Aromatase inhibition during adolescence reduces adult sexual and paternal behavior in the biparental dwarf hamster Phodopus campbelli

Previous studies have failed to identify an activational role for estradiol in the paternal behavior of Phodopus campbelli fathers. However, none of these studies addressed a developmental role that estradiol might play in establishing paternal behavior in this species. Males were orally administered the aromatase inhibitor letrozole (1 mg/kg/day) for three days at 18, 34, or 90 days of age. As adults, males were tested for paternal and sexual behavior. Letrozole treatment at 18 days resulted in males that spent less time huddling over pups during the birth, and had higher pup losses and male-biased pup survival for the first litter. Letrozole treatment at 34 days resulted in males that had altered sexual behavior; males had a longer interval between mounts and between intromissions, and took longer to achieve ejaculations over the first three ejaculatory series. Furthermore, these males sired smaller first litters and produced second litters with a male-biased sex ratio. Males treated with letrozole as adults showed a modest increase in paternal care during the birth, but pup development and survival were not altered. There was no effect of treatment on attack or retrieval behavior either as sexually naive adults or as new fathers. Thus, the results of the present study suggest that estradiol acts during adolescence to establish the normal expression of midwifery behavior and sexual behavior during adulthood.     

Paternal responsiveness in biparental dwarf hamsters (Phodopus campbelli) does not require estradiol

Males of the biparental hamster species Phodopus campbelli act as midwives and are responsive to an experimentally displaced pup. Males also have peripheral estradiol concentrations that are similar to conspecific females. Castration reduces peripheral estradiol, yet does not affect paternal responsiveness despite the known role of estradiol in maternal behavior. Synthesis of estradiol within the central nervous system, however, might not be affected by castration. Males received implants of osmotic pumps containing the aromatase inhibitor letrozole to reduce both peripheral and central estradiol concentrations. Though estradiol was effectively reduced, it had no effect on paternal responsiveness or reproductive success. Neither testosterone nor aggression directed towards an intruder was altered. Results support the emerging conclusion that estradiol is not required for the exceptional paternal behavior of male P. campbelli.     

Estradiol and progesterone in paternal and non-paternal hamsters (Phodopus) becoming fathers: conflict with hypothesized roles

Phodopus campbelli has an extensive paternal behavior repertoire whereas the closely-related Phodopus sungorus is not paternally responsive to a displaced pup. For the first time in a naturally paternal mammal, male estradiol and progesterone were determined during two critical phases: (1) the transition from sexually naive male to paired, expectant father that occurs in the absence of stimuli from pups (sexually naive males, paired males on G8, G12, G15, or G17 of the 18-day gestation) and (2) after pup stimuli became available to the males (paired males on days L1, L3, L5, or L12 of pup development). Hormone concentrations in naive males and between G17 and L1 (as stimuli from the birth and the pups became available to males) were also compared. Paternal responsiveness was tested on L3-L5 and confirmed species differences. Hormone concentrations in naive males were similar in the two species and males of both species had estradiol concentrations as high as fertile adult females. However, in direct contrast to predictions, estradiol concentrations were stable in P. campbelli males but increased before the birth, fell across the birth, and increased over pup development in P. sungorus males. Progesterone concentrations in P. campbelli males increased from G17 to L1 whereas a decrease had been predicted. Testosterone dynamics were consistent with previous studies. Either hormonal facilitation of paternal behavior is a hyper-variable trait that has evolved differently in different species, or, more probably, peripheral hormone concentrations are inadequate to explain the role of sex steroid hormones in paternal behavior.     

From here to paternity: Neural correlates of the onset of paternal behavior in California mice (Peromyscus californicus)

In a minority of mammalian species, including humans, fathers play a significant role in infant care. Compared to maternal behavior, the neural and hormonal bases of paternal care are poorly understood. We analyzed behavioral, neuronal and neuropeptide responses towards unfamiliar pups in biparental California mice, comparing males housed with another male (“virgin males”) or with a female before (“paired males”) or after (“new fathers”) the birth of their first litter. New fathers approached pups more rapidly and spent more time engaging in paternal behavior than virgin males. In each cage housing two virgin males, one was spontaneously paternal and one was not. New fathers and paired males spent more time sniffing and touching a wire mesh ball containing a newborn pup than virgin males. Only new fathers showed significantly increased Fos-like immunoreactivity in the medial preoptic nucleus (MPO) following exposure to a pup-containing ball, as compared to an empty ball. Moreover, Fos-LIR in the bed nucleus of the stria terminalis (STMV and STMPM) and caudal dorsal raphe nucleus (DRC) was increased in new fathers, independent of test condition. No differences were found among the groups in Fos-LIR in oxytocinergic or vasopressinergic neurons. These results suggest that sexual and paternal experiences facilitate paternal behavior, but other cues play a role as well. Paternal experience increases Fos-LIR induced by distal pup cues in the MPO, but not in oxytocin and vasopressin neurons. Fatherhood also appears to alter neurotransmission in the BNST and DRC, regions implicated in emotionality and stress-responsiveness.     

Testosterone response to courtship predicts future paternal behavior in the California mouse, Peromyscus californicus

In the monogamous and biparental California mouse (Peromyscus californicus), paternal care is critical for maximal offspring survival. Animals form pair bonds and do not engage in extrapair matings, and thus female evaluation of paternal quality during courtship is likely to be advantageous. We hypothesized that male endocrine or behavioral response to courtship interactions would be predictive of future paternal behavior. To test this hypothesis, we formed 20 pairs of California mice, and evaluated their behavior during the first hour of courtship interactions and again following the birth of young. We also collected blood from males at baseline, 1 hr after pairing, 3 weeks paired, and when young were 4 days old to measure testosterone (T). We found that male T-response to courtship interactions predicted future paternal behavior, specifically the amount of time he huddled over young when challenged by the temporary removal of his mate. Males that mounted T increases at courtship also approached pups more quickly during this challenge than males who had a significant decrease in T at courtship. Proximity of the male and female during courtship predicted paternal huddling during a 1-hr observation, and a multiple regression analysis revealed that courtship behavior was also predictive of birth latency. We speculate that male T-response to a female in P. californicus is an honest indicator of paternal quality, and if detectable by females could provide a basis for evaluation during mate choice.     

Castration reduces male testosterone, estradiol, and territorial aggression, but not paternal behavior in biparental dwarf hamsters (Phodopus campbelli)

Biparental male hamsters, Phodopus campbelli, act as midwives during the birth of their litter and are highly responsive to an experimentally displaced pup. They also have high peripheral concentrations of estradiol, a hormone with known roles in maternal behavior. Surgical castration during the gestation of their first litter was used to investigate the source of that estradiol and the functional role of testicular sex steroids in paternal responsiveness. In Experiment I, castration reduced both testosterone and estradiol concentrations, confirming that the testes were the primary source of estradiol. However, neither paternal responsiveness nor multiple measures of reproductive success were altered by the castration. Aggression directed towards an intruder, however, was reduced by castration. In Experiment II, removal of prior experience with birth or pups also failed to alter paternal responsiveness in castrated males. Although the present results do not preclude a role for local estradiol synthesis in the brain, results do not support an association between high circulating estradiol in males and their paternal behavior.     

Effects of repeated pup exposure on behavioral,neural, and adrenocortical responses to pups in male California mice (Peromyscus californicus)

In biparental mammals, the factors facilitating the onset of male parental behavior are not well understood. While hormonal changes in fathers may play a role, prior experience with pups has also been implicated. We evaluated effects of prior exposure to pups on paternal responsiveness in the biparental California mouse (Peromyscus californicus). We analyzed behavioral, neural, and corticosterone responses to pups in adult virgin males that were interacting with a pup for the first time, adult virgin males that had been exposed to pups 3 times for 20 min each in the previous week, and new fathers. Control groups of virgins were similarly tested with a novel object (marble). Previous exposure to pups decreased virgins' latency to approach pups and initiate paternal care, and increased time spent in paternal care. Responses to pups did not differ between virgins with repeated exposure to pups and new fathers. In contrast, repeated exposure to a marble had no effects. Neither basal corticosterone levels nor corticosterone levels following acute pup or marble exposure differed among groups. Finally, Fos expression in the medial preoptic area, ventral and dorsal bed nucleus of the stria terminalis was higher following exposure to a pup than to a marble. Fos expression was not, however, affected by previous exposure to these stimuli. These results suggest that previous experience with pups can facilitate the onset of parental behavior in male California mice, similar to findings in female rodents, and that this effect is not associated with a general reduction in neophobia.     

Mongolian gerbil fathers avoid newborn male pups, but not newborn female pups: olfactory control of early paternal behaviour

We examined effects on the parental behaviour of male Mongolian gerbils, Meriones unguiculatus, of the sex and number of pups in a litter. Recent fathers interacting with foster litters consisting entirely of newborn males decreased the time that they spent in contact with a litter the greater the number of pups it contained. However, fathers that interacted with litters composed entirely of newborn females showed no change in the time they spent in contact with a litter as a function of its size. Fathers responded similarly to litters of 1- and 3-day-old female pups, but their responses to male pups changed from avoidance to approach as the age of males increased from 1 day to 3 days, and fathers made anosmic by intranasal administration of zinc-sulfate solution did not avoid neonatal litters. Results of a correlational study revealed that the more time males spent with newborn young during a 30-min test, the greater their latency to mate with their partners in postpartum oestrus and the shorter the duration of their mating effort during the 24 h immediately after parturition. We discuss these findings as consistent with the view that androgen-mediated olfactory stimuli produced by newborn male Mongolian gerbils make them unattractive to fathers, possibly functioning to increase the time that recent fathers mate-guard while their partners are in postpartum oestrus.     

Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles (Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.     

Hormonal stimulation and paternal experience influence responsiveness to infant distress vocalizations by adult male common marmosets,Callithrix jacchus

Parental experience and hormones play a large role in the common marmoset (Callithrix jacchus) father's care of their offspring. We tested the effect of exogenous estradiol or testosterone on the responsiveness of common marmosets to respond to infant distress vocalizations and whether males who haven't become fathers yet (paired males) would have increased responsiveness to infant distress calls with either steroid or whether parental experience is the most important component for the onset of paternal care. Sixteen male marmosets (8 fathers, 8 paired males) received a vehicle, low dose or high dose of estradiol and additional 16 males were tested with testosterone at three doses for their response either to a vocal control or a recording of an infant distress call for 10 min. Without steroid stimulation fathers were significantly more likely to respond to the infant distress stimulus than paired males. Low dose estradiol stimulation resulted in a significant increase in fathers' behavioral response towards the infant distress stimulus but not in paired males. Fathers also showed a significant increase in infant responsiveness from the vehicle dose to the estradiol low dose treatment, but not to the estradiol high dose treatment. Testosterone treatment did not show significant differences between infant responsiveness at either dose and between fathers and paired males. We suggest that neither steroid is involved in the onset of paternal care behaviors in the marmoset but that estradiol may be involved in facilitating paternal motivation in experienced fathers.     

Prolactin and paternal behavior in the biparental California mouse, Peromyscus californicus

Relatively little is known about hormonal mechanisms underlying paternal behavior in mammals. Male California mice, Peromyscus californicus, display extensive parental care toward their young. Parental behavior of fathers, expectant fathers (males living with their pregnant partner), and virgin males was assessed in a 10-min test with a 1- to 3-day-old alien pup. Few virgin males acted parental (19%) compared to fathers one day postpartum (80%) and expectant fathers (56%). Plasma prolactin levels were significantly elevated in fathers 2 days postpartum compared to expectant fathers and virgin males. Paternal prolactin levels were similar to those of mothers. There were no differences between groups in levels of plasma testosterone. These data suggest, contrary to other reports, that prolactin is a likely correlate of paternal behavior in rodents.     

Prolonged use of letrozole causes morphological changes on gonads in Galea spixii

Letrozole is used as a therapeutic agent in reproductive disorders caused by high estrogen levels. Letrozole inhibits cytochrome P450 aromatase and reduces estrogen levels. However, the effects of long-term use on reproductive traits are unknown. The aim of this study was to evaluate the prolonged use of letrozole in the gonads of rodents (Spix''s yellow-toothed cavy; Galea spixii). Forty-eight rodents (24 males and 24 females) were randomly divided into the treated and control groups. Letrozole administration started at 15 days of age and continued weekly until 30, 45, 90, and 120 days of age. The body, testis, and ovary weights were analyzed, as well as the morphological progression of spermatogenesis and folliculogenesis. Macroscopically, body weight gain and gonads weight were increased in the letrozole group. Microscopically, the ovaries of treated females showed stratified epithelium and a cellular disorder of the tunica albuginea. In the testes of treated males, the development of seminiferous tubules was delayed and sperm was absent. The collective findings indicate that the prolonged use of letrozole alters secondary sexual characteristics, and causes weight gain, reproductive changes, and male infertility.     

Chronic variable stress in fathers alters paternal and social behavior but not pup development in the biparental California mouse (Peromyscus californicus)

Stress and chronically elevated glucocorticoid levels have been shown to disrupt parental behavior in mothers; however, almost no studies have investigated corresponding effects in fathers. The present experiment tested the hypothesis that chronic variable stress inhibits paternal behavior and consequently alters pup development in the monogamous, biparental California mouse (Peromyscus californicus). First-time fathers were assigned to one of three experimental groups: chronic variable stress (CVS, n = 8), separation control (SC, n = 7), or unmanipulated control (UC, n = 8). The CVS paradigm (3 stressors per day for 7 days) successfully stressed mice, as evidenced by increased baseline plasma corticosterone concentrations, increased adrenal mass, decreased thymus mass, and a decrease in body mass over time. CVS altered paternal and social behavior of fathers, but major differences were observed only on day 6 of the 7-day paradigm. At that time point, CVS fathers spent less time with their pairmate and pups, and more time autogrooming, as compared to UC fathers; SC fathers spent more time behaving paternally and grooming the female mate than CVS and UC fathers. Thus, CVS blocked the separation-induced increase in social behaviors observed in the SC fathers. Nonetheless, chronic stress in fathers did not appear to alter survival or development of their offspring: pups from the three experimental conditions did not differ in body mass gain over time, in the day of eye opening, or in basal or post-stress corticosterone levels. These results demonstrate that chronic stress can transiently disrupt paternal and social behavior in P. californicus fathers, but does not alter pup development or survival under controlled, non-challenging laboratory conditions.     

Acute effects of corticosterone injection on paternal behavior in California mouse (Peromyscus californicus) fathers

Glucocorticoids are thought to mediate the disruption of parental behavior in response to acute and chronic stress. Previous research supports their role in chronic stress; however, no study has experimentally tested the effects of acute glucocorticoid elevation on paternal behavior. We tested the prediction that acute corticosterone (CORT) increases would decrease paternal behavior in California mouse fathers and would lead to longer-term effects on reproductive success, as even short-term increases in CORT have been shown to produce lasting effects on the hypothalamic-pituitary-adrenal axis. First-time fathers were injected with 30 mg/kg CORT, 60 mg/kg CORT or vehicle, or left unmanipulated. Interactions between the male and its pup(s) were recorded 1.5–2 h after injection and scored for paternal and non-paternal behavior. Treatment groups were combined into control (unmanipulated + vehicle, n = 15) and CORT (30 mg/kg + 60 mg/kg, n = 16) for analysis based on resulting plasma CORT concentrations. CORT treatment did not alter paternal or non-paternal behaviors or any long-term measures (male body mass or temperature, pup growth rate, pup survival, interbirth interval, number or mass of pups born in the second litter). Fathers showed a significant rise in body mass at day 30 postpartum, followed by a decrease in body mass after the birth of the second litter; however, this pattern did not differ between the CORT and control groups. In summary, acute elevation of plasma CORT did not alter direct paternal behavior, body mass, or reproductive outcomes, suggesting that acute CORT elevation alone does not overtly disrupt paternal care in this biparental mammal.     

Interactions among paternal behavior, steroid hormones, and parental experience in male marmosets (Callithrix kuhlii)

Male black tufted-ear marmosets (Callithrix kuhlii) contribute to the rearing of their offspring. Here we evaluated predictions of hypotheses suggesting that (1) T and E2 influence infant-care behavior in male marmosets, (2) levels of T and E2 are modulated by paternal experience, and (3) paternal behavior and levels of T and E2 in male marmosets covary with stress. We observed the behavior of marmosets in their family groups following the birth of infants and evaluated urinary concentrations of T, E2, and the stress hormone cortisol (CORT) among fathers before and after the birth of young. Urinary levels of T, E2, and CORT were lower among males who carried infants at high rates than males who carried at low rates, and T and CORT levels were negatively correlated with carrying rates across all males. Males had significantly lower T levels while carrying the second compared to the first litter and slightly lower rates of infant-carrying, possibly due to assistance provided by offspring of the first litter. There were increases in CORT levels of fathers after the birth of the first litter, but decreases in CORT after the birth of the second. Our results suggest a relationship in C. kuhlii between paternal behavior, hormones, and paternal experience. Rates of infant-carrying appear to be linked to hormone levels, and hormone levels in turn are affected by experience caring for young. Our data also suggest that T, E2, and CORT have synergistic influences on infant-carrying behavior or alternatively that associations between T and E2 and rates of infant-carrying are influenced by stress or other glucocorticoid-related variables. Finally, we propose a hypothesis suggesting that experience-related changes in hormones reinforce the commitment of males to successful breeding partnerships.     

Acute effects of corticosterone injection on paternal behavior in California mouse (Peromyscus californicus) fathers

Glucocorticoids are thought to mediate the disruption of parental behavior in response to acute and chronic stress. Previous research supports their role in chronic stress; however, no study has experimentally tested the effects of acute glucocorticoid elevation on paternal behavior. We tested the prediction that acute corticosterone (CORT) increases would decrease paternal behavior in California mouse fathers and would lead to longer-term effects on reproductive success, as even short-term increases in CORT have been shown to produce lasting effects on the hypothalamic-pituitary-adrenal axis. First-time fathers were injected with 30 mg/kg CORT, 60 mg/kg CORT or vehicle, or left unmanipulated. Interactions between the male and its pup(s) were recorded 1.5–2 h after injection and scored for paternal and non-paternal behavior. Treatment groups were combined into control (unmanipulated + vehicle, n = 15) and CORT (30 mg/kg + 60 mg/kg, n = 16) for analysis based on resulting plasma CORT concentrations. CORT treatment did not alter paternal or non-paternal behaviors or any long-term measures (male body mass or temperature, pup growth rate, pup survival, interbirth interval, number or mass of pups born in the second litter). Fathers showed a significant rise in body mass at day 30 postpartum, followed by a decrease in body mass after the birth of the second litter; however, this pattern did not differ between the CORT and control groups. In summary, acute elevation of plasma CORT did not alter direct paternal behavior, body mass, or reproductive outcomes, suggesting that acute CORT elevation alone does not overtly disrupt paternal care in this biparental mammal.     

Estrogen-dependent modifications to hippocampal plasticity in paternal California mice (Peromyscus californicus)

In many biparental species, mothers and fathers experience similar modifications to circulating hormones. With these modifications come alterations in neural structure and function suggesting that neuroendocrine mechanisms may underlie postpartum plasticity in both males and females. In the biparental California mouse (Peromyscus californicus), adult neurogenesis is maintained and anxiety-like behavior is attenuated in fathers during the mid-postpartum period. Given a causal relationship between estrogen and regulation of both adult neurogenesis and anxiety, we aimed to elucidate the role of estrogen-dependent mechanisms in paternal experience-related modifications to hippocampal neuroplasticity in California mice. In Experiment 1, hippocampal estrogen receptor beta (ERβ) mRNA expression, along with circulating estradiol concentrations, were determined throughout the postpartum period. An upregulation in ERβ expression was observed in postnatal day 16 males compared to virgins. Additionally, a rise in circulating estradiol concentrations was detected on postnatal day 2 compared to virgins; levels began to decline toward virgin levels on postnatal day 16 and postnatal day 30. In Experiment 2, we determined the role of estrogen-dependent mechanisms in adult neurogenesis and anxiety-like behavior by treating virgin and paternal males with saline or the selective estrogen receptor modulator, tamoxifen (TMX), during the time of axon extension (i.e., one week after bromodeoxyuridine injection). While TMX failed to alter elevated plus maze performance, TMX treatment inhibited survival of adult born neurons but only in paternal mice. These findings highlight the potential for estrogen-dependent pathways to mediate hippocampal adult neurogenesis in paternal mice.     

Hormonal Responses of Male Gerbils to Stimuli from Their Mate and Pups

Following copulation and cohabitation with a pregnant female, male gerbils show high levels of parental behavior toward their pups. The initiation of male parental behavior may be the result of neuroendocrine changes induced by cohabiting with the pregnant female or by pup stimuli. Experiment 1 examines the changes in androgen and prolactin levels in male gerbils cohabiting with females over the reproductive cycle. Gerbils were mated and blood samples taken from males for hormone analysis 1, 10, and 20 days after pairing and 3, 10, and 20 days after pups were born. A group of unmated male gerbils served as controls. Plasma prolactin levels of males were elevated throughout the female's pregnancy and lactation periods, but were only statistically significantly higher than those of unmated males 20 days after pups were born. Androgen levels rose during pregnancy and dropped significantly after the birth of the pups. These hormonal changes are similar to those found in males of monogamous birds and differ from those found in males of polygynous rodents such as the rat. Experiment 2 examined the hormonal responses of male and female gerbils to pup replacement after 4 hr of parent–pup separation. Female gerbils showed a significant elevation of prolactin levels 1 hr after pup replacement, but males did not. Males with pups returned showed no difference in androgen levels from males who did not have pups returned. Thus, male gerbils show neuroendocrine changes following long-term cohabitation with their mate and pups, but do not show acute hormone responses to pup removal and replacement. These results indicate that parental males have neuroendocrine changes associated with parental behavior and these differ from the neuroendocrine changes underlying female parental behavior.     

Autonomic Substrates of the Response to Pups in Male Prairie Voles

Caregiving by nonparents (alloparenting) and fathers is a defining aspect of human social behavior, yet this phenomenon is rare among mammals. Male prairie voles (Microtus ochrogaster) spontaneously exhibit high levels of alloparental care, even in the absence of reproductive experience. In previous studies, exposure to a pup was selectively associated with increased activity in oxytocin and vasopressin neurons along with decreased plasma corticosterone. In the present study, physiological, pharmacological and neuroanatomical methods were used to explore the autonomic and behavioral consequences of exposing male prairie voles to a pup. Reproductively naïve, adult male prairie voles were implanted with radiotransmitters used for recording ECG, temperature and activity. Males responded with a sustained increase in heart-rate during pup exposure. This prolonged increase in heart rate was not explained by novelty, locomotion or thermoregulation. Although heart rate was elevated during pup exposure, respiratory sinus arrhythmia (RSA) did not differ between these males and males exposed to control stimuli indicating that vagal inhibition of the heart was maintained. Blockade of beta-adrenergic receptors with atenolol abolished the pup-induced heart rate increase, implicating sympathetic activity in the pup-induced increase in heart rate. Blockade of vagal input to the heart delayed the males’ approach to the pup. Increased activity in brainstem autonomic regulatory nuclei was also observed in males exposed to pups. Together, these findings suggest that exposure to a pup activates both vagal and sympathetic systems. This unique physiological state (i.e. increased sympathetic excitation of the heart, while maintaining some vagal cardiac tone) associated with male caregiving behavior may allow males to both nurture and protect infants.