after the last injection. Statistical Analyses. Dependant variables (based on behaviors we have observed in the lab and field): Ratio of time spent near nest (within 50% core home range)/territory boundary (50% outer home range), time spent within 1 m of stranger, time spent within 1 m of partner, time spent chasing/fighting, rate of USV production, spectral and temporal parameters of USVs as described in Kalcounis-Rueppell et al (2010). Independent variable will be the categorical T manipulation (close to nest, close to boundary, or control). All data will be examined for violations of parametric statistics and transformed or non-parametric statistics will be used. Standard statistical packages will be used. The influence of T manipulation on behaviors and USVs will be examined using ANOVA approaches. Expected Outcome: We expect that the social context/location will impact a male?s behavioral responses to T-injections. This would reveal a new role for T as a hormone 8 underlying the ability to rapidly adapt to changing environments through allocation of time to behaviors that can contribute to reproductive success. We have data suggesting that the development of the winner effect is weaker when fewer resources are available, therefore our expected outcome could be extended to resource availability in the territory if linked with T release. The induction of site specificity from T-injections would be a significant finding for a large variety of species and extend our understanding of the function of the ?Challenge Effect?. Potential Problems and Alternatives: The two T-injection groups will be combined if no differences are found based on location at time of injection. We could still investigate a variety of other variables including changes in social interactions, spacing with neighbors and exposure to visual predators. It can also be determined if males leave their territories in response to the manipulations. If the T-injections alone do not induce strong enough CPPs, then these will be combined with behavioral experiences through playbacks of conspecific males.

Visit #26765 @Hastings Natural History Reservation

Approved

Under Project # 24480 | Research

Social Context and Functions of Testosterone Pulses in Wild California Mice

faculty - University of North Carolina

Reservation Members(s)

Mary Timonin Aug 30 - Dec 18, 2012 (111 days)
Tracy Burkhard Aug 31 - Dec 19, 2012 (111 days)

Reserve Resources(s) | Create Invoice

Red House #2 1 Jan 3 - Mar 2, 2013
Red House #2 1 Aug 30 - Dec 18, 2012
Red House #2 1 Aug 31 - Dec 19, 2012