Current Zoology, 2016, 62(3), 285–291 doi: 10.1093/cz/zow032 Advance Access Publication Date: 29 March 2016 Article

Article Biparental behavior in the burying : a role for dopamine?

a, b a S. Carmen PANAITOF *, Jazmine D. W. YAEGER , Jarod P. SPEER , b and Kenneth J. RENNER aDepartment of Biology, University of Nebraska at Kearney, Kearney, NE 68849, USA, and bDepartment of Biology, University of South Dakota, Vermillion, SD 57069, USA

*Address correspondence to S. Carmen Panaitof. E-mail: [email protected].

Received on 11 December 2015; accepted on 17 February 2016

Abstract Burying Nicrophorus orbicollis exhibit facultative biparental care of young. To reproduce, a male–female burying beetle pair bury and prepare a small vertebrate carcass as food for its altricial young. During a breeding bout, male and female behavior changes synchronously at appropriate times and is coordinated to provide effective care for offspring. Although the ecological and evolutionary factors that shape this remarkable reproductive plasticity are well characterized, the neuromodulation of parental behavior is poorly understood. Juvenile hormone levels rise dramat- ically at the time beetle parents accept and feed larvae, remain highly elevated during the stages of most active care and fall abruptly when care is terminated. However, hormonal fluctuations alone cannot account for this elaborate control of reproduction. The biogenic amines octopamine (OA), dopamine (DA), and serotonin (5-HT) mediate a diversity of reproductive and social behav- iors. In this study, we measured whole brain monoamine levels in individual male and female bury- ing beetles and compared OA, DA, and 5-HT profiles between breeding (parental) and nonbreed- ing, unmated beetles. Remarkably, after 24 h of care, when parental feeding rates begin to peak, DA brain levels increase in breeding beetles when compared to nonbreeding controls. In contrast, brain OA and 5-HT levels did not change significantly. These results provide the first evidence for a potential role of DA in the modulation of burying beetle parental behavior.

Key words: dopamine, Nicrophorus, neuromodulation, octopamine, , serotonin.

Extended biparental care is a reproductive strategy that is rarely ex- (Ketterson and Nolan 1992; Buntin 1996; Adkins-Regan 2005). By hibited by or by most vertebrate species. Among vertebrates, comparison, many aspects of reproductive behavior and physiology, biparental care is ubiquitous in but unusual in mammals, with including the hormonal basis of parental care, remain poorly under- primates and carnivores, for example, serving as notable exceptions stood in insects (Trumbo 2002; Riddiford 2012). Remarkably, the (Clutton-Brock 1991). In insects, utilization of rich and ephemeral neuroendocrine system of insects bears fundamental similarities to reproductive resources like dung or carrion is thought to have pro- the hypothalamo–hypophyseal axis of vertebrates (Scharrer 1987; moted selection for unique behavioral and physiological adaptations Hartenstein 2006; Wirmer et al. 2012), further opening a unique av- leading to subsociality and biparental behavior (Tallamy and Wood enue for comparative studies of the physiology of reproduction. 1986). Given the complex underpinnings of caregiving, the study of The burying beetle, Nicrophorus orbicollis,providesanexperi- parental care can provide critical insights into the interplay among mentally accessible model for studies of insect parental care (Trumbo ecological, social, and neuroendocrine factors in the regulation of 1996, 2012). The natural history of burying beetles is well described behavior. The pervasive roles of hormones in the control of repro- (Pukowski 1933) and the ecological and evolutionary processes that ductive social behavior have been explored extensively in vertebrates have contributed to molding their intricate reproductive behaviors are

VC The Author (2016). Published by Oxford University Press. 285 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 286 Current Zoology, 2016, Vol. 62, No. 3 well understood (Trumbo 1996, 2012; Eggert and Muller€ 1997; Scott and serotonin (5-hydroxytryptamine; 5-HT), represent the most 1998). During reproduction, a male–female beetle pair will cooperate likely candidates for contributing to the neuromodulatory control of in burying and preparing a small vertebrate carcass as food for its al- parental behavior in the burying beetles. Since the discovery of their tricial larvae. Upon discovery, the carcass is quickly concealed under- synthesis and release by the central nervous system of insects, the di- ground, fur or feathers removed and the carrion mass rolled into a verse functions of biogenic amines and their receptor subtypes have ball. Within 48 h following carcass discovery, the female lays eggs in received considerable interest (Roeder 1999, 2005; Farooqui 2012), the soil nearby and 3–4 days later larvae hatch and make their way to with functional studies revealing that the invertebrate octopaminer- the carrion ball. Larvae beg and are fed, typically by both parents, gic system shares fundamental properties with the adrenergic system regurgitated predigested food. In N. orbicollis, the offspring are en- of vertebrates. tirely dependent on parental regurgitations for survival soon after Variation in OA levels has been implicated in the modulation of hatching (Trumbo 1992). During a breeding bout, male and female behavioral state in a number of insects. In the highly eusocial honey behavior has to change at appropriate times, from passive to aggres- bees Apis mellifera, rising OA levels are linked to behavioral sive responses to conspecific intruders, and from infanticidal to paren- maturation and age-related division of labor, illustrated by the tal, a few hours before larvae hatch and arrive on the carcass (Muller€ transition from nursing (brood care) to foraging activities (Wagener- and Eggert 1990; Scott 1990; Oldekop et al. 2007). Male and female Hulme et al. 1999; Schulz and Robinson 2001; Schulz et al. 2002a, burying beetles also need to coordinate their reproductive tasks during 2002b). Brain OA levels of foragers are higher than those of nurses larval care, the most demanding stage of the reproductive cycle (Wagener-Hulme et al. 1999; Schulz et al. 2002a, 2002b) and OA (Fetherston et al. 1990). Parental activities center on provisioning and treatment results in precocious expression of foraging behavior in protecting the larvae, maintaining the brood chamber, as well as pro- workers (Schulz and Robinson, 2001; Schulz et al. 2002a, cessing and treating the carcass with antimicrobial secretions to delay 2002b). Octopamine also facilitates nestmate recognition (Robinson its decomposition (Fetherston et al. 1990; Robertson 1993; Scott et al. 1999) and has been similarly implicated in the discrimination 1998; Arce et al. 2012). of foraging-related stimuli and olfactory learning (Hammer and While many of the costs and benefits associated with reproduct- Menzel 1998; Barron et al. 2002; Scheiner et al. 2002). Recent ive cooperation and conflict have been thoroughly explored, both in studies of the octopaminergic system in the burying beetle, the field and in the laboratory (Scott 1998), we have still an incom- , show differential gene expression of plete understanding of the physiological factors, neural circuits, or several OA receptor subtypes across reproductive and social con- molecular pathways involved in promoting the highly plastic repro- texts thus linking OA signaling to behavioral flexibility during ductive and parental behaviors of burying beetles. In previous work, breeding (Cunningham et al. 2014). we found that in N. orbicollis, hemolymph titers of juvenile hor- Biogenic amines have also been implicated in the modulation of mone (JH) rise dramatically at the onset of parental behavior, are aggression in a number of invertebrate models (Kravitz and Huber very high during the most active stages of larval care when rates of 2003; Stevenson and Schildberger 2013; Alekseyenko and Kravitz parental feedings peak, and decline rapidly before care is terminated 2014; Bubak et al. 2014a). The roles of OA, the invertebrate “fight (Panaitof et al. 2004). Importantly, in both males and females, JH or flight” neurotransmitter, and 5-HT have been extensively levels are responsive to social cues, such as the presence or absence explored, especially in the social context-dependent initiation and of a mate and brood age or size (Panaitof et al. 2004; Scott and escalation of fighting behavior and establishment of dominance. In Panaitof 2004). Despite the remarkable correlation between changes crustaceans, 5-HT was repeatedly linked to aggression motivation, in circulating JH levels and the well-timed behavioral shifts that presumably by altering the behavioral threshold for retreat (Huber male and female burying beetles undergo during reproduction, the et al. 1997; Kravitz 1988, 2000). In territorial nonsocial insects like precise neurophysiological mechanisms responsible for the initiation the cricket, Gryllus bimaculatus, OA acts to restore fighting readi- and maintenance of care remain elusive. Direct treatment with flu- ness following defeat (Hofmann and Stevenson 2000) and 5-HT has vastatin, a robust inhibitor of JH biosynthesis (Debernard et al. been functionally linked to the modulation of fighting behavior and 1994), while significantly lowering JH levels (Panaitof and Scott expression of behavioral features associated with dominant social 2006), does not seem to interfere with either the acceptance of lar- status (Dyakonova and Krushinsky 2013). The genetic tractability vae or initiation of parental feedings (Panaitof 2006). Thus, it is un- of the fruit fly, Drosophila melanogaster, has further enabled re- clear whether JH has a direct or an indirect role in the transition to markable dissection of neural circuits and neuromodulatory path- parental behavior and initiation of care. This raises the likely ways involving both the serotonergic and dopaminergic system in possibility that JH may act in conjunction with other unidentified the initiation and ability to escalate aggressive behavior (Zwarts neuromodulatory pathways. Lending support to this idea, several et al. 2012; Alekseyenko et al. 2013; Alekseyenko et al. 2014). studies in the eusocial Hymenoptera have shown that JH interacts Novel model systems of invertebrate aggression, such as the stalk- with the biogenic amines octopamine (OA) and dopamine (DA) in eyed fly, Teleopsis dalmanni, have also provided important the regulation of reproductive maturation, behavioral development, additional insights into the role of 5-HT in mediating conflict escal- and age-related task specialization in honey bee workers (Schulz ation, contest behavioral outcome and establishment of a winner– et al. 2002a, 2002b; Harano et al. 2008; Sasaki et al. 2012a). loser relationship (Bubak et al. 2014a, 2014b). Moreover, treatment with the JH analog, methoprene, causes eleva- Given the robust links established between OA, 5-HT, and DA tion of OA and DA levels in the honey bee brain (Schulz et al. and the modulation of behavioral state, we sought to similarly 2002a, 2002b; Harano et al. 2008; Sasaki et al. 2012a). explore whether the levels of the three monoamines change signifi- Based on their wide involvement in a variety of invertebrate re- cantly as a result of the transition from nonparental to parental state productive and social behaviors (Roeder 1999, 2005), combined in the burying beetles. Burying beetles, in addition to exhibiting with evidence that JH alters monoaminergic function (Harano et al. behavioral changes toward newly hatched and developing larvae, 2008; Sasaki et al. 2012a), we hypothesized that the biogenic amines including acceptance (decrease in aggression) and provisioning of OA, an analog of norepinephrine in vertebrates (Roeder 2005), DA larvae, also show changes in aggression toward adult conspecifics as Panaitof et al. Biparental behavior in Nicrophorus orbicollis 287 a means of protecting the offspring (Muller€ and Eggert 1990; Scott 2013). The pellet was solubilized in 0.4 M NaOH and analyzed for 1990; Robertson 1993). Thus, it seems likely that changes in central protein using the Bradford method (Bradford 1976). Monoamine monoaminergic function associated with changes in aggression and/ concentrations were determined using a CSW32 data program or motivation to perform parental tasks would be involved in modu- (DataApex, Prague, Czech Republic) set in internal standard mode lating the response of burying beetles to social cues from competi- using peak height values relative to standards. The resulting concen- tors or intruders, mates, and young during breeding. trations were divided by mg protein in the sample yielding pg amine/ In the current study, we test a potential role for central mono- mg protein and corrected for injection versus preparation volume. amines in modulating burying beetle parental behavior by measur- ing whole brain levels of OA, 5-HT, and DA in male and female Data analysis burying beetles prebreeding and during the breeding cycle, 24 h after Data were log-transformed for statistical analysis and means and the initiation of care. We predicted that the behavioral transitions standard errors back transformed for graphical representation. To associated with the acceptance and feeding of larvae for 24 h would test for differences in monoamine levels between nonbreeding (con- be accompanied by significant changes in the brain levels of OA, trol) and breeding (parental) burying beetles, a linear mixed model 5-HT, and DA in breeding but not in nonbreeding controls. (SYSTAT 13, San Jose, CA) was used, with breeding status (2 levels) We interpret these findings in the context of exploring a neuromo- and beetle sex (2 levels) as fixed effects and sampling session as ran- dulatory mechanism that may mediate the behavioral shifts associ- dom effect, and applying a critical alpha level of 0.05. Post hoc pair- ated with the onset of care and organization of parental activities in wise comparisons were performed in R version 3.2.1 (R Core Team the burying beetles during breeding. 2015) using the lsmeans function and the Tukey method for P value adjustment. Materials and Methods Animals and breeding experiments Results Experimental animals were laboratory-reared offspring of wild- Whole brain levels of each monoamine measured in male and female caught beetles trapped in Buffalo County, NE, in May–June 2014. burying beetles in either nonbreeding status or during the breeding Beetles were housed in plastic boxes lined with damp paper towels, cycle, after 24 h of parental care, are shown in Figure 1A (OA), b (5- with up to 10 same-sex individuals, and maintained at 20 Cona HT), and c (DA). OA levels (mean 6 SE; pg/lg protein) measured in 14L:10D cycle. They were fed a diet of beef or chicken liver ad lib- the brains of breeding animals (48.5 6 1.0) after 24 h of care were itum. All animals were previously unmated, sexually mature, rang- not significantly different from levels obtained from nonbreeding ing in age from 21 to 30 days at the time of the experiments. pairs (41.7 6 1.1; Figure 1A). Levels of 5-HT were also similar in To initiate breeding, at “lights-off” each male and female was ran- breeding and nonbreeding beetles (25.6 6 1.0 and 23.0 6 1.0 pg/mg domly assigned to a breeding pair (N ¼ 12 pairs) and provided with protein, respectively; Figure 1B). However, dopamine levels a 20–25 g previously frozen mouse carcass (RodentPro.com, increased from 14.9 6 1.0 pg/mg protein in nonbreeding beetles to Inglefield, IN) in a box (19 14 10 cm3) three-fourth filled with 18.1 6 1.0 pg/mg protein in breeding pairs (F1,43 ¼ 4.74 , P ¼ 0.03; peat moss. Each box was monitored for the presence of eggs and the Figure 1C). There was no statistically significant effect (P > 0.05) of time of oviposition was recorded for each beetle pair. Eggs were beetle sex on DA levels. Similarly, there was no statistically signifi- monitored daily until larvae hatched (typically 3–4 days later). The cant effect of either breeding status or beetle sex, as well no signifi- timing of larvae arrival on the carcass was recorded for each pair. cant interaction of factors (P > 0.05) for either OA or 5-HT levels. The behavior of each beetle pair was then monitored until the first Central OA levels (pg/mg protein; Figure 2A) measured in both parental feeding of larvae was observed. We chose to evaluate cen- breeding males (46.7 6 1.1) and breeding females (50.2 6 1.1) were tral monoamine levels following 24 hours of care, given that paren- comparable to those of nonbreeding males (43.5 6 1.1) and females tal feeding activities are highest at this stage (Fetherston et al. 1994). (39.9 6 1.1). Brain 5-HT levels (pg/mg protein; Figure 2B) also Twenty-four hours following the initiation of parental feedings, showed little change between breeding animals (23.9 6 1.1 for males each beetle pair and brood chamber were briefly observed to ensure and 27.4 6 1.1 for females) and nonbreeding animals (24.2 6 1.1 for that larvae had been fed and developed to second instar stage. males and 21.8 6 1.1 for females). Post hoc pairwise comparisons similarly revealed no statistically significant differences (P > 0.05) Brain dissection and HPLC analysis among brain DA levels between male and female burying beetles in Twenty-four hours after the onset of parental care, the male and fe- either nonbreeding or breeding status (Figure 2C). Brain DA levels male of each pair were sacrificed and the brains were dissected in (mean 6 SE; pg/lg protein) of breeding males (17.1 6 1.1) and fe- ice-cold insect saline (137 mM NaCl, 2.7 mM KCl, 10 mM males (19.2 6 1.1) were similar to those of nonbreeding males (15.6 6 1.1) and females (14.2 6 1.1). Of note, we have nevertheless Na2HPO4, 1.8 mM KH2PO4,pH¼ 7.4) and transferred to an acet- ate buffer (pH ¼ 5.0; 60 mL) containing the internal standard a-me- identified a trend toward higher DA levels in breeding (parental) fe- thyl DA (1 x 10-7 M). Individual male and female brains of males compared to nonbreeding (control) females (P ¼ 0.09). nonbreeding (control) beetles from the same colony were similarly obtained in the same experimental session. The brains were lightly Discussion disrupted by sonication, frozen, then thawed, and centrifuged (17,000 g, 4 C). The monoamines were separated using a In the facultatively biparental burying beetles, N. orbicollis, repro- NOVAPAK C18 column (Waters Associates, Inc.) and detected ductive behavior depends on a complex neuroendocrine control that using an LC 4 potentiostat and a glassy carbon electrode appears to have enabled a vertebrate-like plasticity in the modula- (Bioanalytical Systems) set at 0.5 nA/V with an applied potential of tion of the onset, intensity and termination of care (Panaitof et al. þ0.997 V versus an Ag/AgCl reference electrode (Bubak et al. 2004; Scott and Panaitof 2004). Because this elaborate pattern of 288 Current Zoology, 2016, Vol. 62, No. 3

A A

B

B

C

C

Figure 2. Levels (pg/lg protein) of octopamine (OA), serotonin (5-HT), and dopamine (DA) measured in the brains of male and female burying beetles in either nonbreeding status or after 24 h of parental care (N ¼ 12/beetle sex). Bars represent mean 6 SE of OA (A), 5-HT (B), and DA (C) brain levels. Post hoc pairwise comparisons identified a trend toward higher DA levels in breeding (parental) females compared to nonbreeding (control) females (P ¼ 0.09).

care depends on coordinated changes in the reproductive behavior and physiology of male and female beetle parents, a thorough inves- tigation of the neuromodulatory and endocrine factors involved is critical for discerning the precise neural and physiological mechan- isms enabling such flexible reproductive behaviors. Despite the well-established hormonal correlates of parental behavior, it is not Figure 1. Levels (pg/lg protein) of octopamine (OA), serotonin (5-HT), and clear how changes in reproductive hormones like JH may be respon- dopamine (DA) measured in the brains of burying beetles in either nonbreed- sible, directly or indirectly, for the organization of reproductive ing status (N ¼ 24) or after 24 h of parental care (N ¼ 24). Bars represent events or for the changes in behavioral state associated with the mean 6 SE of OA (A), 5-HT (B), and DA (C) brain levels. A linear mixed model onset of parental care during a breeding bout. Here, we have begun was used to test for differences in brain monoamine levels. Of the three monoamines, only DA showed a significant increase in the brains of parental to explore a potential role for the biogenic amines in the neuromo- beetles compared to nonbreeding controls, as denoted by the * (P ¼ 0.03). dulation of biparental behavior in the burying beetles. Panaitof et al. Biparental behavior in Nicrophorus orbicollis 289

The significant elevation of DA, but not OA or 5-HT levels, in To our knowledge, this is the first study exploring the link be- the brains of parental beetles, compared to unmated controls, is tween central monoamine levels and breeding status in a biparental intriguing, especially in the context of exploring a neural mechanism insect and our findings seem to indicate that DA may be especially for the transition from the nonbreeding to breeding (parental) be- important in orchestrating the behavioral transitions associated with havioral state. During a reproductive bout, the initiation of care, in the initiation of care and/or altering a behavioral threshold respon- particular, appears to be a finely tuned behavior: parents will accept sible for the performance of the specialized tasks that it entails. To and feed larvae that arrive on the carcass when their own are ex- better understand the role of DA in burying beetle reproductive be- pected to hatch but will cannibalize larvae that hatch either too early havior, we have further set out to explore a potential functional link or too late (Muller€ and Eggert 1990; Oldekop et al. 2007). The between JH and DA during breeding, given that both molecules neurophysiological mechanism underlying this striking change in re- show significant increases associated with the initiation of care. sponsiveness to larvae is not yet understood but it seems likely that Similarly, to begin to probe the intracellular effects of the three it may involve neuromodulation of a behavioral response threshold monoamines, we plan to identify and characterize several OA, DA, and subsequent decrease in aggression towards newly hatched and 5-HT receptor subtypes in N. orbicollis. Related studies will use offspring. in vivo pharmacological treatments to alter brain monoamine func- Our findings thus seem to suggest that DA, but not OA or 5-HT, tion, either by directly manipulating DA levels via dietary supple- may be potentially implicated in the onset of care and/or modulation mentation or inhibition of biosynthesis or by administering specific of parental activities in breeding burying beetles. A related question monoamine receptor antagonists or agonists, followed by assays to is whether an increase in DA levels may serve to drive the behavioral uncover any behavioral changes associated with the initiation of switch to the parental state, presumably by altering a behavior acti- care, intensity of parental effort, and duration of care. vation threshold necessary for the initiation of care, or could con- ceivably arise in response to stimuli from young, facilitating the modulation of parental care activities. Intriguingly, the trend for Funding higher brain DA in parental (breeding) females compared to non- This work was funded by a RSC grant (SCP) and a URF award (JPS) from the breeding females, and the lack of a similar trend in breeding males, UNK’s Office of Undergraduate Research & Creative Activity, and by may similarly reflect a role for DA in mediating the fine adjustments National Science Foundation Grant IOS 1256898 (KJR), and a Center for in the intensity of parental care activities between the sexes, and Brain and Behavioral Research pilot Grant, USD (KJR). would be in accord with established behavioral sex differences, given that N. orbicollis females spend significantly more time provi- sioning larvae compared to males (Fetherston et al. 1990). The ab- Acknowledgments sence of a significant change in either OA or 5-HT brain levels in parental beetles is surprising, given that OA has been implicated in We thank Dr. Wyatt Hoback for generous assistance with field trapping and collection of burying beetles. altering the threshold for task-related behavioral transitions (Schulz et al. 2002a, 2002b), while 5-HT linked to the modulation of social context-dependent aggression in a number of insects (Stevenson and References Schildberger 2013; Alekseyenko and Kravitz 2014; Bubak et al. Adkins-Regan E, 2005. Hormones and Social Behavior. Princeton: 2014a). Princeton University Press. A potential role for DA in mediating the transition between con- Alekseyenko OV, Chan YB, Fernandez Mde L, Bulow T, Pankratz MJ et al., flicting behavioral states, such as the switch from nonparental to 2014. 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