MICHlG \W W 1th WW 3 1293000 3726 fl IIT““"L A 2"; Heb-Sada?“ Michigan Side La"- New"? 4* “‘1i This is to certify that the dissertation entitled REPRODUCTIVE INHIBITION IN YOUNG FEMALE PEROMYSCUS LEUCOPUS: ANALYSIS OF MECHANISMS AND EFFECTS presented by Gale R. Haigh has been accepted towards fulfillment of the requirements for PH.D. degree in Zoology @(JMQW Major professor Date October 14, 1986 MSU i: an Waive Action/Equal Opportunity Institution 0-12771 )V1531_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from .—r_—. your record. FINES will be charged if book is returned after the date stamped below. {Ti ‘35 7 WM INHIBITION OF YOUm FEMALE PERGGYSCUS LEIEOPUS: ANALYSIS OF MECHANISG AND EFFECTS Gale R. Haigh A DISSERI‘ATIN Submitted to Michigan.$tate university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1986 ABSTRACT WM INHIBITION OF YOUNG FEMALE PERIMYSCUS LEUCOPUS: ANALYSIS OF mans AND arms by Gale R. Haigh The first hypothesis tested was that young fanale white-footed mice (W M) were reproductively inhibited by the presence of an adult female. Reproduction of groups containing an adult male and 21 day old f‘le were cmpared to pairs of mice with an adult fmle present. Both treatment groups were observed until the young females were 150 days old. Yomg f‘le white-footed mice were prevented from reproducing by the presence of an adult female conspecific. Recovery of reproductive function was possible after removal of the adult female. Additional tests characterizing the inhibitory stimuli revealed that olfactory exposure in the absence of physical or chanical contact is sufficient to inhibit young female mice from reproducing. Exposure times of as little as three hours per day proved sufficient to prevent reproduction. A chemical constituant of the urine from adult females was found to be the source of the inhibitory stimuli. Stimulus fran adult female gem maniculatus has no effect on young female 13. m or visa versa indicating that the stimulus is species-specific. The physiological effects of inhibition were contradictory to my initial hypothesis that inhibition was an extended maturational delay. Inhibited females have normal estrous cycles, number of corpora lutea and ovarian weights. Sperm present in the reproductive tracts of a number of inhibited females revealed that copulation occurred and that inhibition was therefore a postcopulatory effect. Behavioral 1y, inhibited females showed a preference for adult male odors Just as their control couterparts did in 5-minute y—naze trials. However in both the W trials and 24 hour nestbox selection experimt, inhibited fules avoided the odors of adult fules conspecifics. Juvenile females who are susceptible to the influence of the pheranone also avoided adult female odors when tested against either water or adult male odors. Evaluation of the pwsible effects of inhibition on population dynamics and the selection process that brought it to near fimtion in thethreespeciessofarstudiedismade. Themostparsimoniuos explanation is that this phmomenon evolved as a result of selection pressure on adult females to reduce the reproductive success of their counterparts to increase their own relative reproductive success. In This text is dedicated tomywife Marcia, whose support and strengthmadethisstudypossibleandtonr. JohnA.King, whohad anighfaitharxitmderstarxiingtogivemeachanceinthebegiming. ii WW I thankDr. JohnA. Kingforhisguidanceandassistance throughout my graduate education. The time and effort of my cannittee members, Drs. Martin Balaban, John Gill, and Donald Straney, are greatful 1y acknowledged. Robert Haigh provided his time and technical expertise in the design and construction of the behavioral testing apparatus. Dr. Bruce Cushing, my co-graduate student and cfficemate, was very helpful in his ccmments and criticisms during the preparation of this study, and with his moral support during our years at Michigan State. To the many undergraduates who assisted during various portions of this study: Tobey Gordon, Barbara Jo Hoppe, Patricia Byrnes,‘ Laurie Vargo, Mary Jo Burghard, Jeanne Corbett, Linda Hagerman, and in particular to Dawn Lamsmryandxathi Cowlinggomydeepest thanksandbestwishes. This study was supported by a Diwertation Inprovanent Grant from the National Science Foundation (Grant Number BNS-B3-14017) . To my wife, Marcia l-laigh who endured late nights, strange dinner conversations. and at times a short-tempered and less than perfect husband my deepest gratitmie and thanks. This study and my graduate education could never been completed without her. iii TABIEOFCONTENI‘S Page LIST OF TABLES ...... . v LIST OF FIGURES .......................... vii INTRODIETION . . . . ............... 1 CHAPTER 1 LITERATURE REVIEW ..... . .............. 6 CHAPTER 2 EVIDENCE FOR REPRODUCTIVE INHIBITION .......... 22 GeneralMethods...... ..... ..... 23 atperiment I-Reprcductive Inhibition And. Recovery In Young Fanale Peromyscug leucogis ................ 24 Experiment II-Phercmonal Stimuli As The Inhibitory Mechanism 25 Experiment III-Temporal Dose-Response To The Inhibitory Pheromone ........... . 31 Ehrperiment IV-Interspecific Effect Of The Inhibitory Stimulus . 32 Discussion . ........... . ............. 32 CHAPTER 3 PHYSIOLOGICAL CHANCES ASSOCIATED WITH INHIBITION ..... 39 Experiment V—Determination Of Estrous Cycling. 40 Experiment VI-Effects Of Inhibition On Reproductive Organ Weights . . ................... 42 Experiment VII-Effects Of Inhibition On Number Of Corpora Lutea........ ...... ...45 Discussion...... ......... ..... 47 CHAPTER 4 BEHAVIORAL CHANGES ASSOCIATED WITH INHIBITION ..... 51 Experiment VIII-Short-Term Behavioral Responses To Conspecific Odors ................... . . 52 Experiment IX—Long-Tem. Behavioral Responses To Conspecific Odors ...................... 56 Discussion ...................... . . . . 61 GENERAL DISCUSSION 66 BIBLIOGRAPHY . 78 iv Table 1 . LISTOFTABLES Reproduction of young female (y) g. M exposed to an adult female (F) or control with only an adult male(M)present.... .................... 26 Recovery from reproductive inhibition by young female _. Mwnhtheadult fanalesfromtheesgewhen the young females were 150 days old (M—y—Flso) or With the adult famle remaining until the end of the experiment (M—Y—Fam) ......................... 27 Percentage of parous and nulliparous young fanale g. l_eu_c_o& in a test for pheromonal components of repro- ductive inhibition. M—y denotes an adult male and 21 day old female. Subscripts are: none = control, ch a 100cc of clean bedding every two days, H20 = 1cc of water sprinkled on the cage bedding every two days, sb = 100cc of soiled bedding francegescontainingtwoadult femalemiceeverytwodays, olf = 100cc of soiled bedding as above, but placed under a screen floor so that only airborne stimulus is given, u = 1cc adult fenaleurineaddedtothecagebeddingeverytwodays, andM-y—F= anadultfalalepresentinthecage ....... 29 Paired comparisons of odor treatments with M—y and M—y—F mofz-Wusimaan-ifemnixzarmwis... so Temporal dose-reproductive response of young female P. m to adult female stimuli. Subscripts indicate the number hoursper day ofacposuretoanadult female ........ 33 Paired canparisons of temporal dose-response with M—yo control group versus groups receiving adult female exposure .M using a Bonniferroni X2 analysis ...... 34 Ability of adult female non-conspecifics to inhibit reproduction of young fatale P. maniculatus (Pm)and .Wl)..... . 35 Results of single vaginal lavages performed on inhibited and same-age isolated fanale g. leucopug for determination of estrous cycling ...................... 43 10. Weights of uteri and paired ovaries (mg) of inhibited female 3. m and same—age isolated females in eachofthestagesoftheestrouscycle(n=20/group) . . . Corpora lutea counts of inhibited and control female gig—zooms ....................... vi. 45 47 LISTOFFIGURES Figure Page 1. Photograph of the y—maze test apparatus .......... 54 2. Results of five minute y—maze test for response to adult conspecific odors by control (uninhibited) 75-150 day old adult f‘les, inhibited fanale g. leucogg 75-150 days old and 21-25 day old prepubertal females. The Y—axis is the frequency of preference for the odors listed at the ends of the bars. (0 = significant at the 0.05 level using a x2 Goodness of Fit Test) .............. 57 3. Photograph of the nestbox selection apparatus ......... 59 4. Results of 24 hour nestbox selection test for response to adult conspecific odors by control (uninhibited) 75-150 day old adult females, inhibited female 3. leucogg 75-150 days old and 21-25 day old prepubertal fmles. The Y—axis is the frequency of preference for the odors listed at the ends of the bars. (0 = significant at the 0.05 level using a X2 Goodness of Fit Test) .............. 61 vii." WON The number of animals found in any given location is determined by the birth and death rates within the papulation and the movement of animals intoandout of theareaof concern. Eachof thesegeneral factors can cane under the influence of the social envirornnent. Aggression of resident animals toward transient and unfamiliar animals may limit the number of permanent residents in a given area (Chitty, 1967; Healey, 1967; Metzgar, 1971; Oren, 1982). Aggression of adults toward Juveniles also my precipitate dispersal and act to lower population numbers (Fairbairn, 1978; Gaines and McClenaghan, 1980). Inbreeding avoidance has been described as a factor in dispersal and lowering the number of potential mates (Hill, 1974; Slcryja, 1978; Wolff, 1986a) . Stress associated with crowding or overabundance may cause physiological changes in small manuals that can affect population growth (Christian, 1978; Terman. 1969, 197Sa,b). However, the variable most susceptible to the influence of the social environment is reproduction. Sufficiently regulated reproduction would lessen the selective pressure to develop post-natal forms of control. Whatever mechanisms control the reproduction of individuals within the population precedes the impact of post-natal mechanisms and lowers their necessity. Behaviors that regulate animal numbers may be a secondary effect or epiphenomemn of selection at a more basic level, an individual's potential to reproduce. The overabundance of animals has obvious 2 negtive implications for the individual and population alike. However, the major selective forces on individual members of a population are concerned with mimizing their om reproductive success relative to that of other individuals in the population. The ultimate outcane of this selective process may be the buffering of populations against overamndance. However, the selective forces are more proximal, increasing an individual's own relative reproductive success. Selection will favor individual females that are able to maximize their own relative reproductive success either by increasing their own reproduction (i.e. lowered gestation times, increased litter size or increased survival rates of their own offspring) or by being able to decrease the reproductive output of other females in the population. Lack (1966, 1968) and his contemporaries stated that some female manuals are physiologically constrained and unable to increase their own reproductive output. This probably is not true for all “is, but temperate species such as W M and Perauyscus maniculatus may fal 1 into this category. Physiological constraints of female m can result from either a limitation in energy intake by the female or an inability to provide sufficient maternal care and energy output to place more offspring into the next generation than is possible with smaller litters. Several studies have dommmted that while male 2. AM do not respond to supplemental feeding, females increase in number and decrease the size of their home ranges. This indicates a possible energy constraint for females (Fordham, 1971; Hansen and Batzli, 1978; Gerzoff, 1984; Wolff, 1986b). Additional evidence on litter survival rates in the laboratory shows that for g; naniculagus and 3. W larger than average litters are canposed of lower weight individuals at birth and have a reduced survival to weaning (Fleming and Rauscher, 1978; Myers and Master, 1983). Individuals from larger litters also have reduced fertility conpared to the young from smaller litters (Leary, 1981). Gmmdie and Vessey (1986) have reported an average litter size of 4.21 for females in natural populations. This value falls well within the range of 3.83-4.8 reported in laboratory studies (Svihla, 1932; Drickaner and Vestal, 1973; Planing and Rauscher, 1978; Lemy, 1981). Females reproducing in a laboratory setting are not energetically constrained by food limitations or the physiological costs of climatic conditions and food searching efforts. Failure to be able to maintain larger litters under the "ideal" conditions of the laboratory, canbined with the possibility of an energy input limitation. suggests that physiological constraint, in natural populations is plausible. If the assmnption of physiological limitation, either due to ecological conditions or evolutionary constraint is correct, then the selective mechanisms operating on fanale reproduction that result in oranges in population growth would be mechanisms that allow a female to reduce the reproductive output of other females in the population. No major social mecmnisms have been described that result from adult fmle stimuli and have the capability to reduce the reproductive output of ccnspecific young fanales. Maturational delay of prepubertal fules results from the odors from grouped adult fmles (Vandmbergh, 1973). Reproductive inhibition (prevention) results when young female W are exposed to the odors of individual adult fanales (Skryja, 1978; Haigh, 1983b). The fact that fanales exert inhibitory influences 4 on each other's reproduction is not new. Maturation delays in prepubertal fanale manuals due to chemcsignals or social contact with other feualee has been well documented. (Whitten, 1958; Bronson and Desell, 1968; Bronson. 1971; Channplin, 1971; Cowley and Wise, 1972; Vanienbexgh, 1973; Drickaner, 1974: McIntosh and Drickaunr, 1977; Ternan, 1979, 1984; Mmey and Vandenbergh, 1980). This phenomenon has beenpumedas: 1) amechanismfordecreasingthemmberof potentially reproducing fueles in an area, 2) a mechanism for inbreeding avoidance and 3) as an adaptation by young famles that al love them to delay their first pregnancy. While a conclusive answer as to the selection process and function of maturation delay awaits further study, the phernnencnhas been well described in terms of the stimulus that causes the delay, the control of the pheramne production in the adult female and the physiological changes that result in affected young fanales. The same cannot be said for reproductive inhibition. The lack of reproduction by young fmle Peronyscus resulting fran stimuli from adult female conspecifics has been described in only two studies (Skryja, 1978; Haigh, 1983a). In these studies, only data demonstrating the phenanenon were presented. If the questions of the functim and selective processes involved in the acquisition of reproductive inhibition are to be addressed, additional information on the medianisms must first be collected. The duration of inhibition must be lmown. Is it permanent in affected fanales or will they recover once the stimuli is removed? What is the type and specific source of the stimulus responsible for inhibition? What is the frequency and duration of the stimulus sufficient to case inhibition? This issue must be addressed to determine if, based on what we know of W social structure, it can affect natural populations. “at are the physiologiesl changes resulting in the affected fules? This will allow canparisons to other effects of the social environment and formation of hypotheses about the possible relationships between reproductive inhibition and other social factors affecting fanale reproduction. Are ther behavioral affects associated with inhibition? This nnist be determined as behavioral changes could affect both the likelihood of fanales becaning inhibited and also the social structure of the population. Thepurposeof thisstudyistoanswerthequestionsstatedinthe preceding paragraph. With these answers available, the questions of the function and the evolutionary seqzence that resulted in the acquisition of this trait may be better understood and testable hypotheses formed for future work on reproductive inhibition in young fenale Pe_rg_n'x§cus. m 1 LITERATURE REVIEW Mmmmtaimbetwemmardflspeciesofmrth am Central American rodents (Hall and Kelson, 1959; Hooper, 1968). Time are all snail male that feed on seeds, vegetation or insects and have a relatively short lifespan (Hamilton, 1941; Jameson, 1952; Baker, 1968; Eisenberg, 1981). Correlated with this relatively short life expectancy is the ability of these mice to mature at a relatively early age and immediately begin producing offspring unless prevented by environmental stimuli (Millar, 1977). All female deermice, and indeed met rodents, are capable of producing a large number of offspring over one or several years unless physiologically sterile. This is evident franflaelaboratorybmeedingrecordsofammberofgemspecies (Drickaner and Vestal, 1973; Skryia. 1978; Myers and Master, 1983). In natural popilations fenale deermice do not reproduce continuously, which raises the question: what environmental constraints prevent a female fran reproducing constantly as she would under laboratory conditions? Three general enviromnental factors act as basic modifiers of the reproductive capacities of falale small manuals: 1) the physical envimnent; 2) the caloric availability and nutritional characteristics of food and 3) the influence of social stimuli. Since my concern is with the ability of social influences to perturb the reproduction of female W, I will discuss only one example of the effects of the physical environment (photoperiod) , briefly eiamine dietary influences 6 on reproduction and concentrate on the variation in reproduction (physiological and behavioral) attributable to the social environment. The physical environment (topography, rainfall, temperature, l'nrmidity, photoperiod, etc.) is the ultimate factor that limits reproduction in small ”is (Baker, 1938) . In order for a particular species to occur, anareamustbewithincertainminimumandmaxinm limits for physical characteristics. Animals with extensive distributions often exhibit a wide range in the duration and timing of reproduction. Climate and food may ultimately determine when particular animls reproduce, but photoperiod serves as a proodnial cue dictating thebeginningandendofthebreedingseason. Exposuretodaylengthsof minimal duration is necessary for functional mintenance of the reproductive systems in a number of rodent species; hamsters, voles and deemloe (Elliot, 1976; Johnston ard Zucker, 1980; Grocock, 1981; Dark e_t g" 1933). Daylengths below the critical minimum result in cessation of the estrous cycle and non-receptivity (see reviews by Reiter, 1980; Zucker gt _a_l., 1980). Responsiveness to photoperiod is not species-specific. A latitudinal gradient in the response to photoperiod has been observed in both 3. maniculatus and 2. 1m (Desjardins, 1981; Lynch e_t _a_l_., 1981; Dark e_t 92., 1983). For erample, while 14h / day of light was sufficient to cause naturation in g. maniculatus from Mexico, 90% of the mice from Canada did not develop functional gonads under this scheduale (Desjardins, gt gl_., 1987). The responsiveness to photoperiod in these mice appears to be controlled by a simple genetic system as only a few generations of selection are necessary to create a population that is 8 rnearly finned (> 95%) either as photoresponsive or rnornresponsive (Desjardins gt gl_., 1987). The same study also revealed that populations across the rarnge of the species contain individuals of both types. The frequency of photoresponsive and rnonresponsive individuals is dictated by the latitude of the population. Food along with the physical envirormnent are'the most important factors limiting reproduction. Producing offspring is an energy consuming activity. The energy used in the production of offspring is limited to that available after expenditures for growth, maintenance arnd food searching are already satisfied (Barnett, 1973). Speculation on the importance of food availability in controlling the reproduction of Wiesbeenintroducedinseveral studiesexaminingtheeffects of supplenental food on population dynamics (Fordham, 1971; Hansen arnd Batzli, 1978; Gerzoff, 1984; Wolff, 1986). These studies do not show a conclusive link between food availability and reproduction, but several laboratory studies indicate that this is the case. Female house mice are able to successfully raise litters in temperatures as low as 9°C when the amournt of work required to gather food is low (Perrigo and Bronson, 1985). Honever when required to use a running wheel in order to obtain food, reproduction was curtailed and even arrested entirely when large amount of running were required. Prepubertal female house . mice restricted to only the food necessary for weight mintenance do not mature (Hamilton and Bronson, 1985). Restricted food did not affect reproductive developne'nt of prepnlbertal males. These results are a contrast to studies on other species in which both males and females were prevented fran becaning sexually mature when kept on a weight maintenance diet (Kennedy and Mitras, 1963; Moustgard, 1969; Glass and Swerloff, 1980; Sisk and Bronson, unpub. data). Restricting food intake to weight maintenance dose not have the same type of effect on adults. Copulatory behavior is unaffected in nale rats even when given no food for several days (Sachs, 1965). The same can be said of adult females rats provided that sufficient food is available for weight mintenance. The enount of available reproduction energy is modified by the tenperature and humidity of the animals microfabitat. Snell naturals, such as Lem, are extremely susceptible to even minor changes in temperature with respect to their energetic costs due primarily to their high surface to volmne ratio (Hart, 1971) . Low tennperature sumress both naturation arnd reproduction in female mice (Porter and Gates, 1969 ; Barnett, 1965, 1973; Bronson and Pryor, 1983; Hill, 1983). Mice may spend much of their day in thermally buffered nests, but are required to leave then in order to forage (Harland arnd Miller, 1980). The enonmt of energy that an animal can assimilate per day even under optimum condition, such as in the laboratory, are limited. As food becanes scarcer arnd / or temperature becone lower, a larger percentage of assimilated energy must be used for nan-reproductive costs. This nnay be one of the factors responsible for thernormal lackofbreedingbdeuringwintermonths in tenperate area. Although winter breedirng has been observed in temperate regions it's occurrence is variable (Brown, 1945; Bronson, 1987) . Even in the laboratory where the physical environment and food availability can be maintained at optimal levels, other external stimuli can come into play entancing or reducirng a fennales ability to reproduce These stimuli that affect reproduction are subtle, fine grained and a 10 less well understood set of influences: the social environment. The social relationships among individual mice nay account for as much variationn in the reproductive performance of fenale mice as either the physical environment or food. These relationships have been found to act at all stages of an individual's life from before birth to aiulthcod. (van Saal, 1979,1983; van 8331 and Bronson, 1980; Nbssey and Vardenbergh, 1980; Terman, 1984; Schadler, 1985). They can either enlance or retard the reproductive physiology and behavior of fenles (Christian, 1978; Temn, 1984; Haigh, 1987). As with energetic costs and the physical stimuli, the effects of the social environment on reproduction are different for males and females of the same species. Thisreviewcoversenlythcsefactors impingingonthereproductive performance of tennis mice, although social stimuli can modify male reproductive perfornance (Bediz and Whitsett, 1979; Vardenbergh, 1983; Bronson, 1983) . This enamination of social influences on fenale reproduction might have been approached fron a mmnber of perspectives. A conparison of the relative impact of each of the social influences on reproduction could yield a thorough account of the pertinent literature. Honever, too little is known about the actual effects of these phenomena in natural populations and would therefore only be a sumnary of speculations. The fornat that I will use is to discuss each social effect as a separate process. The effects of the social environment on reproduction and the mechanisms for transmitting social stimuli in mice have been the subject ofnanystudiesoverthepastthreedecades (seereviewsbyBronson, 1983, 1985; Vandenbergh, 1983). As each new process is discovered, the question is asked whether it involves differences in behavioral 11 interplay and individual contact, a chemosignal transmitted by'a given sausage class of animals, or a combination.of both. Differences in belavioral interplay have been associated with several changes in the reproduction of young mice. Terran (1968, 1973) noted cessation of reproduction in adult mice and inhibition of young mdce in.high.density laboratory populations of g, maniculatus. Similar findings have been reported in house mice (gt musculus) (Southwick, 1958; Vessey, 1967). no specific population density is associated with this effect (Terman, 1973). All experimental populations eventually reached asymptotic levels, but the»density at which.each.population reached this level varied widely. Emotional stress mociated with heightened.aggression.at elevated densities has been.hypothesized as a predoninant factor affecting reproduction in small manual populations, particularly house mice and.microtine populations (Chitty, 1967; Healey, 1967; Metzger, 1971; Grau, 1982). Increased aggression.resulting in an elevation of circulating corticosteroids provides negative feedback on the production of gonadal hormones, thus reducing reproduction (Christian, 1978; Terran, 1969, 1973a, 1973b). Testing by Terran (1974) showed this phenomenon.was not associated with.aggressive behavior as suggested for populations of house mice (Christian, 1971) or Peromyscus (Healey, 1967). Behavioral observations by Terman (1979) on.populations of g. maniculatus from founding urntil breeding ceased showed that 4496 of the aggression observed was during the first seven.days after founding the populations. .More recent studies have shown reproductive arrest in these populations to be the result of tactile and olfactory cues (Terran, 1979, 1984). 12 In looking at population level pl'nennea such as the cessation of reproduction at high densities, several problens arise. The number of possible social stimuli and the interactions between then rake assessment of the overall causal nnechanisms extremely difficult to measure. Eveniftheseproblems couldbeoverccmeaccountirg forthe selection and spread of such population level traits necessitates the assnmnption of population or interdenic selection occurring. Although this type of selection process is possible, I think it more plausible to consider that reduced reproduction within an entire population is an accunmlation of more subtle social stimuli affecting the individual that come increasingly into play as densities increase and social contacts becone more frequent. The reproduction of juvenile and adult mice is able to be manipulated with olfactory stimuli from both nale and ferale conspecifics. Male nnouse chenosignals are the principle esusal agents for the Bruce Effect, Written Effect and acceleration of female natmation (Written, 1957; Bruce, 1965; Whitten e_t _a_l_., 1968; Vardenbergh, 1969; Drickamer, 1982a). Female chemosignals are associated with estrous suppression, and naturational delays of young ferale mice (Whitten, 1958; McIntosh and Drickamer, 1977; Drickamer. 1981) . Pregnancy block or the "Bruce Effect" is the failure to establish pregnancyinarecentlyinseminated fenalemouseas theresult of exposure to a strange nale during the first four days after copulation (Bruce, 1961). The phenanenon has been described for g. musculus, g. naniculatus, Microtus mlvanicus and Microt'us W (Bruce. 1959, 1960; Parks and Bruce, 1961; Bronson gt _a_l., 1964; Clulow nd 13 Clarke, 1968; Clulow and Langford, 1971; Stehn and Richmmd, 1975). Pregnancyblockishoweverabsentinsmneinbredstrainsoflabmice (Inbreden and Bronnson, 1965; Clnipnan and Bronson, 1968). A male's physical presence is unnecessary as urine fraln an unfamiliar adult male, yields the same result (Danninic, 1964). Even though more than one male has proved to be no more effective in blocking pregnancy than a single male (Bruce, 1963) , both behavioral familiarity and genetic similarity were found to be important (Parkes annd Bruce, 1961). Grouping recently inseminated females during exposure to male stimuli however, resulted in a higher proportionn of pregnancies than with individual exposure but not equivalent to central females (Bruce, 1963) . 'nne effectiveness of the mle was greatest when the exposure was extended to 48-72 hours, but Chipman gt _a_l. , (1966) showed that multiple short-term exposures were sufficient to sense pregnancy block. More recently studies of mmmmt shortexposures (2h) toastrangemale immediate after copulation result in pregnancy failure (Dewabury, 1979b, 1981, 1982, 1983; Devebury annd Baumgartener, 1981) .. nus block negates the previous insemination and muses the affected female to cane into estrus. Dewabury (1983) claims that this pregnancy block caused by short- term exposure just after copulation is different from the Bruce Effect because a) it occurs arounnd the time of copulation, b) exposure to the first male is brief and c) exposure to the second male is brief. The evidencepresentedbynewaburyappearstobeweakinthisregani. The classification of the results of Dewsbury (1983) as a distinct phenannencn based solely on a temporal bmis (exposure to the strange 14 nale 20 minutes or 24 hours after copulation) I believe is unnfounnded. y. W is not affected by the short—term exposure imnediately following copulation (Dewsbury and Banmngartener, 1981) but is subject to pregnancy block when exposure is longer and delayed until several hours after copulation (Stehn and Richard, 1975). If this is true for other species, it renainns to be tested as in all of the earlier studies females were given 24-72 hours before exposure to the strange nale and herminedwithornearthefemale foranentenndedperiodof time (> 24 hours). Until such time as additional studies can be made, assigning theseresultsasaseparatephememispreature. Raolution of the above results nay be of little concern other than in terms of anninal husbandry. The "Bruce Effect" recently has been considered by sane investigators as a laboratory artifact that will have an insignificant effect in natural populationns (Bronson, 1979). With the exception of the studies by Devabury cited above a 24-48 hour exposure to a strange male is required for an effective block and the affected female does nnot cane into estrus for three to seven days. Adultmaleandfennalemareseldom found tooccupythesame nestsite (Trudeau e_t 31., 1980; King, 1983; Goundie and Vessey, 1985). Those that are found together in the same nestsite are seldann found together again even 24 hours later. If the female receives a sufficient amounnt of stimulus to have a pregnancy block occur during the period whentheyaretogether, themalewouldhave to findthe femaleagain after several days to be able to mate with her. Multiply-sired litters of Famous have also been found in natural populations (Birdsall and Nash, 1973) . Given the unlikely occurrence of this being advantageous to the male in terms of cost/benefit and the occurrence of multiply- 15 sired litters, the Bruce Effect may be an encaptation, simply still found as it has become a neutral trait and nnot subject to selectionn. Chennnosignals fran adult males are also associated with the synnclnronizatien of the estrous cycle (whitten Effect) and shortening of its length. Gmnped female mice in the absence of adult male stimuli becane annoestrous (mitten, 1959) or in sane cases pseudopregnnannt (van der LeeandBoot, 1955). mequent exposureof thaegrouped annoestrous females to olfactory stinnuli frann adult male conspecifics not only reinitiates estrous, but fennales in the group have synnchronized cycles of a shorter duration than prior to the annoestrous conditionn developed (Whitten, 1958; Bronson and Marsden, 1964). Exposure to male chemosignnals isnotnnecessaryforrecoveryfrantheanoestrous condition. Isolation of single annoestrous mice from olfactory stimuli of any kind will result in eventual initiation of her cycle, but nnot in synchronization with other members of the groups she was in. The synnchronization effect is lnowever transitory and re—exposure to a male is needed during each subsequent cycle for synchronization to be mintainned (Bronson and mrsden, 1965). The substannce or substannces responsible for the Wnitten Effect are volatile and androgen-dependent as urine from castrated or prepubertal nales has nno effect (Wnitten, Bronson and Greestein, 1968) . The variation in the effectiveness of this phencnnensn is a funnction of group size (Paries and Bruce, 1961; Champlin, 1971). This annoestrous syndrane can be artificially created by placing a single adult female into a cage previously innhabited by a group of females (Bronson, 1966; Champlin, 1971) . A mixture of soiled bedding from several individual fenales is 16 inneffective and thus verifies the physiologiml changes occur in female mice after grouping. Suppression of the estrous cycle in fenale mice could have onne of several funnctions in natural populations. During the breeding season, at least in tennperate regions adult feales usually occupy nestsites alone. Only during late fall and winter are adult females observed to share nestsites in large enough number that estrous suppression would occur. This clme association with other fenales may be associated with the photoperiodic response seen in nnorthernn populations of W. Regression of reproductive organs during periods of short daylegth have been reported for several species of 2m (Desjardins and Lopez,1980: Lynchgtglu 1981; Dark, 3311., 1983; Forger and Zucker, 1985). While this process of gonadal regression takes place over several weeks, suppression of estrous mnn occur in a matter of days (Bronson and Clnapnan, 1968). If short days or sane other stimulus (lack of green plant material, tennperature, etc.) muses females to be less territorial and nest together, the result would be a rapid inability to becanne pregnant with it's and lactattion's energetic risk during colder and more energy depleting environmental conditions. The hypothesis that estrous suppression reduces the possibility of pregnancy during more energetimlly risky times of the year would only be effective if male olfactory stimuli were absent. Studies by Trudeau e_t_ a_l_., (1980) and Wolff, (1986) omerved groups of mice containing up to 14 individuals occupying a single nestsite, however, in only a very small mmberofmsesdidthesegroupscontainadultmales. Juvenile as well as adult fenales are affected by the chewsignnals franadultmales. Fenslemiceraisedinthepresenceofanadultmale 17 attainned puberty at an earlier age than those isolated frann adult male olfactory stimulus based on either vaginal opening (Castro, 1967) or first estrus (Vandenbergh, 1967, 1969). Additional studies have shownn this effect to result frann a chemosignnal given off as a constituent of 8311.11: nale urinne (Kennedy and Brown, 1970; leey and Wise, 1972; Colby and Vandenbergh, 1974; Bronson and Marunniak, 1975; Drickamer, 1981. Tactile stimuli frann a male's presence enhannces, but is insufficient to muse accelerationn (Vandenbergh, 1967) . Drickamer (1984a, 1986) found that while two hours per day of contact with adult male urinne was effective, onlyonehourwasnecessarywhentheadult nalewasalso present. This manipulation of female reproductive physiology as with theestrousinductionisandrogen—depedentasearlyandrogenimd fenales also muse accelerated maturation of Juvenile female mice (Drickamer, 1974; lanbardi gt gt” 1976). Urine fran prepubertal males or mstrated males does nnot possess pheranonnal activity in the urine and replacement by testosteronne propionnate in mstrated adult nales restores activity in a @e-dependent fashion (Lombardi gt gt. , 1976; Drickamer and Mnrphy, 1978) . The active agent for accelerating maturation of young fenale mice appears to be present in bladder urine fran all adult nale mice regardless of testosteronne level (Drickamer, 1978) . In mstrated males or those with low testosterone levels, it is removed or redered inert prior to excretion. The actual synthesis of the accelerator ingredient is nnot depedent on testosteronne, but the process for its removal prior to accretion is linked to testosterone. The link between the effectiveness of adult male urinne and testosterone levels suggests that other stimuli , such as dominance or 18 unotoperiod that are knam to affect testosterone level could affect pheromone activity. Indeed Iombardi and Vandenbergh (1977) observed that adult males determined to be subordinate after 7 days of paired encounters with other males did not possess the ability to accelerate maturation. This does nnot war to be linked to adrenal activity as urine fran adrenalectanized males was still effective. Chennimlly, the active substannce responsible for this effect has yet to be characterized. Preliminary studies by Lombardi gt get, (1976) and Vandenbergh gt _a_l.,(1976) suggest that the substance is a small peptide or associated product. Interestingly, the same urirne fraction containing the accelerating substance also contains the active ingredient responsible for the Bruce Effect (mrchlewska-Koj, 1980) . In spite of a lack of chemiml characterization, the potency of this substannce is high. 0.0001 cc per day of adult male urinne applied to the external hares of young females for three days is sufficient to hasten the time to naturity (Drickamer, 1982a, 1984c) . The effect of accelerated maturity on the reproduction of young female mice is unclear. while becoming able to reproduce at an earlier age would seen to be an advantage in such short-lived annimals, the risks innvolved nay outweigh then. Myers and Master (1983) found age and weight to be negatively correlated with litter size, offspring weights and survival to weaning. Much of this correlation attributed to the fact that young, low body weiglnt females are still utilizing energy for growth that is unavailable for reproduction. These results were collected in the nontaxing environment of the laboratory. Additional energetic requirenents found under natural condition are only likely to annplify what was observed in the lab. Field data necessary to evaluate 19 theactual costsand/orbenefits toyourgfenalemicewhoreproduce earlier than the rest of their cohort have nnot been collected primrily due to the logistin problems of monitoring the animal's actual reproduction. Field studies by Massey and Vandenbergh (1980, 1981) have however shown that acceleration pheranone is present in the urine of males in natural populations. Live-traps containing filter paper were used to collect urine senples from adult males at high and low population densities. Urine frcm males living in both high and low density population were found to accelerate maturation when used as the stimulus on young fenale mice in the laboratory. A similar acceleration of puberty is observed enong young feels mice exposed to the urine of lactating or pregnant females (Drickamer, 1983). This, like the pherunone found in adult male urine, requires a three day exposure beginning prior to the affected fetale being 30 days old to accelerate maturation (Drickamer, 1984a) . The presence of active agents in both the bladder and excreted urinne of lactating and pregnant fenales indicates that is not a product of post-bladder accessory glands. This accelerating ability disappears fran the urinne when pregnnant or lactating females age are aged together (Drickamer, 1983) . Wnetherthis, likethephemneproducedbytheadultmalse is indepedent of gonadal honnonss, with the excreted urine regulated by a gonadal hormone-dependent secondary process is nmknown (Drickamer, 1986) . Sensitivity to accelerated maturation is seasonally regulated as the W is ineffective on juvenile mice during winter months (Drickamer, unpub. data). Accelerated maturation by lactating and 20 pregnant females is annother eemple of a physiology-depedent effect. Chenosignals from adult females that are neither lactating nor pregnnant delay the maturation of young fennales (Whitten, 1958; Denzel and Bronson, 1968; Champlin, 1971; Vandenbergh, 1973; McIntosh and Dridmr, 1977; Ternan, 1979, 1984; Drickanner, 1982c). This is also true for females of any age vinen caged at sufficient densities. This, unlikeeitherof thetwochenosignalsdiscussedaboveisdepedenton adrenal hormones for both its production and excretion (Drickamer and McIntosh, 1980) . Injection of hydrocortisone or corticosteronne resulted in recovery of pherunone production (Drickamer and Shapiro, 1984) . The absence of functional ovaries does nnot prevent either production or encretion of the delaying substannce (Drickamer e_t _a_l_. , 1978). The genetic relatedness of the stimulus fenale to the young fenales affected does nnot influence maturation delay (Drickamer, 1984) . The delaying effect of adult fenale urine has been hypothesized to function as a mechanism that 1) assists fenale offspring to avoid inbreeding, 2) allows yang fenales to avoid pregnnancy until they are older and possibly more capable of the stress associated with pregnnannoy andlactationanda) asamechanismusedbyadult fenales toreduce the number of potentially reproducing fenales in the area surrounding then. The viability of these hypotheses are discussed by Haigh (1987) and in the General Discussion of this tent and so will nnot be evaluated here. Recently, it has been determined in Perauysous erenicus and g. maniculatus that the presence of a single adult fenale severely inhibits thereproductionofyanngfuleswhentheyhavebeenecposedsince birth (Sharia, 1978; Haigh, 1983a). In these studies, juvenile (21 day old) fennale mice were placed with an adult male and an adult fenale or 21 just an adult male until the young fennale was 180 days old. In both of these studies the reproduction of the yang fenale was almost entirely inhibited as only 8 of 33 g. erenicus and 0 of 30 g. maniculatus produced litters. The adult females in all cases cantimsd to reproduce nnormally. Tlnisfailure toreproduceentendsfarbeyondtheaverageage of first reproduction for either of these two species (104 days old and 84 days old, respectively). This phenomenon appears to be new in tents of rodent reproductive biology, but a similar facultative if nnot obligate reproductive failure has been reported for wolves, dwarf mngooses, gibbons and sienngs (Mech, 1970; Rasa, 1971, 1972; Zienan, 1975; Kleinan, 1977; Seal _e_§ £11., 1979). Evidence for reproductive inhibition in yang fensle W seensclearfranthetwostudiesinwhichithasbeenenamined. A description of this effect only annswers a 91le portion of the questions that arise. The database canpiled so far by Skryja (1978) and Haigh (1983a) yields a starting point fran which to begin asking questions about the communication of the inhibitory stimulus and its behavioral and physiological effects on inhibited fenale mice. The pages to follow in this volume expands and enteds our knnawledge about reproductive inhibition and how it may funnction as a reproductive manipulator in natural population of Perggyscus W. CHAPIERZ DESCRIPTION OF WM INHIBITION INYCUNGWPERCMYSCUSIEUCOPUS We Adult feneles will inhibit the reproduction of yang fenele 2- im- Reproductive inhibition is nnot peruennent. Chenosignals fran the adult fenele are responsible for inhibiting reproduction in yang feneles. The inhibitory chencsignal is received by the yang feneles thragh olfactory receptors The inhibitory chemosignal is a camanent of adult feels urine. Continuous exposure to the inhibitory pheromone is nnot necessary for inhibition. The inhibitory effect is the result of a species—specific stimlus. Predictions Yang feneles will fail to reproduce when exposed to an adult fenels. Yang fenneles will regain reproduc- tive function when the adult fenels stimulus is removed. Young feneles will fail to reproduce when exposed to soiled bedding fran cages containing adult feneles. Yang females will fail to reproduce when exposed to soiled bedding from adult fenelss, but nnot allowed contact with it. Yang fenales will fail to reproduce when exposed to adult fenele urine. Young fenales will fail to reproduce when exposure to an adult fenele is less than continuous. Exposure to conspecific fenals odors will nnot ceuse reproductive inhibition in yang fenneles. The existence of reproductive inhibition in yang _E. erennicus and g. nenniculatus has been previously documented (Skryja, 1978; Haigh, 1983a) . Implicit in evaluating this phenomenon is an understanding of; 1) howthisphenanenon is coumuniceted to theyang female, 2) the enount of stimulus necessary to elicit the inhibition and 3) the time required for inhibited feneles to begin reproducing once the stimulus is 22 23 removed. Chennosignals or pheromones have been described as the causative agent for a large number of the conspecific effects on the neturation and reproduction of yang mice (See Literature Review). A review of the possible mechani- by Haigh (1983a) conncludsd that the most plausible explanation for the inhibitory stimulus was a pheromone given off by the adult fenale. Eacperiments in this chapter evaluate the hypotMes by testing: a) if the inhibition is preent in g. M. b) if the inhibitionn is due to a chsnnsignnal, c) if the chenosignnal is cannunieated to the yang fenale by olfactory signals, d) if the pheramne is conntained in urinne from adult fuls mics, e) the amount of daily exposure necessary to cause innhibition and f) if the pheromone is sufficiently similar between congeneric species that inhibition will occur inayangfenelemexposed to theodors of females of other species. M m Aninnels used in the following experiments were Peromyscus lsucggng nnot more than three generations renoved from the wild. Mice in the colony from which the experimental aninels were drawn are maintained in clear plastic cages (20 x 28 x 20 cm) with screen tops. Food (Wayne Breeder Blocks.) and water were provided ad _L-‘Q- with woodchips and Nestlets (Ancers, Innc.) used as bedding. Lighting was neintained at 15L:9n (0500-2000h). Temperature was maintained at 21°C : 2°C and humidity was that ambient to the building. Unless otherwise specified in the fol lowing protocols, all experiments were conducted under the above conditions. Since infertility ansng adult aninels used in the experiments could confound the data, all adult animals had parented at least onne litter. 24 Juvenile females used in this study were 21 days old at the initiation of the enpsriments. Cmnparisons of father-daughter and unrelated pairs of W maniculatus (Haigh, 1983a) and g. M (Haigh, unpub. data) failed to reveal any qnenntitativs differences in percentage of females reproducing, age at first reproduction, litter size or mmbsrof litters produced. Therefore, nno distinction was neds in treatment groups between related and unrelated groups of mice. Unless otherwise specified in the individual protocols, all treatment groups had a senple size of n =- 15. figment _I.- Reproductive Inhibitionn and Recovery In Yang Pale W Lassa. Test anninels were divided into three treatment gralps. In treatment group 1, an adult male and a juvenile female were paired as controls (M—y) and observed nnntil the yang fenels was 150 days old. Treatment groups 2 and 3 contained an adult male, an adult fuels and a Juvenile female. In gralp 2, the adult feuele remained in the cage until the juvenile fenale was 300 days old (M-y-Faoo)- Tlns adult fenels ingranp 3was rennovedwhenthe juvenile fenelewas 150daysoldandthe reneining pairs of aninels were observed until the yang fenele was 300 days old (M-y-F15o). Sample sizes in the M-y and M-y-F groups were 45 and 120 respectively. These large sample sizes result from pooling of anninels from other experiments where reproductionn was nnot the variable of interest, such as behavioral avoidance of inhibition (see Chapter 4). The variable measured in this experiment was whether or nnot a fenele reproduced by the age of 150 days. Results of this experiment evaluated if fenels g. M were inhibited by the presence of an adult female 25 annd if the inhibited fmles would recover reproductive fnnnction once the inhibitory stimulus was renoved. Results indicate that young feels g. 1_eu_cog_n§ were inhibited by the presence of an adult feels (M-y-F groups pooled; Table 1) as 2/120 yonng feeles produced a litter, while in the M-y group 28/45 yonng feeles reproduced (x2 = 76.56, df = 1, p < 0.001). This inhibition was effective as long as the older feels was present. In the M'Y'Fiso group with the adult feels removed, nine daughters began to reproduce. In contrast, no offspring were produced by young feeles in the M—y—Fsoo groupuptoaoodaysofagewhenthsstudywasdiscontimed (x2 = 15.6, df - 1, P < 0.001)(Table 2). The reproduction of the yonng fmlss in the M-y-Faoo group after removal of the older female were camparable to the young females in the M-y group (X2 = 0.260, df = 1, n.s.). Young fmle 2. m were inhibited by the presence of an adult fmle, however, the inhibition is nnot psrnennent as renoval of the adult feels resulted in initiation of reproduction. The recovery period of 49 days is substantially longer than the recovery time observed in other socially mediated effects on reproduction such as maturation delay, pregnnanncy block, etc. The recovery time is the average time fran renoval of the adult female to first successful copulation based on backdating 25 days fran the birth of the first litter. £3th _I_I.- Pherenonal Stimuli As me Inhibitory Mechanism. Test animals in Encperinnent II were pairs of mice consisting of an adult male (M) annd a 21 day old feels (y). Each pair of aninels was subjected to one of the following seven treatments: 1) no treatment (control; M-Y); 2) an adult feels (F) presnt (M-y—F); 3) 100 cc of 26 Table 1. Reproduction of young female (y) g. lggggpg§_exposed to an adult feels (F) or controls with only the adult male (M) present. GROUP . N PAROUS NUILIPAROUS M—y 45 28 17 M—be 120 3 117 x2 = 76.56 df = 1 p 5 0.001 27 Table 2. Recovery fran reproductive inhibition by young feels g. mmmtheadultfeelerenovedfranthecagewhentheyonng fem-+13 were 150 days of age (M—Y_F150) or with the adult feels reeinirg until the end of the experiment (M—y—Faoo) . GROUP N PAROUS NULLIPAROUS ”Hr-F150 15 9 6 M—y—Faoo 15 0 15 x2=15.60 df=1 p60.001 28 clean bedding (cb) were placed in their cage every two days (M—ycb)’ 4) 100 cc of soiled bedding (sb) fram a cage containing two adult feeles were placed in their cage every two days (M—ysb): 5) the pair was placed in a cage with an elevated screen floor, under which 100 cc of soiled bedding from a cage containing two adult females were placed every two days (Id-You); 6) 1 cc of distilled water (1120) was sprayed on their bedding every two days (Id-yum); or '1) 1 cc of adult female urine (u) was sprayed an their bediing every two days (M-yu). Groupiservedasacontrol for bothhanndlingof themiceanndthe effectsofthesoiledbedding. Groups3and4had100ccofbedding removed from their cages immediately prior to either clean or soiled bediing being placed into their cages. This ensured that an equivalent eeuntofbeddingwasalwaysprssent intheirege. Group6servedasa control for the urine protocol of Group 7. All pairs of mice were maintained until the young fuels was 150 days old or had her first litter. The variable measured was the proportion of f‘les that produced a litter in each treatment. This experiment was designed to show if the inhibition is due to the physiel presence of the adult fuels or a chemical oamponent given off by her and if the caumnnicetion mode of inhibition is olfactory or by chemical contact. The first camparison made in Experiment II was to look for a W1 effect using a x2 test for overall significannce of the seven groups involved. Results of this test shmed a significant departure from expected values (P < 0.001) (Table 3). The M-ycb, M-szo, M—ysb, M'Yolf and M-yu groups were then compared to the M-y annd M-y-F groups using a mfem X2 (Gill, 1978) (Table 4). Comparisons of the M—y versus M-ycb annd M-y versus "WI-120 showed no significant differences, 29 Table 3. Percentage of parsz annd nulliparous young feels P. leucopus in a test for pheromonal components of reproductive inhibition. M—y denotes an adult male end 21 day old feels. Subscripts are: none = control, cb = 100cc of clean bedding every two days, 820 = 1cc of water sprinkled on the cage bedding every two days, sb = 100cc of soiled beddingfrolmegescontainingtwoadult feelemiceeverytwodays, olf = 100cc of soiled bedding as above, but placed unnder a screen floor so that only airbonnne stimulus is given, u = 1cc of adult feels urine addedtothecegebeddingeverytwodays, andM-y-F=theadu1t feels preent in the cage. GROUP N PAROUS NULLIPAROUS M—y 45 62.2 37.3 14-ch 15 60.0 40.0 M‘Ymo 15 66.7 33.3 M—ysb 15 13.3 86.7 M'Yolf 15 13.3 86.7 M.“ 15 20.0 30.0 M—y-F 15 0.0 100.0 x2 = 31.25 df = 5 p 5 0.001 30 Table 4. Paired comparisons of odor treatments using a Bonferroni x2 analysis. mmsous :12B nr p M-Y VB "-ch - 0.56 1 ns M—y vs M'YHZO 0.09 1 ns "-ch vs M—ysb 7.03 1 0.05 M—ycb vs M—yolf 7.03 1 0.05 M'Yfizo vs M-yu 6 . 65 1 0 .05 M"'l’sb V3 M"Yolf 0'00 1 ns M-Y-F vs M'Ysb 2.14 1 ns ”.y.p V3 M'Yolf 2.14 1 ns M—y—F vs M—yu 3.34 1 ns 31 indicating that there were no effects of the experimental manipulation or ertra handling. The M-y versus M‘Ysb, ".3, versus M‘Yolf and M-y versus M—yu comparisons showed a signnificant lack of reproduction among fnles exposed to the soiled bedding or urine of adult fmle mice. Comparisons of the M-y—F versus ’4st: M-y—F versus M-yo 1t and M—y-F versus M—yu groups were all nonsignificant. These results indicate that the inhibition of young fule g. Len__ncogn_s; is due to a chemosignal or pherumne given off by the adult fule in her urine annd received through olfaction by the young fuels. The lack of significarncs between tl'egroupswiththsadult fulepresentandthesoiledbeddingand urine groups inndicates that the phenomenon is primarily the result of olfactory cues annd occurred in the absence of tactile cenponents. 325th III.- Temporal Me-Response To The Inhibitory Pteromone. This experiment was designed to investigate the minimum amonnnt of daily emosure that is sufficient to elicit the inhibition of reproduction in young fmls mice. Test animals were divided into seven treatment groups. Each set of test aninels consisted of an adult male and 21 My old feels. Treatment groups were exposed to an adult feels for a given amount of time each day. This was accomplisl'ed by placing tl'e adult male annd yonng fule in the adult feele's cage for the stinmllus period and then removing them to their home cage. Stimulus periods were designed so that exposure occurred during both the light annd dark portiorns of the cycle in equal amounts. Exposure times were 0 h (no contact): M-yo), 1 h (M-yl), 3 h (M-ya), 6 h (M-ye), 12 h(M—y12). 16 h (M—yle), and 24 h (continual contact, M-y24)- This protocol was continued nnntil each young feels was 150 days old or had had her first litter. The variable measured was the proportion of young feeles that 32 had a litter in each treatment group. Results of Experiment III are given in Table 5. Analysis for a difference between the seven groups showed a significant overall effect. Comparisons Of the“"370gruulawiththeotl'ner sixgroupsinpairwise fashion using a Bonniferroni x2 test inndicated that all of the treatments except the M—y1 group were significant in their inhibitory effect on the yonng false (Table 6). These results establish that between one annd three hours is the minimum daily exposure sufficient for inhibition to occur. Qgriment LY.- Interspecific Effects Of The Inhibitory Stimulus. Test animals in Experiment IV were pairs of mice consisting of an adult male annd a 21 day old feels of either 3. M (Groups 2,4,6) or g. maniculatus (Groups 1,3,5). Each pair of animals was subjected to one of the following seven treatments: 1) a g. meniculatus pair with nno treatment (control); 2) a 3. 1m. pair with no treatment (control); 3) a g. maniculatus pair exposed to an adult feels conspecific (inhibition control); 4) a g. m pair exposed to an adult fmle conspecific (inhibition control) ; 5) a g. maniculatus pair exposed to an adult feels g. M; annd 6) a g. leucoms pair exposed to adult feels g. maniculatus. Results of Experiment IV are shown in Table 7. From data collected in this experiment, tl'e pheramene responsible for inhibition in g. meniculatus and 3% is species specific. Comparisons of reproduction of false exposed to non-conspecific adult feels odors to time feeles not exposed to any odors were almnost identical. Likewise, comparison of the non-conspecific group to feeles exposed to 33 Table 5. Temporal dose-reproductive response of young feels g. M to adult feels stimmnli. Subscripts inndicate tie nnunmber of hoursperdayofexposuretoanadult feels. M"1’0 45 28 17 M—yl 15 7 8 "fig 15 3 12 M—y6 15 3 12 "-le 15 2 13 M—yla 15 1 14 M‘Y24 15 o 15 x2 = 27.01 df = 6 p £- 0.001 34 Table 6. Paired comparisons of temporal dose-response with the M-yO control group versus groups receiving adult feels exposure in 13. M, using a Bonniferroni X23 analysis. MARISONS x2B DF P M-Yo vs M—y1 1.12 1 ns M—yo vs M—y3 8.03 1 0,05 M'Yo VB M-Ys 6.03 1 0.05 M-vo VB M-Y12 10.75 1 0.01 M-Yo vs M—yla 13.90 1 0.01 35 Table 7. Ability of adult feels non-conspecifics to inhibit reproduction of young feels g. maniculatus (Pm) and 2. 153m (Pl). Group No. Young Feels Adult Female No. Parous Feelss l Pm - 12/20 2 Pl — 14/20 3 Pan Pm 0/20 4 P1 P1 0/20 5 Pm P1 13/20 6 P1 Pm 11/20 Comparison 1:28 df P 1 vs 3 17.14 1 0.01 1 vs 5 0.11 1 ns 3 vs 5 19.26 1 0.01 2 vs 4 21.54 1 0.01 2 vs 6 0.96 1 ns 4 vs 6 15.17 1 0.01 36 conspecific adult f-nle odors were highly significant. Thus the exposure of yourg f-le W to adult females of other species known to have the inhibitory pheranone have no effect on their reproduction. DISCUSSION The effects of the social environment amorg rodents, particularly with respect to chenidsl commisstion, have been well documented in the literature (see reviews by Doty, 1976; Bronson, 1983; Vandenbergh, 1983). This body of research has sham the reproductive systen of yourg rodents to be particularly susceptible to the effects of conspecific: or .theirodors. Delaysinreproductimofymnganimalscausedbythe presence of, or odors from, same-sex conspecifice have been sham in several studies (Kipps and Terman, 1977; Lombardi and Vandenbergh, 1977; Bediz and Whitsett, 1977; Drickamer, 1979; Vandenbergh, 1980; Haigh, 1983a,b). The results of the present study again show this effect to occur, but in a more dramatic fashion than in most previous studies. Yourg fmle g. was exposed to odors from adult f‘le conspecific. were almost completely inhibited from reproducing. The time required for inhibited fmlss to recover and reproduce (24-92 days) once the adult female is removed adds to the possible effects of inhibition on lifetime reproductive success. In addition to the extended effects on yourg fanale mice, the results to Experiment III of this study show that as little as two hours of exposure per day is sufficient to cause inhibition. This not only shows the sensitivity of the young fenales to the pheranone, but also gives credibility to this effect as a naturally occurring phencmenon. It is unlikely that, after weaning, a young fanale would be in contact with an adult female for lorg periods of time 37 each day. I-bwever, if only a short period of stimulus is necessary then this ptenanenon would have a significant impact on reproduction of yourg female mice and the population as a whole. The results of this study seen to be inconsistent with those of Temn (1984), who found that yourg female 2. m exposed from 21 to 40 chys of age to adult males or their urine had lower body, ovary and uterine weights than females exposed to adult females, their urine or distilled water. Adult f‘le g. m apparently had no effect on the maturation of yourg fanales. It is possible that the reproductive inhibition described in this paper is not the result of maturational arrest, but rather sane post-copulatory process that Ternan's methods failed to reveal. Evaluatirg the maturation of fmle mice on the basis of body weight, reproductive organ weights and corpora lutea (CL) counts at 40 days of age my not reflect eventual maturity. Studies by Clarke (1938) and Rogers and Beauchamp (1974) showed the mean date of first estrus in g. M to be 46 and 51 days of age. respectively. Thus Teman's measurements were made an average of 7-10 days before the first estrous cycle, with less than 596 of the fmles cyclirg acoordirg to the distribution given by Rogers and Beauchamp (1974). These facts are consistent with the low numbers of corpora lutea found by Terman (1.33-3.00 CL/female). While organ weights may measure whether the process of maturirg has begun, simple weights do not reveal if puberty or any specific matuational landmarks have been acheived. In contrast, age at first estrus (Vandenbergh, 1967, 1973; Cowley and Wise, 1972; Drickamer, 1974, 1982) or backdating fran the birth of the first litter to determine when conception occurred (Hill, 38 1974; Haigh, 1983a,b; this study) does indicate the age of potential or functional sexual maturity respectively. tong-term reproductive inhibition in a short-lived animal such as 3. 3121—002! would rave a significant impact on lifetime reproductive success. Several field studies have shown that only a small percentage of the adult fenales perform the majority of the reproduction in natural populations of Peranyscus and other rodents (Metzger, 1981; Myton, 1974; Tallarin, 19773.1); Bronson, 1979; Trudeau _e_t_ a_.'l_., 1980). The inhibitory phememn described here and in earlier studies (Skryja, 1978; Haigh, 1983a,b) could be at least one of the (misstive agents for the lack of reproduction seen in many of the adult females in natural populations. This view is reinforced by the short duration of stimulus necessary for inhibition to occur. Dependirg on how lorg a time is required for the initial inhibition to occur and the frequency with which the stimulus needs to be reinforced, this effect could render many potentially parous females in the population functionally incapable of reproducirg. A reduction in the overall population reproduction rate would be realized and growth of the population buffered. The response of the potentially inhibited fanale to the pheromone may take either of two paths, each havirg different effects on the social structure of the population. If yourg feuales have the ability to detect the pheranone, then they should avoid prolonged exposure to it by not oohabitatixg with or by stayirg may form a nest site recently occupied by an adult fuele. This would lead to the spacirg observed in fetale mice (Metzger, 1971), particularly in areas that have a limited number of nest sites. Inability of the yourg females to detect the {immune would make them subject to inhibition. Thus the females in 39 the population muld include a small number of reproductively active females and a proportion of yourger fenales that are inhibited. Trudeau _t al. (1980), in a nestbox study of the social relationships in g. m, found very few incidents of two or more adult fenales occupying the sane nest site except during the winter when no reproduction ves occurrirg. They also found that only a fraction of the adult female mice ever reproduced. This might be expected if the meme described in this study has a similar effect in natural populations. Studies in the field are necessary to elucidate the actual effectiveness of this and otter rodent phemaes under natural situations before their influence on social structure and population regulation can be known CHAPTERS PHYSIOWICAL EFFECTS OF MINE INHIBITION mmm Predictions The effect of the adult feels Inhibited feeles will exhibit inhibitory pherancne is to arrest diestrous indicative of a lack Ieturation. of an estrous cycle. Inhibited feeles will have seller ovaries and uteri than uninhibited mice of the same age. Inhibited feeles will lack corpora lutea . Young feels mice of the genus W are reproductivsly inhibited by the presence of an adult feels (Skryja, 1978; I-Iaigh, 1983a,b; Haigh, gt. _a_l_., 1985; Chapter 2 this text). This result couldbedustoabehavioral effectsuchasrefusal ofthefeeleto show the appropriate behavioral responses to male advances or an millirgness to copulate. Alternately, the mechanism of reproductive inhibition may be through physiological charges caused by stimulation frcm the adult feele's pheranone. Physiological charges could result in a feels failing to mature, ovulate, implant or an interruption durirg fetal developent. In terms of physiological effects, the reproductive tract would show the most observable response to stimuli from adult females. Although the inhibition may have its fundmtal roots in the release of ovarian and uterine stimulatory hormones fren the pituitary, these hormonal charges should be reflected in follicular developent, growth of the ovaries and uterus and the estrous cycle. The experiments in this chapter were undertaken to emine the 40 41 effects of reproductive inhibition on the reproductive tract and the estrous cycle of inhibited feels white-footed mice (3. M) . Previous studies on the effects of adult feels presence and odors on young feels mice have shown that age at first estrous is delayed and reproductive organs are reduced in size (Cowley and Wise, 1972; Drickmer, 1979; Terman, 1979, 1984a,b; My arxi Vandenbergh, 1980) . The most logical mechanism for the inhibition seen in feels 2. 1m would be a long-term neturational delay or arrest. If neturation is delayed, then inhibited feeles should be found to be acyclic with sueller reproductive organs than controls and the ovaries lackirg corpora lutea. Epgiment y.- Determinatim 0f Estrous Wolirg. Test animals in the first experiment were either isolated control or inhibited adult feeles 120-150 days old. Inhibited feeles for purposesof thisandthereeiningexperiments inthisstudyaredefinsd as feeleg. Mthathavsbssnl'uesdwithanadult feelsfrom 21 days of age until tested at 120-150 days of age. Feeles in each of the two groups (n = 75/group) were lavaged once, usirg a 0.9% saline solution injected into the lumen of the reproductive tract and then withdrawn. Each lavage sample was then placed on a labeled slide, stained with Toluidine Blue, dried and examined umier the microscope. Criteria used for determinirg estrous stage were those of Vandenbergh (1967) . If, the inhibition is due to a lack of reproductive returation, then the inhibited feeles should be in a diestrous state. However, if the inhibited feeles have matured, they should resenble the estrous states of the isolated adult controls. Inhibited and control adult feeles in the first experiment were 42 found tobecyclixg (Table 8). Anixels inbothgroupswerefoundinall ‘0‘“ stages 01‘ “m- A X2 test of the feeles found no differences inthenunberof feelesinanyofthestages. Theseresultsshowthat inhibited feeles were not in maturational arrest as had been hypothesized, but were showixg a nonel estrous cycle. In addition, sperm was founi in smears from five inhibited feeles. Inhibited feeles, in addition to cycling normally were copulatirg with the adult neleintheege. Thefive feeleswersneintained forat least 35days after the lavages were taken to see if any litters would result. None ofthefeeleshadlittersbyasdayspost lavage. Theanimalswere then sacrifimd and their reproductive tracts examined. No evidence of placental scars or eilargene'xt of the uterus was evident to indicate a recent or orgoirg pregnancy. Given that inhibited feeles were cyclirg, the only conclusions that could be dram were that the feeles had a physiological nelfunction that prevented fertilization (lack of ovulation or sperm transport) or that fertilization occurred but implantation was blocked. when E: Effects Of Inhibition 0n Reproductive Organ Weights. Mice in the second experiment were divided into five groups. Groups 1-4 were adult feels mice in diestrous, mstestrus, proestrus or estrus respectively. As there are no data available on the effects of estrous state on weights of the tissues that were examined, these four groups served as controls. Group 5 consisted of inhibited feels mice at equivalent ages to their control counterparts (120-150 days). Feels mice in all five groups were sacrificed and their ovaries and reproductive tracts renoved. For purposes of this experiment, the 43 Table 8. Results of single vaginal lavages performed on inhibited and same-age isolated feels g. M for determination of estrous cyclirg. Grcmp Diestrus mtestrus Proestrus Estrus Spam Present Isolated females 24 17 15 19 - Inhibited feeles 27 13 19 16 5 x2 = 1.438 df = 3 n.s. 44 reproductive tract was considered to be the oviducts and uterus to a point just cranial to the urinary bladder. Prior to removal of the organs, total body weights and body lergths were taken to test for size biasbstweenaninelsinthsvarious treatmentgroups. Thetissues tobe emimd were placed in Perfix (Fisher Scientific Co. , Philadelphia, PA) for48hoursandtl'enplacedin708EtCX-Iforatleastseve1days. The fatwasthe'xrenoved, tremariesseparatedfranthereproductivetract and both tissues were weighed to the nearest 0.1 mg with a Mettler sirgle pan balance. This procedure provided a gross emiretion of the feeles's reproductive development. . The effects of inhibition on the gross morphology of the reproductive organs was also examined. Weights of the uteri and ovaries are seen in Table 9. A Kruskal—Wallis test of the uterine weights revealed tlet the uteri fran the inhibited feeles were signifiently heavier than any of the four control groups (P _<_ 0.05). At this point it is unclear whether the enlarged uteri in inhibited feeles are the result of the inhibitory stimrlus or the presence and/or stimulation fran copulation with the male. Control feels g. le_uc_ogg§ were isolated and not exposed to reles either physielly or olfactorally. AnANOVAanalysis of thepairedovarywsightswas found tobe significant (F = 4.31, df = 4, P 5 0.005). An a $193.31. orthogonal contrast was the) used to test the hypothesis that the inhibited feeles have szeller ovaries than any of the control feels groups. This test sl'rowed no significant differences between inhibited and control adult feeles in the weight of their ovaries with respect to the hypothesized contrast. In view of the results of the first experiment thisresultwastobeexpectsd. Theestrouscycleisdeperxientonthe 45 Table 9. Weights of uteri and paired ovaries (mg) of inhibited female 3. ms and same-age isolated fanales in each of the stages of the estrous cycle (n = 20/group). Inhibited Diestrus Metestrus Proestrus Estrus Uterine Wts. 148.97 69.55 84.52 68.40 64.60 SD 147.23 57.52 79.08 58.56 38.60 Paired Ovary Wts. 21.23 16.17 28.07 18.61 24.57 SD 10.08 9.15 8.93 7.35 14.18 Uterine Wts. (Kruskal-wallis): H = 9.619, P < 0.05 Ovarian Wts. (Orthogonal Contrast 1 vs 2-5): f = 0.06, n.s. 46 presence of ovarian.hormones for proper function. The females in.the first experiment appeared to be having normal estrous cycles and therefore functional ovaries would be expected. The ability of the ovaries to produce estrogen.and.progesterone does not necessarily indicate that ovulation was taking place. To make a deteminatim of whether or not ovulation.is occurring it was necessary to compare the number of corpora lutea present in the ovaries of inhibited and control females. §§p§giment 2;;.- Effects Of Inhibition.0n.NUmber 0f Corpora.Lutea. In.this experiment, the number of corpora lutea in the ovaries of inhibited fanales and 20 randcmly selected fanales fran the four control groups in.Experiment VI was determined. Corpora lutea counts were made by external examination under a dissecting microscope. This procedure has been shown to be nearly as effective as histological examination by serial sectioning in rabbits (Allen g al_., 1947), voles (Snyder. 1969 and Davis e_t 11., 1974) and 3. 1m (Haigh, unpub. data). Ewaluation of the reproductive organs, both.in.terms of gross weights and the number of corpora lutea, gave a good indication of how the inhibition.has affected the development and.function of the uterine and ovarian tissues. Failure to ovulate as indicated by an absence or low'number of corpora lutea or atrophy of the reproductive organs would indicate the inhibitory pheromones was affecting these organs via the pituitary gonadotrophic hormones. The corpora lutea count data are summarized in Table 10. Analysis of these data revealed no differences in the number of corpora lutea present (t = 0.6328, df = 1, ns). Inhibition, therefore, does not affect either the frequency of ovulation or the number of ova shed 47 Table 10. Corpora lutea counts of inhibited and control fenals g. 1%- Group n x + 150 Control fanale 20 12.25 + 5.40 Inhibited females 20 11.53 + 5.35 t = 0.6328, df = 38, n.s. pr of 0th, C1111l 48 during each cycle. A change in either of these variables would be reflected in the corpora lutea counts. These results, alorg with those of the first two experiments, indicated that the cause of the inhibition was post-copulatory. Discussion These experiments reveal that the reproductive inhibition oberved in young fenals g. m is a post-copulatory phamerm. The presence of similar stages of the estrous cycle in the inhibited fenales wtmcanparsdtocontrol falalesandthepresenceofspsminseveralof the lavages taken indicates that inhibited females were receptive to nale advances. No atrophicetion of either the uteri or ovaries was omerved to indicate dysfunction. In fact, the uteri of inhibited fanales were larger than their control counterparts, possibly a state of pseudopregnancy resulting fran male copulatory stimulation. The presence and similarity in the number of corpora lutea preclude ovulatory dysfunction as the mechanism for inhibition. Although not quantified, representative serial sections of the ovaries showed the presence of maturing follicles. This post—copulatory inhibition of reproduction is a unique effect of the social environment. Bruce (1959, 1960; 1965) described a pregnancy block associated with exposure of a recently inseminated femalehousemousstothepresenceorodorsofastrangeadultmals. Affected fanales fail to implant and return to estrus within 72 hours. MorerecsntworkhasshowmtheBruceEffect tooccurinanumbsrof other rodent species including: 3. maniculatus, Microtus We; and Microtus pinetorum (Brcmson g 3;. , 1964; Clulow and Clarke, 1968; Clulow and Iangford, 1971; Schadler, 1983). Dewebury (1982) also has 49 reported a pregnancy block in g. maniculatus fanales that were either sequentially rated by more than one male or exposed to a strange male for a short period of time inmediately following copulation. As with the Bruce Effect, females returned to estrus within a few days and will then successfully copulate. In both of these cases, the cause of the post-copulatory blockage was an adult male and only resulted in a short- tsrm delay in reproduction. The inhibition described for g. M here is the result of olfactory stimuli fran adult fanales and the effect persists as long as the stixmrlus remains. This effect differs fran other post-copulatory inhibitory effects both in terms of the stimulus source and the resultant physiological changes. These experiments do not enable an exact physiological explanation, however, several possibilities exist that merit discussion. Examination of the reproductive tracts fran a number of inhibited females revealed no evidence of placental scars or pregnancies in progress. This would seem to preclude reabsorption of the embryos or spontaneous abortion as the cause for lack of offspring. The only possibility for one of these mechanisms to be involved is if reabsorption took place very soon after implantation occurred. A more likely possibility is that the blocking process cams into play prior to or during implantation. One mechanism that could have caused the observed post-copulatory results is a lack of sperm transport facilitation. Without the proper conditions in the reproductive tract of the fenals, sperm might be very shortlived, tlmsnothavingenoughtinetoreachtheovafor fertilization. An imbalance in the hormones could prevent muscle contraction in the uterus that normally assist the transport of sperm. 50 Oxytocin is involved in facilitating sperm transport by increasirng the contractile motion of the uterus. If the effect of the adult female _ pheranone were to lower the mated female's ability to respond to copulatory stimulation by reduced output of oxytocin, the results described in these experiments would be deemed. Other hormones such asestrogsnshavebssnstnwntoaffsctthechemicalbalancsofths uterus. A change in the pH of the uterine secretions could result in an inhospitable environment for the sperm, causing high sperm mortality and lowering the probability of successful fertilization. Another possibility is implantation failure. Failure of a fennale to implant the blastocysts in the wall of the uterus has been shown to occur in several previous studies (Bruce, 1965; Dewebm'y, 1982). The Bruce Effect is apparently the result of an implantation failure. Likewise, the pregnancy block observed by Dewebury (1982) has been shown to result from a similar process. In the Bruce Effect, implantation failure results frcm lowered levels of prolactin (Bruce and Parkes, 1960) . Exogenous administration of prolactin during the exposure psriodmtsfleblockfrmxmrimasdoestheprmof functional ectopic pituitary graft (Dominic, 1966) or the fanals nursing a previous litter (Bruce and Parkes, 1961) . Coupled with this failure of the hypothalamus to release prolactin is the release of FSH and LH from the pituitary that results in resumption of the estrous cycle and ovulation (Dominic, 1966) . The physiological mechanism for the effect described by Dewebury (1982) has yet to be revealed, but the stimulus is similar to the Bruce Effect and except for the temporal variable, it can, with reasonable certainty, be assumed to be of a similar nature. The inhibition observed in this study is the result of adult female 51 stimuli rather than that of a male. The duration lasts as long as the adult fanale is present wdnerea in these other enamples the duration is only a few days. Therefore, it is reasonable to postulate that the underlying physiological charges are different as well. Any or all of the above mentioned mechanisne my play a role in the post—copulatory inhibition observed in this study. No data are available at this time to evaluate the validity of these hypotheses. Further work is needed to determine both the time that post-coitus inhibition occurs and the hormonal changes that resultf CHAPTER 4 BEHAVIORAL EFFECTS OF REPROIHCTIVE INHIBITION gymtheses Pre‘diction Those feeles that either are Inhibited and prepubertal feeles inhibited or can potentially will avoid the odors of adult be inhibited (prepubertal) feels conspecifies in both the will avoid stimuli that results five minute y-maze and 24 hour in a loss of lifetime repro- nestbox selection trials. Control ductive success. adult feeles will not show such an avoidance. The failure of feels g. m to reproduce or to experience a leg-term delay in having offspring represents a serious loss in lifetime reproductive success. This is especially true for sell mls such as g. _l_gu_c_omg that seldom survive through two bresdirg seasons (Gonmdie and Vessey, 1986). It would ,therefors, be advantageous for yourg fuels mice to avoid being inhibited. Avoidance behavior is dependent an recognition of the inhibitory or correlated stimuli and the ability of affected or susceptible feeles to move away frcmn it. The inhibitory stimulus was shown in Chapter 1 to be a chenosignal with reception occurring throxgh olfaction and the associated neural pathways. A yourg f‘le could have two types of encounters with the inhibitory stimulus, 1) a short-term encounter, such as mestirg or movirg into an area recently traversed and marked by an adult fmle, or 2) a leg-term encounter, such as cohabitation with an adult feels or utilizing a nest that recently has been abandoned. These two types of encounters differ in the quantity arnd duration of stimulus and may elicit different behavioral responses: dependent on the fmls's ability 52 53 to recognize the inhibitory signal, the strength of the signal and the physiology of the feels receiving the stimulus. Experiment VIII examined the behavioral responses of control and inhibited adult females to short-term encounters with the inhibitory stimulus. Experiment I)! evaluated the responses to long-term encounters based on nestsits selection of the same classes of false. Results fran these two experiments will yield information on a feele's ability to recognize and avoid the innhibitory signal. 993th V_I_I;.-Short-Tsrm Behavioral Response To Conspecific Odors. Experiment VIII dealt with the belevioral response of prepubertal, isolated "nonel" feeles and inhibited feels to conspecific odors. In this experiment, fuls mice were given a choice betweentwo odors in a Y—mazs to determine if they would avoid the inhibitory stinmrlus found in adult feels odors. Three types of ffils mice were used in this experiment as test subjects: 1) 21-25 day old prepubertal feels mice; 2) isolated adult females 75-150 days old annd 3) inhibited fmle mice 75-150 days. old. The Y-mazs used in this experiment was constructed of 0.64 cm thickness Plexiglass (Fig. 1). The arms of the apparatus had dimensiorns of 601:. x 10.2w x 10.21! cm annd were set at equidistant angles to one another. At the distal end of each arm was a renovabls release box (15.25 x 15.25 x 15.25 cm) with a 4.5 cm high window covered with 0.64 cm mesh hardware cloth forming the near wall. A 5.1 cm diameter hole in the far wall allowed placement of a sell fan just beyond the odor source. Each fan motor (1/500 hp) was equipped with a 3.8 cm diameter blade that gave a neximum air movenent rats of 0.66 m3/minute. Figure 1. Photograph of y—maze test apparatus. 55 This relates to an air velocity rate of 2.48 kin/hr or 1.42 exchanges per minute. Each of the two Y-nnazes used was pretested with water sprinkled on clean bedding placed in the odor boxes in both stimulus arms. Each apparatus was tested 20 times to verify that no unnknam external factors were biasing the choice of anus. Pretests of both Y-mazes indicated thattherewasnnobiasinthesystenn. Eadnmousewasplacedinthereleaseboxandrestrained from entering the maze by a Plexiglass partition for five minutes. After the five minmte adjustment period, the partition was lifted annd each meme was given ten minutes to choose between the two odors being blownn down the arms not originally occupied by the subject. Odors used were adult mle soiled bediing, adult fennale soiled bedding or clean bedding sprinkled with distilled water. Af‘lewassaid tohavenadeachoicewhenshepaweda linne 2O cmfranthedistalendofoneoftheodorarms. Anymmnsethatdidnnot mks a choice in the allotted time was called a "no test" annd the mouse was allowed at least 72 hours before retesting. A mouse that had two "no test" trials was disguarded and replaced. At the connclusion of each test run, the entire apparatus was washed with 95% EtOH, distilled water annd then dried to assure that no residual odors were present. Each fennale was tested using only one odor canbination. Sample sizes were 25 subjects per odor combinnation or a total of 75 juvenile, 75 control annd 75 inhibited fenales in the experiment. Analysis of these data provided information on the behavioral response of fenales to the inhibitory stimulus arnd two alternnate familiar odors. 56 Results of this experiment are shown in Figure 2. Juvenile feeles ted nno preference in the adult nels odor versus H20 dyad. A significant avoidannce of the adult feels odors was observed in both the adult feels odor versus H20 annd adult fuels odor versus adult male odor dyads. Theseresultswereasecpectedbasedonthsreproductivs neturity of the young feeles. The young feeles used in the first group, as stated previously, were prepubertal annd should nnot show the attraction to adult nels odors shown by their older, reproductivsly mature counnterparts. Control feels mice fed a significant preference for adult nels odors when tested against both water and adult fuels odors. No preference was observed in the central feeles in the adult feels odor versus water test. Inhibited feeles had a similar positive response toward adult male odors in tests versus adult feels odors or water. Inhibited feeles signnificantly avoided the adult ffile odors when tested againnst water, a result similar to that observed in tests of juvenile feeles. These data indicate a strong avoidannce on the part of the inhibited feeles for adult feels odors in a short-term enposure situation. Results from the Y—maze experiment inndicated tlet inhibited feels were identical to the isolated adult control fennales except that in both the adult feels odor versus water annd adult fule odor versus adult nele odor tests the inhibited feeles had a significant avoidance of adult fmls odors. This inndicates that the innhibited feeles were able to avoid the inhibitory stimulus. Prspubertal and inhibited feels avoidannce of the adult feels odors, whether because they avoided the Figure 2. Results of 5-minute y-nezs test for response to adult conspecific odors by control (uninhibited) 75-150 day old adult feeles, inhibited feels g. leucopu_s 75-150 days old and 21-25 day old prepubertal feels mice. The Y axis is the frequency of preference for the odors listed at the end of the bars. (0 = significant at the 0.05 level 118139 a X2 Goodness of Fit Test) Y-MAZE SELECTION BY FEMALE P. bucopus O c’ 376' . me_=_:________e___ E: _: _: sees . . _i____2:2:__:___:__=_:_:__:____:_.__eH. W.. o. ..--,----....- _ :E :EZZEEE : .-- e”. . . -- ._ :3: . as. 3:._:___.______._:______E_em. 58 inhibitory phereene or a separate but correlated stimulus are as yet unknown. Ineithsr case, theresult isthssams. Feeleswhoevade innhibition are able to successful 1y reproduce, while their inhibited counterparts expend energy annd time in finding a nele annd copulating with little or nno dance of success. Egriment at: Leg-Term Behavioral Response To Conspecific Odors. The second behavioral test was performed to observe long-term behavioral responses of fmle g. m to the inhibitory pherenons produced by adult feeles. Feels mice in this experiment, as in the short-term experiment, were either 1) 21-25 day old prepubertal feeles, 2) isolated adult fmles or 3) inhibited feeles 75-150 days old. Each meewasgiventreopportmitytodeossbetweentwonestbonesinwhich to construct a nest. The selection apparatus consisted of two Plexiglass cages (12.7 x 12.7 x 12.7 can) each connected by a 3.75 x 3.75 cm tunnnnsl, 50 on long to the release box (Fig. 3). A Nestlet (Ancare, Inc.), food annd water were placed in each cage. As in Bnperiment VIII, neutral tests of the six nestbox selection apparatuses were performed, with results showing nno choice bias in the systen. Each cage was also scented with adult nele soiled bedding, adult feels soiled bedding or cleann bedding sprinkled with distilled water. Each fmle was tested only onnce to insure tl'et "practice effects" did not come into play. SamplssizeswerethesamsasintheY-nezestudydescribedabove. Testing connsisted of placing each feels into the central release boxthroughadoorinthstop. Themousswasrestrictsdtotherelease box for five minutes to adjust to being moved. The guillotine doors at eitherendof thsrslsassboonweretheopenedanndthemousswas lsftin the apparatus for 24 hours. At the ennd of the test period the aninel Figure 3. Photograph of the nestbox selection apparatus. 60 wasrennovedandthecage innwhichanesthadbsenconstructed nnoted. The type of odor-stimulus put into any given nestboun was randomized to insure that unknown factors in the roan would not bias the results. This experiment was performed to test whether adult feels mics fren different social environments show any avoidance to the inhibitory pherannens relative to alternate familiar odors. An avoidance of the innhibitory stimulus would result in social spacing of fules annd possible population regulation. Experiment I)! examined the see odor dyads encspt in a long-term (24 h) setting. Results of the nnestbox selection test are shown in Figure 4. As in the Experiment VIII juvenile feeles had nno preference in the adult nele odor versus 320 dyad. A significant avoidanncs of the adult feels odors was observed in both the adult fmle odor versus H20 annd adult feels odor versus adult nele odor dyads. These results as in the previous experiment were expected based on the reproductive maturity of the young feeles. The control adult feels group, as in Experiment VIII, had a significant preference for male odors when tested against adult feels odors. Control feeles in this experiment showed nno preference in both adult male versus water and adult feels versus water comparisons. Inhibited ffiles were similar, showing nno preference in the adult nele versus water test. In the adult male versus adult feels and adult feels versus water inhibited feeles showed a significant avoidannce of adult feels odors. The data obtained using adult male odors is more difficult to explain. Inhibited annd control adult feeles in Experiment VIII were observed to rave the typical preference for adult nele odors tret was expected. The lack of preference by these feeles for adult Figure 4. Results of 24 hour nestbox selection test for response to adult conspecific odors by control (unninnhibited) 75-150 day old adult fules, inhibited feels g. lsucog 75-150 days old annd 21-25 day old prepubertal feels mice. The Y axis is the frequency of preference for the odors listed at the end of the bars. (0 = significant at the 0.05 1M1 using a X2 Goodness of Fit Test) NESTBOX ssLecnou av FEMALE P. loam CONTROL INHIBITED JUVENILE 62 nele odors in Experiment IX nay inndicats differences in long annd short— term betavioral responses to male odors. When deciding on a nestsite, an adult feels may revs a preference hierarchy with respect to if annd when she is wants to coinnkabit with. Results of Enqasriment IX inndicate this preference hierarchy is: 1) nno other animal present as the preferred situatien, 2) an adult nele being the intermediate choice annd 3) an adult feels being the least preferred cohabitant. Discussion Inhibited as well as conntrol feeles show a preference for adult nele odors, while prepubertal feeles do not. The results for the control feeles were expected. In several previous studies adult feeles lave been shown to prefer adult nele odors in odor choice experiments (Bronson, 1971; Vandenbergh, 1975). The inhibited feeles' preference for adult nele odors was also predicted based on the results of previous experiments. Previous studies on adult feels odor preferenceslevesrmthisphemnemtobsdependententrepresence of ovarian mnennes (Bronson annd Caroann, 1971; Johnston and Schmidt, 1979; Flening and Tambasso, 1960). The inhibited feeles, because of the presence of a functional estrous cycle annd nornel number of corpora lutea, were expected to have rnornel levels of estrogens annd progestegens annd therefore show the preference for adult male odors. Prepubertal feeles lave loner levels of ovarian hormones than adults end the lack of attraction to adult nele odors was predicted. The one unexpected finding was the lack of a preference for adult nele odors when tested against water in Experiment IX. This was nnot an effect of inhibition, as both control and innhibited feeles showed this lack of choice in the test. This discrepancy'was nnot apparent in the 63 shorter term Y—neze tests (Experiment VIII). These results ney reflect an important belavioral difference in the nennnner in which adult fmles respond to an adult male or their odors under different environmental circumstannces. A feels mouse could encounnter a nele under two basic sets of tennporal conditions. She might encounter a nele or his odor while traversing the habitat. This would be a short-term meeting, anelogonis to the Y—mazs experiment. A fennale mouse could also encounter a male or his odor while in the confines of a nestsite. If the feels stays for an extended period of time, a long—term encounter similar in duration to the nestbox selection experiment occurs. Males may be aggressive toward intruders into their nests, regardless of the sex or physiological state of the animal. Injuries to the fules attenpting to colabit the nestsite, or interference with acceptanncs of a fuels searching for a nte ney neke a feels occupying a nele nestsite unadvisable. Two adult 3. m only occasional 1y inrabit the same nestsite, preferring a solitary honesits (Nicholson, 1941; Trudeau gt _a_l_., 1980). The lack of adult nele preference when canpared to water in the nestbox selection experinnent nay be the result of a fmls preferring to inhabit an unnoccupied site rather that to either share it with a male or deal with the consequences of intruding. The preference for a nels's odors over feels odors in this case ney be a reflection of a hierarchy of cohabitation preferences. While a feels ney prefer to utilize an unoccupied nnestsits over staring one with a nele, she ney prefer to associate with a nele rather tnan annother feels. Studies by Mstzger (1971), Myton (1974) and Trudeau gt fl- 64 (1980) l'avs shown trat fmls g. M rave exclusive heeranges. The avoidannce of feels odors when tested against adult nele odors may be a related phenomenon. The nejor outcee of these two experiments was the avoidance of adult fule odors by prepubertal and inhibited feeles in all tests. These results clearly show an avoidance of adult feels odors by both classes of feeles. This is in contrast to the isolated control adult ffiles wlno showed nno preference in the adult fmles versus water tests in both experiments. This is an important belavioral change associated with reproductive inhibition. Feeles who are susceptible to the inhibitory stimulus with the ability to distinguish the odors of other adult feeles should move away fren the stimulus if they can associate the odor with the resultant inhibitionn. Movenent away frann the odor would result in the inhibited fules' recovery of their reproductive abilities or prevent prepubertal feeles from becanirg inhibited. The association of adult feels odors with inhibition by inhibited feeles would be sufficient to cause this result. An actual ability to recognize the pheromone responsible is not a necessity as long as the anniinel is able to recognize correlated olfactory cues. The results of Experiments VIII and IX show this to be true. Until such time tnat the pheromone is chenielly isolated and can be tested in its pure form, the discriminatory factor used by the mice will reein uncertain. Regardless of the discriminatory cues used by inhibited feels mice, the influence of avoidance on social structure and population regulation are clear. Movenent of inhibited fules out of an area containing odors from other feeles would result in a dispersal of prepubertal or inhibited 65 adult feeles to unoccupied areas of the habitat. This would result in a lowered density of fmles in a given area and a damping of density fluctuations. This process only appears to function in adult feneles that are inhibited. Uninhibitsd adult feels mice do not avoid other femals's odors. The pheromone, if it is only effective when exposure is begun at an early age, could lead to a stable number of reproductive feeles in a given area. Newly weaned fsneles would then be forced to chose between staying in an area and becaning inhibited or dispersing out of the area with the possibility of finding an unoccupied area, maturing and reproducing. GENERAL DISCUSSION Two lmowm social mechaniss activated by adult feels stimuli have the ability to reduce the reproductive success of young feels conspecifics. Maturational delay of prepubertal feeles results from exposure to the odors frcm grouped adult feeles (Vandenbergh. 1973). An inability to successfully bear offspring (reproductive inhibition) results franymngfeelembsingenpossdtotheodorsof individual adult feeles (Skryja, 1978; Haigh, 1983a,b). Maturational delays of prepubertal feeles due to chenosignals or social contact with other feeles has been well documented. (Written. 1958; Bronson and Dszsll, 1968; Bronson. 197i; Gremlin. 1971: Cowley and Wise, 1972 ; Vandenbergh, 1973 ; Drickamer, 1974; McIntosh and Driclemer, 1977; Tsnen, 1979. 1984; Massey and Vandenbergh, 1980). Maturational delay of young feels mice has been proposed as: 1) a mechanism for decreasing the number of potentially reproducing feeles in an area, thus increasing the relative reproductive success of the feels emitting the stimulus, 2) a mechanism for inbreeding avoidance and 3) as an adapetion by young feeles that allows then to delay their first pregnancy, increasing their lifetime reproductive success. This study found that reproductive inhibition is not the result of a long-term maturational delay (see Chapter 3) , but a postcopulatory block during the first third of gestation. However, an inability to reproduce regardless of its duration is subject to the same types of selection pressure. The duration of the reproductive delay modifies the strength of selection, but the fundamental forces acting in selection 66 67 are the same. The present study is concerned with a more detailed description of reproductive inhibition, not maturational delays. An eminetion of the three above hypotheses as they relate to both phenncnena yields a clearer picture of the effects of the social environment on the reproductive biology of feels mics and the roles of social stimuli in affecting the population dynamies of Peromyscus. The hypothesis that maturational delay functions to give adult feeles a selective advantage over young feeles recruited into the population fits both laboratory studiss and data from the field. Reproduction in natural populations is achieved by only a sell proportion of the feeles. Only 4496 (83/189) of the adult 2. leucggg monitored by Gcmndis and Vesssy (1986) ever produced a litter. This contrasts with studies in the laboratory where 65-70% of females producing litters is cannon and 75-80% is not unusual (Skryja, 1978; Leamy, 1981; Haigh,1983a,b; Myers and Master, 1983). Delaying initiation of a feele's estrous cycle for as short a time as a week can result in a significant advantage in reproduction to those feeles that are already cycling. Vssssy and Haigh (unpub. data) found an average life expectancy value of 84 days for field dwelling feeles once they entered the trappabls population (12-14g) . If we assume that a feels weighing 12-14g is 21 days old (Layne, 1968), the average lifespan of a feels under natural conditions is 105 days. Assuming that feeles begins cycling when 40 day old, then a one week delay would reduce the number of fertile periods by 1196. Subtraction of a 25 day gestation period and a 21 day lactation and maternal care period from the prepubertal lifespan reduces the impregnation window to 19 days. A 68 seven day maturation delay reduces the inpregnation window to 12 days or results in a 37% reduction in the number of usable fertile periods. Feeles already cycling therefore gain two fertile periods (3 or 4 day cycle) over that of yang females who will be nnnabls to conceive offspring or utilize reproductively active males due to nonreceptivity of the yang feeles. ’ The hypothesis that neturational delay is a mechanism preventing inbreedingisweakerthantheargunent that itreducesthsnumberof fenale competitors. Delaying the maturation of yang feels mice may prevent inbreeding as the probability of maturing prior to dispersal is reduced (Skryja, 1978). Mnils the premise that dispersal occurs prior to maturity is probably true, analysis of currently available data on dispersal indicate that distances moved by yang animals are insufficient to avoid relatives and support the inbreeding avoidance hypothesis. Shields (1982) stated that g. maniculatus have an average dispersal distance of 1.0-1.6 home range diameters based on data from Dice and Howard (1951). Similar calculations show that 3. 1m have an average dispersal distance of 1.9-2.2 home ranges diameters (Goundie and Vessey, 1986; Wolff. 1985). These figures are both well within within the 10 hane range diameter limit for classification as a philopatric species (Shields, 1982) . While any amannt of dispersal may reduce the potential for inbreeding, these distances are insufficient to prevent its occurrence. Field studies by Howard (1949) and Spevak (psrs. cam.) of §_._ maniculatus have reported that a number of observed litters resulted from inbreeding. With this evidence available it seee unlikely that maturational delays function for inbreeding avoidance. A third hypothesis regarding nturational delay is that it has 69 evolved as an adaptation to prevent feeles from becaning pregnant nnntil they are older (Skryja, 1978) . Myers annd Master (1981) have stated that a positive correlation exists between the weight of a feels annd the size of litter and weight of the offspring she will bear. The weights ofyang feeleg. Whammtreachedthat of olderadultswnhen she begins cycling. Yang feeles who avoid becaning pregnant until they are older and heavier may be at a selective advantage over those yang feeles who do nnot. A neturational delay as stated previansly lowers the number of post-pubertal days that a fennale has available for breeding. To be advantageous for the feels to be maturationally delayed, either the life expectancy of the feels must be increased or the litter size and offspring survival rate inncrsased sufficiently to surpass the lifetime reproductive success of fennales that are not delayed. Of the three hypotheses that attempt to explain the fnnnction of the maturation delay of yang feels Perennyscus none of the hypotheses can be conclusively ruled out. The feels cannpstition hypothesis is supported by field and laboratory evidence, but not to the exclusion of the other two. The inbreeding avoidanncs hypothesis has the least annannt of evidence to support it but cannot be discarded. The yang feels reproductive advantage nnodsl has some ancillary laboratory support, but field experimentation and additional data are needed before a complete evaluation can be nnnade. Although the fnnnction of maturational delay to the inndividual reeins unclear, its effect on population dynamies is evident. Delaying the maturation of inndividuals in the population lengthens the generation St‘ 70 time and lowers population growth. The najor question that reeinns from a population perspective is nnot whether it has an effect, but the frequency and magnitude of the effect. Mnils maturational delays have the potential to serve several fnnnctional roles in individuals and affect population dynmics, annothsr effect of the social environment may enert an even greater influence on feels reproduction. In studies of yang §_._ srsmicus (Skryja, 1978), P_. nanniculatus annd _P_._ M (Haigh, 1983a,b), reproduction by yang females in the mence of an adult feels was almost cannpletsly inhibited, while those egedwithanadult nelsalonnehavsanornel numbsrof litters. The inhibitory effect lasted as long as the adult feels was present to reinforce the stimulus. In evaluating its potential to decrease reproduction, determination of the ennamt of time nneeded for inhibited females to recover was required. In social nnnits containing the adult feeles continnuously, yang feeles reeinned inhibited. Those young feeles freed from exposure to adult females at 150 days old recovered and began to have litters. The average recovery time was 49 days, calculated by backdating the 25 days of gestation frann the date of the first litter. As previously discussed, a maturationnal delay of seven days in the time of first estrus has a significant impact on potential reproduction. A recovery time of 49 days even if begun on the first day of inhibition would eliminate reproduction for the lifetime of a large percentage of feeles under field conditionns. Evaluation of the effect of inhibition on the inndividual or the population requires that we unnderstand the trannsnnission mechanism of the stimulus to the yang feeles annd the amannt of enposurs sufficient to in is 71 invoke the inhibitory response. Although the inhibitia'n is robust in its duration, if it requires an unrealistic amount of contact between yang feeles and the inhibitory stimnlus, it is nnnlikely to influence reproduction in natural populations. Soilsd bedding fran adult feeles, either in direct contact with the yang feels or the odors frann it produwd the innhibitionn. Urine from adult feeles was show to be the effective inhibitory agent. Inhibition in the absence of an adult feele's presence indicates that aggression or daninancs are not necessary for inhibition to ocanr. A chencsignalorpheranonepresent intheurinneofthsadult feels isthe stimulus responsible. The effectiveness threshold for inhibition is between one end three hours per day. The low amannt of daily exposure (3 hrs/day) sufficient for innhibitionn to ocanr denonnstrates the potency of the pheranone. A yang feels spending only three hours per day either sharing a nestsite with an adult feels or using a nnestsits recently ocanpied by one is vulnerable to being inhibited over a substantial portion of her potential reproductive life. Mnile adult feeles benefit from the lack of reproduction by their yanger competitors, selection anneg young feeles in this situation should favor behaviors that lead to avoidannce of inhibition, allowing reproduction to occur. It is possible that the yang feeles caild benefit from inhibition, mt due to the duration of inhibition relative to the lifespan of mice unnder natural conditions, it is nnnlikely. The inhibitory pheranone can be avoided either directly by avoiding encounters with the inhibitory stimulus or indirectly as a coincidental 72 effect of a separate process. Behaviors such as dispersal by yang feeles or their exclusion by the territorial defense of the adult fenale thatarenottheresultofaresponsetothepheromonewouldbe indirect or passive avoidance. Yang females, or those already inhibited, that are able to recognize the odor and avoid it would be slnciwing active avoidannce, the result of selection for avoidance of the phsranone. In the Y-naze test both control and inhibited feeles shoved a significant preference for the arm containing adult male odors. Inhibited feeles avoided the odor of adult feeles when tested againnst either adult nele odors or water. In the nestbox selection experiment, both control annd inhibited fenales showed a preference for the adult nele odor, but only the inhibited feeles showed the avoidance of adult feels odors. These results indicate that inhibited females are able to recognize the odors of adult females and actively seek to avoid then. Feeles both in a short-term setting such as encanntering the odor while moving thraigh the habitat, or on a longer time frame, such as setting up a residence, actively avoid the inhibitory stimulus. Tests with prepubertal feeles (21-25 days old) gave similar but not identical responses to those of the inhibited feeles. Young feeles avoiding the odors are less likely to become inhibited annd lose their own reproduction. Movement art of areas, or non-use of nestsites containing the odor, alleviate potential competition with those feeles in residence. If avoidance by yang feeles precipitates their movenent out of an area already ocaipied by adult feeles, it will decrease the growth rate of the population annd therefore act as a buffering mechanism. The question still renains 73 whether this phenomenon is effective in natural populations. Feels m M have been reported to maintain exclusive home ranges in a number of. studies (Metzgar, 1971, 1973; Myton, 1974; Hansen and Batzli, 1978; Wolff et. al., 1983). The exclusivity of these areashasbeenhypothesizsd to result fromaggrsssiononthspartof resident feeles (Vestal annd Hellack, 1978; Wolff e_t. al., 1983, 1985). Hans ranges of g. _l_e_u__com§ range fran 242 m2 (Cranford, 1984) to 3000 - m2(Stickls, 1968; Mstzgar,1973). The amount of time and energy that a feels would have to devote to maintaining a territory of this area would be inlnense. If a resident adult feels were able to distribute a chemosignnal in an area that prevented transient mice from establishing residence, she would be energetically more efficient in maintaining her territory annd avoid the risk of injury from physical confrontationns. This exclusivity would be dependent on the duration of the chemosignals' effectiveness and the quantity nneeded to sufficiently cover the area. Adult feels g. m aner the reproductive portion of the year are rarely fannd to ocanpy the same nestbox (Trudeau e_t. a_l_, 1980). Must feeles are fannd to regularly occupy several adjacent nestboxse on the study site (15.2m spacing). Similar results have also been doanmented for g. manniculatus (King, 1983). Resident fennales maintaining exclusive use of an area would be more effective in doing so if they occupied several nestsites depositing their odors in each. This would make more of the nest sites unfit for yang feeles in the population than if each adult fennale utilizsd'a single Inset. Distribution of the odor in nestsites would also be more effective by being in a more contained area. Wolff (1985) has stated that surplus 74 nestsites do not affect populationns of white—footed mice. However, if resident feeles inncrease the number of nestsites that they utilize and deposit odors in, the number of feeles in the population would be less likely to inncrease. The number of nestsites might be a limiting factor to yang feeles even though they are not connstantly occupied. Conspecifics ney nnot be the only canpetitors that a feels en encamter. The inhibition nnnder disanssion is present in both 3. neniculatus annd g. M. In much of their range, both species utilize the same or adjacent areas. In Michigan, while the former species is prinerily fannd in open grassy areas and the later in wooded habitat, overlaps exist (Master, 1977). In Virginia a different subspecies of deer mouse g. neniculatus nubiterrae is also resident in the woodlots that P_. lsn_ig_ogn_s_ are founnd in (Wolff, 1986). Is it possible that feeles frcnn one of these species might have the ability to inhibit yang females of the other? In this study, 21 day old feels micsweregroupedwithanadultneleof theirsamespsciesandanadult feels of the alternate species. Comparisons of yang feeles exposed to interspecific stimuli with granps containing 1) nno adult feels present and 2) connspscific feeles present indicete that the pheromone produced by either species is nnot effective in inhibiting yang feeles of the other. The chemosignal that has been described is not a usable tool in interspecific scenarios. Either the cheniel composition of the pheromone or receptors of the yang feeles are sufficiently diverged to preclude their effectiveness. neturational delay and reproductive inhibition of yang feels Psron_nnysa.ns are both braght abait by chenosignnals from adult feeles. In each case, the benefit to the feels that releases the chenosignal is 75 reduced reproductian by other feeles in the populatian. In view of the physiological constraints preventing feeles from increaing their litter sizes or reducing the duration of gestation, selection for nechanieethatreducsthereproductionofother feelesmaybsthecnnly process to maximize a feele's lifetime reproductive success. 0n the other lend it is possible that selection for these mschaniens was due to an advantage to the yang feeles being affected. If this is the we thenthslossinreproductiveoutpntdustothsdslaymnstbssurpassed by inncreased future reproduction. While this is a plausible hypothesis for delayed neturation, reproductive innhibition is so long lasting that it is highly unlikely that a benefit cen be shown. The tenporal evolutionnary sequence that produced the neturationnal delay phennanenon is ennbiguous. whether 1) a mutation occurred that resulted in the ability of adult feeles to produce the pheromone with the yang feeles already having the receptive pathways in place or 2) a mutation occurred in yang feeles that delayed neturation while the phsranonewesalreadybsingproducedasabyproduct ofsomeother process aweits mmbiguous evidence of the costs annd benefits of maturation delay. In the cess of reproductive inhibition the evolutionery process is more easily defined. A mutation occurred which allowed adult feeles to affect the reproduction of their yangsr cannterparts thrangh an already present physiological pathway. Given the implausibility of any benefit to the inhibited feels annd the potential cost in lifetime reproduction, a mutation that occurred allowing yang feeles to be inhibited by a stimulus already present in the urinne of adult feeles would be quickly lost from the gene pool. 76 If the above sequence is correct, the hypothesis that maturationn delay annd reproductive innhibition are separate events in the evolution of Pemans nmnst be connsidered true. For species such as g. leucopug where 98% of yang feeles exposed to adult feeles are inhibited the abilitytoproducethepheromoneneybsfixed. Otherspeciessuchasg. erennials that only show 65-70% inhibition may still be in the selection processorareversal oftheselectionpressureneyhaveoccurred. However the original occurrence of the inhibition is sufficiently distant that divergence has occurred between species so that the pheranone given off by adult feeles of one species is ineffective in ceusing innhibition in another. Intermof futm‘eworkinthisarea, threegenneraltypesof studies are needed. First, too few studies have taken the comparative approach. While a substantial body of lmowledgelhas acanmilatsd on the influence of the social environment on reproduction in a few species. thsyrment lessthannofthsldmspsciseofnenmals. Partiallarly, very little is known about either the behavior or reproduction of those uennels that either reside in tropical areas or have limited ranges. With such a small sample size of species that have been studied in detail, the effects of chenosignnals as they effect behavior annd reproduction have barely begun to be exploited. A rennewed effort needs to be nede nnot only to evaluate those specie that have been studied in more detail, but to look at other species that have at present been largely ignnored. The second area that merits additionnal effort in the future is that of field vsrificetion of laboratory studies. Many researchers, including myself, have bennoannsd the difficulties of working with 77 chenosignals in netural populationns. More creative metlnods of field studies, incorporating nestboxes, radiotslennstry, isotope labeling of aninels, etc. nneed to be need in addition to the standard nerk-recepture data collections. Experimental nenipulations and evaluation Of behavioral interactions in natural populations will always be probl‘tic , but they are possible. Our ability to distinguish between hypotheses concerning the functions of these phenomena will renain only speallations until tests nnndsr field conditionns have been nede. Finally, little attention has been given to looking at the evolution of social influences,in partiallar chenosignnals, on reproduction. 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