Caste differences in the association between dopamine and reproduction in the bumble bee Bombus ignitus

Accepted Manuscript Caste differences in the association between dopamine and reproduction in the bumble bee Bombus ignitus Ken Sasaki, Hinako Matsuyama, Naruaki Morita, Masato Ono PII: DOI: Reference:

S0022-1910(17)30245-7 https://doi.org/10.1016/j.jinsphys.2017.10.013 IP 3720

To appear in:

Journal of Insect Physiology

Received Date: Revised Date: Accepted Date:

10 June 2017 29 October 2017 31 October 2017

Please cite this article as: Sasaki, K., Matsuyama, H., Morita, N., Ono, M., Caste differences in the association between dopamine and reproduction in the bumble bee Bombus ignitus, Journal of Insect Physiology (2017), doi: https://doi.org/10.1016/j.jinsphys.2017.10.013

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Caste differences in the association between dopamine and reproduction in the bumble bee Bombus ignitus

Ken Sasaki1,2*, Hinako Matsuyama1, Naruaki Morita2 and Masato Ono1,2

1

Graduate School of Agriculture, Tamagawa University, Machida, Tokyo 194-8610, Japan

2

Department of Bioresource Science, Tamagawa University, Machida, Tokyo 194-8610, Japan

*Corresponding author: K. Sasaki E-mail: [email protected]

Abstract A society of bumble bees is primitively eusocial, with an annual life cycle, and can be used as a physiological model of social bees for comparative studies with highly eusocial hymenopterans. We investigated the dynamics of biogenic amine levels in the brain, meso-metathoracic ganglia, terminal abdominal ganglion, and hemolymph in queens 1 day after mating (1DAM), during diapause (Dp), and during colony founding (CF) in the bumble bee, Bombus ignitus. Dopamine levels in the brain of CF queens were significantly lower than in 1DAM and Dp queens, and the levels in the thoracic ganglia and hemolymph in CF queens were lower than in 1DAM queens, but did not differ from other groups in the abdominal ganglion. Octopamine levels in the brains were higher in Dp queens than in 1DAM queens. Serotonin and tyramine levels did not differ between the groups in different compartments of the central nervous system (CNS) that we examined. The dopamine levels in the brains were significantly positively correlated with those in the thoracic ganglia, abdominal ganglion, and hemolymph, suggesting the regulation of dopamine levels among three different compartments of the CNS. In isolated virgin queens, there were no significant correlations between the brain levels of biogenic amines that we examined and the lengths of the largest terminal oocytes, whereas, in isolated workers, the brain dopamine levels were positively correlated with oocyte lengths. These results suggest that dopamine is associated with ovarian activity in reproductive workers, but not in either virgin or mated queens.

Keywords: biogenic amines; eusociality; Hymenoptera; reproductive caste; social insects. 2

1. Introduction Division of labor for reproduction is an essential property of eusociality in animals. In highly eusocial hymenopterans, including honey bees and ants, queens and workers are specialized for each task morphologically and physiologically. In primitively eusocial species, including bumble bees and paper wasps, apart from size, there are no evident morphological differences between queens and workers and the division of labor is more complicated. In bumble bees in temperate climate regions, the new queens that mated during autumn hibernate under low temperatures and begin to found a colony during the following spring (Michener, 1974; Goulson, 2009). During colony founding (CF) and before the emergence of the first worker brood, the founding queens construct egg cups for egg laying, care for the brood, forage outside of the nest as workers, and lay eggs. After the emergence of the first worker brood, the queen engages in egg laying without caring for the brood or foraging. Such a behavioral transition from mating to CF via hibernation in the queens has been observed in not only bumble bees, but also several species of paper wasps and ants (Hölldobler and Wilson, 1990; Hunt, 2007). Endocrine systems associated with caste-specific behaviors and physiology at the adult stage might differ between castes in female social hymenopterans. For example, honey bee queens maintain low levels of juvenile hormone in their hemolymph, whereas the levels in workers increase as they age (Sasagawa and Kuwahara, 1988; Robinson et al., 1991; Robinson and Vargo, 1997; Bloch et al., 2002). In the hemolymph, such a hormone generally circulates throughout the body and acts on target cells in the central and peripheral nervous systems (CNS and PNS) and other peripheral tissues. In the CNS, biogenic amines have multiple overlapping functions, acting as neurotransmitters in locally confined interneuronal signaling, as neuromodulators in 4

postsynaptic cells in several restricted regions of the CNS, or as neurohormones when released into the hemolymph and transported via the circulatory system to target tissues (Evans, 1980; Burrows, 1996; Lange, 2009). Dopamine is a biogenic amine and its levels in the brain of honey bee queens decrease after mating (Harano et al., 2005, 2008), whereas the levels in normal workers increase as they age (Taylor et al., 1992; Kokay and Mercer, 1997; Schulz and Robinson, 1999) and those in reproductive workers in queenless colonies increase as the ovaries are activated (Harris and Woodring, 1995; Sasaki and Nagao, 2001). Thus, the dynamics of hemolymph hormones and biogenic amines might correspond to important events in the life of each caste, resulting in the promotion of tasks and behavioral changes. In bumble bees, associations between hemolymph hormones and reproduction in reproductive workers and queens have been studied (Robinson and Vargo, 1997; Bloch et al., 2002; Hartfelder and Emlen, 2005). Juvenile hormone has a gonadotropic role in reproductive workers in Bombus terrestris and levels are correlated with ovarian activity (Rössler, 1977; Bloch et al., 2000a), but are not strongly correlated with reproductive states in queens; this is because of the lower biosynthesis of juvenile hormone in mated queens compared with reproductive workers (Bloch et al., 2000a). Ecdysteroid is also a candidate hormone promoting reproduction, and ovarian ecdysteroid levels are correlated with ovarian activity in reproductive workers and mated queens (Geva et al., 2005). However, the hemolymph titer of ecdysteroid is not correlated with ovarian activity. For biogenic amines, Bloch et al. (2000b) measured the brain levels in workers and queens of B. terrestris that exhibited different behavioral and reproductive states. In workers, octopamine levels were positively correlated with social dominance, whereas dopamine levels were positively correlated with ovarian activity. In queens, the levels of 5

dopamine, octopamine, and serotonin in the brains did not differ between virgin and mated individuals (Bloch et al., 2000b). However, there are no reports of biogenic amine levels in queens from mating to CF in bumble bees. To explore the neuroendocrine system of biogenic amines associated with reproduction in queens and reproductive workers in primitively eusocial bees, two different experiments were performed. First, biogenic amine levels between different behavioral and reproductive states in mated Bombus ignitus queens were quantified in several compartments of the CNS and hemolymph. If dopamine has reproductive roles in the mated queens, it is expected that dopamine levels in the CNS or hemolymph would be higher in egg-laying queens than in others. Second, we determined correlations between biogenic amine levels and ovarian activities in isolated virgin queens and workers. If dopamine promotes ovarian activities in both queens and workers, it is expected that dopamine levels in the brains in both castes would be positively correlated with ovarian activities. Thus, the association of biogenic amines with reproduction was compared between castes.

2. Materials and methods 2.1. Animals Commercially reared bumble bee (B. ignitus) colonies were kept in wooden boxes (length 30 cm × width 20 cm × height 12 cm) with transparent windows at the top and steel nets at the bottom at 28°C in constant darkness. The wooden box was divided into two sections: one (length 18 cm × width 20 cm × height 12 cm) was for the nest and the other (length 12 cm × width 20 cm × height 12 cm) was for feeding with sugar (fructose and sucrose) solution. Bees were given ad libitum pollen kneaded with sugar solution in 6

the nest room and sugar solution in the feeding room.

2.2. Experiment 1: mated queens of different behavioral and reproductive states Newly emerged queens taken from three mature colonies that had produced new queens, males, and workers (competition phase) (Asada and Ono, 2000) were isolated in plastic containers (length 16 cm × width 9 cm × height 10 cm) with pollen and sugar solution. After 7–10 days following emergence, virgin queens were transferred into a net cage (length 35 cm × width 35 cm × height 56 cm) with males for mating. The queens that mated with males were removed from the net cage and kept in a plastic container until the end of mating. After mating, the male was removed from the container and the queen was sampled on the next day (1 day after mating: 1DAM) for the measurement of biogenic amines, or was transferred into a plastic vial (i.d.: 2.5 cm, height: 5 cm), with vermiculite at 5°C in an incubator under constant darkness as a treatment for low-temperature diapause (Dp) (Fig. 1). The low-temperature Dp lasted at least 126 days (approximately 4 months). Several queens were roused from Dp by gradually increasing the temperature to 10°C for 2 h, 15°C for 2 h, 20°C for 2 h, 20°C–25°C overnight, and then maintained at 28°C. Each roused queen was kept in a wooden box (length 15 cm × width 8 cm × height 6 cm) divided into two sections of equal size for nest founding and feeding with sugar solution. In the nest room, a cocoon with wax and pollen was provided to promote nest founding. Generally, this treatment does not reduce activities of founding queens, rather, this might make the queens more active (Kwon et al., 2003; Yoneda, 2008). All queens laid eggs in the wax cup in the nest room for 17 days after arousal and were sampled for measurements of biogenic amines (CF). Queens under Dp were also sampled 143–146 days after mating. The day 7

ages of Dp queens were almost the same as the CF queens (Fig. 1).

2.3. Experiment 2: isolation of virgin queens and workers to allow ovarian activation To determine the correlation between the brain levels of biogenic amines and ovarian activity in virgin queens and workers, individuals were isolated from the study colonies for ovarian activation. Virgin queens do not normally activate their ovaries before mating, but rather begin to activate them approximately 2 weeks after emergence if they remain unmated. Therefore, virgin queens were taken from two mature colonies (competition phase) and then kept individually in a plastic container with a filter paper, pollen, and sugar syrup for 13–15 days or 18–20 days. The virgin queens at these ages showed large variation in ovarian activity and, thus, were appropriate for sampling. They were sampled using liquid nitrogen and stored before the measurement of biogenic amines. Newly emerged workers were taken from developing colonies (during late social phase) that had produced 40–50 workers, but not males or new queens; the workers were then isolated in a plastic container (i.d.: 9 cm, height: 4.5 cm). We selected similar-sized individuals among the newly emerged workers and excluded extremely small workers to decrease the variation in the body size of our sample. The isolated workers without a queen (queenless workers) were kept in the container with a filter paper, pollen, and sugar syrup for 6 or 10 days after emergence, sampled using liquid nitrogen and stored before the measurement of biogenic amines.

2.4. Measurement of biogenic amines To sample hemolymph from the mated queens, the queens were quickly 8

immobilized by freezing the head with a cold spray gun (Spot freeze, Fine Chemical Japan, Osaka, Japan) and 1 µL of hemolymph was taken from the abdomen by removing a small piece of cuticle using a micro glass capillary tube (Ringcaps, Hirschmann-Laborgeräte, Eberstadt, Germany). After removing the hemolymph, the queens were frozen using liquid nitrogen and stored until dissection. Each sample of hemolymph was transferred into 49 µL of ice-cold 0.1 M perchloric acid containing 100 ng mL–1 3,4-dihydroxybenzylamine (DHBA) as an internal standard and mixed well. Samples were then centrifuged at 15,000 g for 15 min at 4°C. The supernatant was transferred to a microvial for analysis with high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The brain including a subesophageal ganglion, meso-metathoracic ganglia, and terminal abdominal ganglion were removed and homogenized in a microglass homogenizer with 100 µL (for brains) or 50 µL (for thoracic and abdominal ganglia) of ice-cold 0.1 M perchloric acid containing 100 ng mL–1 DHBA. Each sample was then transferred into an Eppendorf tube, centrifuged at 15,000 g for 30 min at 4°C, and transferred to a microvial for HPLC-ECD analysis. Measurements of biogenic amines were conducted using similar procedures to those used by Sasaki et al. (2012) and Mezawa et al. (2013). The HPLC-ECD system comprised a solvent delivery pump, a refrigerated automatic injector, a C18 reversed-phase column (250 × 4.6 mm i.d., 5 µm average particle size, UG 120, Shiseido, Tokyo, Japan) maintained at 35°C in a column oven, and an electrochemical detector (ECD-300, EICOM, Kyoto, Japan), with a glassy carbon electrode (WE-GC, EICOM). The detector potential was set at 0.83–0.86 V for the brains and meso-metathoracic and terminal abdominal ganglia, and at 0.7 V for hemolymph 9

samples against an Ag/AgCl reference electrode. The mobile phase contained 0.18 M monochloroacetic acid and 40 µM 2Na-EDTA, which was adjusted to pH 3.6 using NaOH. We added 1.62 mM sodium-1-octanesulfonate and CH3CN (5.3%) to this solution. The flow rate was kept constant at 0.7 mL min–1. Samples were analyzed with external standards before and after the sample runs. External standards [octopamine, DHBA, N-acetyldopamine (NADA), dopamine, tyramine, N-acetylserotonin (NA5HT), tryptophan, and serotonin] were used for the identification and quantification of dopamine, serotonin, tyramine, octopamine, a metabolite of dopamine (NADA), and a metabolite of serotonin (NA5HT). Each peak of a biogenic amine was identified by comparing both the retention time and hydrodynamic voltamograms with those of the standards. Measurements based on the peak area of the chromatograms were obtained by calculating the ratio of the peak area of a target substance to the peak area of the external standard.

2.5. Evaluation of ovarian activity A similar procedure to that reported by Bloch (2000b) and Geva et al. (2005) was adopted for the evaluation of ovarian activity. Ovarian activity was evaluated by measuring the length of the largest terminal (basal) oocytes and classifying the stage of the most-activated oocyte in the ovarioles. Four ovarian stages were defined based on the size and shape of oocyte and trophocyte chambers. Criteria for each stage of ovarian activity were as follows: (1) stage 1: ovaries contain clearly distinguishable but still with small (almost spherical) oocytes and a much larger trophocyte chamber (oval in shape) (largest terminal oocyte length ≤ 0.5 mm); (2) stage 2: oocytes larger than in 10

stage 1 and elongated, but the trophocyte chamber is still larger than the oocyte (0.5 mm < largest terminal oocyte length ≤ 1.0 mm); (3) stage 3: oocytes now oval in shape and as large or larger than the trophocyte chamber (1.0 mm < largest terminal oocyte length ≤ 2.5 mm); (4) stage 4: ovaries contain mature eggs with a clearly visible chorion and the trophocyte chamber has almost entirely degenerated (2.5 mm < largest terminal length). Each pair of ovaries was carefully removed from the abdomen under a dissecting microscope. Photographic images were taken with a digital camera and analyzed with commercially available computer software (Photomeasure, Kenis, Osaka, Japan).

2.6. Statistics Mean values of biogenic amine levels were compared between 1DAM, Dp, and CF queens with a Kruskal–Wallis test following a Steel–Dwass test (P = 0.05 significance level) for multiple comparisons. Proportions of individuals at each ovarian stage between 1DAM, Dp, and CF queens were compared by Fisher’s exact test (3 × 4 table). Correlations of biogenic amine levels between the brain and other tissues (meso-metathoracic ganglia, terminal abdominal ganglion, and hemolymph) in mated queens were examined by a Spearman’s rank correlation test. Correlations between the brain levels of biogenic amines and the lengths of the largest terminal oocytes in isolated virgin queens and workers were also examined by a Spearman’s rank correlation test.

3. Results 3.1.1. Experiment 1: biogenic amine levels in the central nervous system and ovarian 11

activities in queens Biogenic amines, including dopamine, serotonin, tyramine, octopamine, a metabolite of dopamine (NADA), and a metabolite of serotonin (NA5HT), were measured in the brains, meso-metathoracic ganglia, terminal abdominal ganglion, and hemolymph of queens in different behavioral and reproductive states (Figs 2–5). In the brains, levels of dopamine and octopamine were significantly different among the three groups (Kruskal–Wallis test, dopamine: H = 15.598, P <0.001, Fig. 2A; octopamine: H = 7.645, P <0.05, Fig. 2D), whereas levels of serotonin and tyramine did not differ among the groups (serotonin: H = 1.148, P = 0.563, Fig. 2B, tyramine: H = 3.003, P = 0.223, Fig. 2C). Dopamine levels were significantly lower in CF queens than in 1DAM and Dp queens, but were not different between 1DAM and Dp queens (Steel–Dwass test, P <0.05, Fig. 2A). Octopamine levels in the brains were significantly higher in Dp queens than in 1DAM queens (P <0.05), and the levels in CF queens were intermediate between those of 1DAM and Dp queens (Fig. 2D). Levels of serotonin and tyramine did not differ significantly between the groups (Fig. 2B and 2C). Levels of NADA and NA5HT were significantly different among the three groups (Kruskal–Wallis test, NADA: H = 13.173, P <0.01, Fig. 2E; NA5HT: H = 13.277, P <0.01, Fig. 2F). The trends in the differences in both metabolites were similar, with the levels in Dp queens significantly lower than those in 1DAM or CF queens (Steel–Dwass test, P <0.05, Fig. 2E and 2F). In the meso-metathoracic ganglia, dopamine levels were significantly different among the three groups (Kruskal–Wallis test, H = 12.072, P <0.01, Fig. 3A), whereas levels of serotonin, tyramine, and octopamine did not differ significantly (serotonin: H = 5.394, P = 0.07, Fig. 3B, tyramine: H = 3.697, P = 0.158, Fig. 3C; octopamine: H = 12

0.575, P = 0.75, Fig. 3D). Dopamine levels were significantly lower in CF queens than in 1DAM queens, and the levels in Dp queens were intermediate between those in 1DAM and CF queens (Steel–Dwass test, P <0.05, Fig. 3A). Levels of NADA and NA5HT did not differ among the groups (NADA: H = 2.699, P = 0.259, Fig. 3E, NA5HT: H = 0.55, P = 0.76, Fig. 3F). In the terminal abdominal ganglion, tyramine levels were significantly different among the three groups (Kruskal–Wallis test, H = 6.090, P <0.05, Fig. 4C), whereas levels of dopamine, serotonin, and octopamine did not differ significantly (dopamine: H = 3.207, P = 0.201, Fig. 4A; serotonin: H = 0.725, P = 0.696, Fig. 4B; octopamine: H = 1.469, P = 0.48, Fig. 4D). Although tyramine levels were different among the groups, the multiple comparison test did not detect any significant differences between the groups (Steel–Dwass test, P >0.05, Fig. 4C). Levels of NADA and NA5HT did not differ significantly among the groups (NADA: H = 0.023, P = 0.989, Fig. 4E; NA5HT: H = 0.196, P = 0.907, Fig. 4F). In hemolymph samples, dopamine and NADA were detected, but serotonin, NA5HT, and tyramine were not from the volume of hemolymph that we collected. Dopamine and NADA levels were significantly different among the groups (Kruskal–Wallis test, dopamine: H = 6.213, P <0.05, Fig. 5A; NADA: H = 10.736, P <0.01, Fig. 5B). Dopamine levels were significantly lower in CF queens than in 1DAM queens, and the levels in Dp queens were intermediate between those in 1DAM and CF queens (Steel–Dwass test, P <0.05, Fig. 5A). The trends in the hemolymph levels were similar to those in the thoracic ganglia. NADA levels were significantly lower in CF queens than in Dp queens, and levels in 1DAM queens were intermediate between those in Dp and CF queens (Steel–Dwass test, P <0.05, Fig. 5B). 13

The lengths of the largest terminal oocytes were significantly different among the three groups (Kruskal–Wallis test, H = 16.187, P <0.001, Fig. 5C). The length in CF queens was significantly longer than those in 1DAM or Dp queens (Steel–Dwass test, P <0.05, Fig. 5C), indicating that CF queens had activated ovaries whereas the other queens did not. Ovarian activity was also evaluated by classifying the stages of the most-activated oocyte in ovarioles. All CF queens had ovaries at stage 4, whereas 1DAM and Dp queens had ovaries at stages 1–3 and stages 1–2, respectively (Fig. 5D). The proportion of individuals at each ovarian stage differed significantly (Fisher’s exact test, χ2 = 26.22, d.f. = 6, P <0.001). Thus, the ovarian status in mated queens indicated that CF queens had more activated ovaries than the other queens, supporting the results of the lengths of the largest terminal oocytes.

3.1.2. Experiment 1: correlation of amine levels between the brain and other CNS tissues The correlation of biogenic amine levels between brains and other CNS tissues, including the meso-metathoracic ganglia, terminal abdominal ganglion, and hemolymph, was assessed to investigate the temporal relation of amine dynamics between several compartments of the CNS. For dopamine, the brain levels were significantly positively correlated with the levels in the thoracic ganglia, abdominal ganglion, and hemolymph (Table 1). For octopamine, tyramine, and serotonin, the brain levels were not significantly correlated with any of the other CNS tissues examined (Table 1).

3.2. Experiment 2: correlation of brain amine levels with ovarian activity in virgin queens and queenless workers 14

The correlation of dopamine levels in brains with the lengths of the largest terminal oocytes was assessed in both isolated virgin queens and workers (Fig. 6). Several virgin queens began to lay eggs after they were 13–15-days old and had well-activated ovaries (Fig. 6A). In virgin queens, there were no significant correlations between brain dopamine levels and the lengths of the largest terminal oocytes in 13–15-day-old individuals (rs = −0.209, P = 0.419, n = 16, Fig. 6A-1) or 18–20-day-old individuals (rs = 0.287, P = 0.236, n = 18, Fig. 6A-2). However, in isolated workers, the brain levels of dopamine were significantly positively correlated with the lengths of the largest terminal oocytes in both 6-day-old (rs = 0.587, P =0.05, n = 12, Fig. 6B-1) and 10-day-old individuals (rs = 0.418, P <0.05, n = 27, Fig. 6B-2), indicating a caste difference in the correlation between brain dopamine levels and ovarian activity. For the other biogenic amines, including serotonin, tyramine and octopamine, the brain levels were not significantly correlated with the lengths of the largest terminal oocytes (Table S1). The ovarian stages in isolated virgin queens varied from stage 1 to stage 4, whereas those in isolated workers varied from stage 1 to stage 3 (Fig. 6C). Thus, there was larger variation in ovarian stages in isolated queens, but no significant correlations between biogenic amine levels and the lengths of the largest terminal oocytes.

4. Discussion This study compared the physiological states of queens between 1DAM, during Dp and during CF, with a focus on the levels of biogenic amines in several compartments of the CNS and hemolymph (experiment 1) in the bumble bee B. ignitus. This study also examined the correlation between the levels of biogenic amines and ovarian activities in isolated virgin queens and workers (experiment 2). In experiment 1, dopamine levels in 15

the brain, meso-metathoracic ganglia, and hemolymph decreased remarkably from mating to CF via Dp. Brain levels of dopamine were significantly lower in CF queens with well-activated ovaries than in the same-aged queens during Dp, which suggests that dopamine does not function in promoting ovarian activation in CF queens. In experiment 2, brain levels of dopamine were not significantly correlated with the lengths of the largest terminal oocytes in virgin queens, even if the levels in isolated workers were significantly correlated with oocyte length. These results suggest that dopamine is associated with ovarian activity in queenless workers, but not in either virgin or mated queens. The low levels of dopamine in the brains of CF B. ignitus queens were similar to those in egg-laying mated queens of the honey bee Apis mellifera, although the honey bee queens do not hibernate, secrete wax for egg cups, care for broods, or found colonies. In honey bee queens, the levels of dopamine and NADA in the brain and hemolymph decrease after mating and are maintained at relatively low levels during egg laying, although the levels are still higher than in workers (Harano et al., 2005, 2008; Sasaki et al., 2012). It has also been reported that expression of the genes encoding dopamine N-acetyltransferase (Manfredini et al., 2015) and dopamine transporter Amdat (Nomura et al., 2009) in mated queens is downregulated compared with the expression in virgin honey bee queens. The former encodes an enzyme that converts dopamine to N-acetyldopamine, thus inactivating any released dopamine (Evans, 1980; Mir and Vaughan, 1981; Cheng et al., 2012), the latter encodes a protein involved in the uptake of released dopamine by presynaptic dopaminergic cells (Jones et al., 1998; Pörzgen et al., 2001). Expression of the dopamine receptor genes Amdop1, Amdop2, and Amdop3 in the brains of honey bee queens is not altered by mating, whereas expression of 16

Amdop1 in the ovaries is decreased by mating (Vergoz et al., 2012). A similar decrease in the brain levels of dopamine in egg-laying mated queens has been reported in the ant Formica japonica (Aonuma and Watanabe, 2012). In this ant, the brain levels of octopamine and serotonin also decrease during CF. The founding queens of these ants shed their wings shortly after mating, and by resorbing their flight muscle mass they can use these resources to rear the first brood without foraging. Thus, the life histories of the mated queens are slightly different among these bumble bee, honey bee, and ant species. In B. ignitus, lower levels of dopamine were detected not only in the brains, but also in the meso-metathoracic ganglia and hemolymph in CF queens. A decrease in dopamine levels in several parts of a body might be needed during CF. Dopamine can enhance locomotor activities that might promote mating flights and other behaviors of virgin queens in the honey bee (Harano et al., 2008). Similar effects of dopamine on locomotor activities or flight activities have been reported in worker and male honey bees (Beggs et al., 2007; Akasaka et al., 2010; Mustard et al., 2010; Mezawa et al., 2013) and in the large carpenter bee Xylocopa appendiculata (Sasaki and Nagao, 2013). Given the similar behavioral roles of dopamine in other eusocial species, high locomotor activities might not be necessary for egg laying by mated queens of these bumble bee, honey bee, and ant species. Rather, a reduction in locomotion might be beneficial for energy reserves, investment in egg production, maintaining low sexual responses to males, and a low motivation for mating. This ecological background might be associated with the low dopamine levels in CF queens. In experiment 1, there were no significant differences in the levels between groups of any of the biogenic amines in the thoracic ganglia and abdominal ganglion, except for dopamine levels in the thoracic ganglia. Large variation in the levels in groups were 17

recorded and this variation could explain the insignificant results. It is possible that different ganglial volumes between individuals result in variation in the amount of biogenic amines present in those structures. However, we consider that there might be other explanations for this variation. For example, there was a larger variation (standard error) in octopamine levels in thoracic ganglia from 1DAM compared with Dp and CF (Fig. 3D), whereas no such variation of other biogenic amines in 1DAM compared with Dp and CF occurred (Fig. 3). Thus, given that these data are derived from the same ganglion, the variations might not result from different volumes of the individual ganglia, but from individual physiological states. The regulation of dopamine levels might be shared among the brain, and thoracic and abdominal ganglia in mated B. ignitus queens. There were positive correlations of dopamine levels in the brain with those in the thoracic, abdominal ganglia, and hemolymph, suggesting that dopamine is regulated among different compartments of the CNS and hemolymph. There were no significant correlations in the levels of other amines between the brain and other compartments of the CNS, indicating that only dopamine is selectively regulated. It has been reported that D-2 autoreceptors expressed in dopaminergic neurons in mammals can regulate the dopaminergic system by providing feedback inhibition of the synthesis, release, and uptake of dopamine (Ford, 2014). Although we do not know the control mechanisms responsible for dopamine levels in B. ignitus, a possible mechanism is that the hemolymph dopamine acts on autoreceptors in dopaminergic neurons and regulates dopamine levels in several compartments of the CNS. Alternatively, other unknown factors in hemolymph might influence the dopamine levels in these nervous tissues. In isolated B. ignitus workers, dopamine levels in the brains were significantly 18

positively correlated with the lengths of the longest terminal oocytes. Although the effects of dopamine on ovarian activity remains to be determined in the bumble bee, reproductive effects of dopamine have been reported in reproductive workers in the honey bee (A. mellifera) (Dombroski et al., 2003), paper wasp (Polistes chinensis) (Sasaki et al., 2009) and a queenless ponerine ant (Diacamma sp.) (Okada et al., 2015). Given the reproductive roles of dopamine in queenless B. ignitus workers, the role of dopamine might differ between castes. The role of dopamine in mated queens remains to be determined. In B. terrestris, juvenile hormone has a role as a gonadotropin and promotes ovarian activity in reproductive workers, but does not affect task performance or the division of labor among workers (Röseler, 1977; Bloch et al., 2000a; Shpigler et al., 2016). Juvenile hormone biosynthesis was higher in reproductive workers than in queens (Bloch et al., 2000a), which might result in a significant relationship between juvenile hormone and reproduction in reproductive workers, but not in functional queens. Given the reproductive functions in reproductive B. ignitus workers, the relationship between juvenile hormone and brain dopamine should be tested to understand mechanisms underlying regulations of dopamine levels in the brain. It has been reported that juvenile hormone could enhance dopamine levels in the brain in male honey bee (Mezawa et al., 2013) and the large carpenter bee (Sasaki and Nagao, 2013). Octopamine levels in the brain were significantly larger in Dp queens than in 1DAM queens. This might be a response to low temperature stress, although the role of octopamine in the stress response during Dp remains to be determined. Surprisingly, low-temperature Dp did not decrease the brain levels of dopamine, serotonin, or tyramine, even though it can cause the inactivity of various metabolic processes, 19

including the biosynthesis of monoamines by enzymes (Chen et al., 2008; Khodayari et al., 2013). Interestingly, the levels of metabolites of dopamine (NADA) and serotonin (NA5HT) in the brain were significantly lower in Dp queens than in 1DAM or CF queens. This suggests that the low temperature during Dp inhibits the release of dopamine and serotonin, and metabolic processes involving N-acetyltransferase. This study focused on the whole-brain dynamics of biogenic amines and several other compartments of the CNS. This is a first step in the clarification of the entire neuroendocrine system of mated queens and the different roles of dopamine in the brains of different bumble bee castes. However, the detailed function of particular biogenic amines within the bumble bee brain remains to be determined by the quantification of biogenic amines in specific brain regions.

5. Conclusion To explore the neuroendocrine system of biogenic amines associated with reproduction in queens and reproductive workers in primitively eusocial bees, we investigated the dynamics of biogenic amine levels in the brain, meso-metathoracic ganglia, terminal abdominal ganglion, and hemolymph in mated queens in different behavioral and reproductive states. Brain levels of dopamine in CF queens were significantly lower than in Dp queens of the same age. These results suggest that brain dopamine does not have a role in promoting ovarian activity or egg laying in mated queens. Dopamine levels in the brains were significantly positively correlated with those in the thoracic ganglia, abdominal ganglion, or hemolymph, suggesting the regulation of dopamine levels among several CNS tissues. In isolated virgin queens, there were no significant correlations between the brain levels of the biogenic amines 20

that we examined and the lengths of the largest terminal oocytes, whereas, in isolated workers, the brain dopamine levels were positively correlated with oocyte length. These results suggest that dopamine is associated with ovarian activity in reproductive workers, but not in either virgin or mated queens. The neuroendocrine system in this species is similar to that of other highly eusocial hymenopterans and might be evidence of physiological caste differences in primitively eusocial bees.

Acknowledgments This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant numbers 25450483, 26440181, 26291091 to K.S.

References Akasaka, S., Sasaki, K., Harano, K., Nagao, T., 2010. Dopamine enhances locomotor activity for mating in male honeybees. J. Insect Physiol. 56, 1160–1166. (doi: 10.1016/j.jinsphys.2010.03.013) Aonuma, H., Watanabe, T., 2011. Changes in the content of brain biogenic amine associated with early colony establishment in the queen of the ant, Formica japonica. PLoS ONE 7, e43377. (doi: 10.1371/journal.pone.0043377) Asada, S., Ono, M., 2000. Difference in colony development of two Japanese bumblebees, Bombus hypocrita and B. ignitus (Hymenoptera: Apidae). Appl. Entomol. Zool. 35, 597–603. (doi: 10.1303/aez.2000.59) Beggs, K.T., Glendining, K.A., Marechal, N.M., Vergoz, V., Nakamura, I., Slessor, K.N., Mercer, A.R., 2007. Queen pheromone modulates brain dopamine function in worker honey bees. Proc. Natl. Acad. Sci. USA 104, 2460–2464. (doi: 21

10.1073/pnas.0608224104) Bloch, G., Borst, D.W., Huang, Z., Robinson, G.E., Cnaani, J., Hefetz, A., 2000a. Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris. J. Insect Physiol. 46, 47–57. (doi: 10.1016/S0022-1910(99)00101-8) Bloch, G., Simon, T., Robinson, G.E., Hefetz, A., 2000b. Brain biogenic amines and reproductive dominance in bumble bees (Bombus terrestris). J. Comp. Physiol. 186A, 261–268. (doi: 10.1007/s003590050426) Bloch, G., Wheeler, D.E., Robinson, G.E., 2002. Endocrine influences on the organization of insect societies, in: Pfaff, D., Arnold, A., Etgen, A., Fahrbach, S., Moss, R., Rubin, R. (Eds.), Hormones, Brain and Behavior. Academic Press, San Diego, vol. 3, pp. 195–235. Burrows, M., 1996. The Neurobiology of an Insect Brain. Oxford University Press, Oxford. Chen, Y., Hung, Y., Yang, E., 2008. Biogenic amine levels change in the brains of stressed honeybees. Arch. Insect Biochem. Physiol. 68, 241–250. (doi: 10.1002/arch.20259) Cheng, K., Liao, J., Lyu, P., 2012. Crystal structure of the dopamine Nacetyltransferase–acetyl-CoA complex provides insights into the catalytic mechanism. Biochem. J. 446, 395–404. (doi: 10.1042/BJ20120520) Dombroski, T.C.D., Simões, Z.L.P., Bitondi, M.M.G., 2003. Dietary dopamine causes ovary activation in queenless Apis mellifera workers. Apidologie 34, 281–289. (doi: 10.1051/apido:2003024) Evans, P.D., 1980. Biogenic amines in the insect nervous system. Adv. Insect Physiol. 22

15, 317–473. (doi: 10.1016/S0065-2806(08)60143-5) Ford, C.P., 2014. The role of D2-autoreceptors in regulating dopamine neuron activity and transmission. Neuroscience 282, 13–22. (doi: 10.1016/j.neuroscience.2014.01.025.) Geva, S., Hartfelder, K., Bloch, G., 2005. Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris. J. Insect Physiol. 51, 811–823. (doi: 10.1016/j.jinsphys.2005.03.009) Goulson, D., 2009. Bumble bees: Behaviour, Ecology, and Conservation, second ed. Oxford University Press, Oxford. Harano, K. Sasaki, K., Nagao, T. 2005. Depression of brain dopamine and its metabolite after mating in European honeybee (Apis mellifera) queens. Naturwissenschaften 92, 310–313. (10.1007/s00114-005-0631-3) Harano, K. Sasaki, M., Nagao, T., Sasaki, K., 2008. Dopamine influences locomotor activity in honeybee queens: implications for a behavioural change after mating. Physiol. Entomol. 33, 395–399. (doi: 10.1111/j.1365-3032.2008.00644.x) Harris, J.W., Woodring, J., 1995. Elevated brain dopamine levels associated with ovary development in queenless worker honey bees (Apis mellifera). Comp. Biochem. Physiol. C 111, 271–270. (doi: 10.1016/0742-8413(95)00048-S) Hartfelder, K., Emlen, D.J., 2005. Endocrine control of insect polyphenism, in: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Molecular Insect Science. Elsevier, Oxford, chapter 3.13, vol. 3, pp. 651–703. Hölldobler, B., Wilson, E.O., 1990. The ants, The Belknap Press of Harvard University Press, Cambridge, MA. Hunt, J.H., 2007. The Evolution of Social Wasps, Oxford University Press, Oxford. 23

Jones, S.R., Gainetdinov, R.R., Jaber, M., Giros, B., Wightman, R.M., Caron, M.G., 1998. Profound neural plasticity in response to inactivation of the dopamine transporter. Proc. Natl. Acad. Sci. USA 95, 4029–4034. Khodayari, S., Moharramipour, S., Larvor, V., Hidalgo, K., Renault, D., 2013. Deciphering the metabolic changes associated with diapause syndrome and cold acclimation in the two-spotted spider mite Teranychus urticae. PLoS ONE 8, e54025. (doi: 10.1371/journal.pone0054025) Kokay, I.C., Mercer, A.R., 1997. Age-related changes in dopamine receptor densities in the brain of the honey bee, Apis mellifera. J. Comp. Physiol. A 181, 415–423. (doi: 10.1007/s003590050125) Kwon, Y.J., Saeed, S., Duchateau, M.J., 2003. Stimulation of colony initiation and colony development in Bombus terrestris by adding a male pupa: the influence of age and orientation. Apidologie 34, 429–437. (doi: 10.1051/apido:2003039) Lange, A.B., 2009. Tyramine: from octopamine precursor to neuroactive chemical in insects. Gen. Comp. Endocrinol. 162, 18–26. (doi:10.1016/j.ygcen.2008.05.021) Manfredini, F., Brown, M.J.F., Vergoz, V., Oldroyd, B.P., 2015. RNA-sequencing elucidates the regulation of behavioral transitions associated with the mating process in honey bee queens. BMC Genomics 16, 563. (doi: 10.1186/s12864-015-1750-7) Mezawa, R., Akasaka, S., Nagao, T., Sasaki, K., 2013. Neuroendocrine mechanisms underlying regulation of mating flight behaviors in male honey bees (Apis mellifera L.). Gen. Comp. Endocrinol. 186, 108–115. (doi: 10.1016/j.ygcen.2013.02.039) Michener, C.D., 1974. The Social Behavior of the Bees, second ed. The Belknap Press of Harvard University Press, Cambridge, MA. 24

Mir, A. K., Vaughan, P. F. T., 1981. Biosynthesis of N-acetyldopamine and N-acetyloctopamine by Schistocerca gregaria nervous tissue. J. Neurochem. 36, 441–446. (doi: 10.1111/j.1471-4159.1981.tb01612.x) Mustard, J. A., Pham, P.M., Smith, B.H., 2010. Modulation of motor behavior by dopamine and the D1-like dopamine receptor AmDOP2 in the honey bee. J. Insect. Physiol. 56, 422–430. (doi: 10.1016/j.jinsphys.2009.11.018) Nomura, S., Takahashi, J., Sasaki, T., Yoshida, T., Sasaki, M., 2009. Expression of the dopamine transporter in the brain of the honeybee, Apis mellifera L. (Hymenoptera: Apidae). Appl. Entomol. Zool. 44, 403–411. (doi: 10.1303/aez.2009.403) Okada, Y., Sasaki, K., Miyazaki, S., Shimoji, H., Tsuji, K., Miura, T., 2015. Social dominance and reproductive differentiation mediated by dopaminergic signaling in a queenless ant. J. Exp. Biol. 218, 1091–1098. (doi: 10.1242/jeb.118414) Pörzgen, P., Park, S.K., Hirsh, J., Sonders, M.S., Amara, S.G., 2001. The antidepressant-sensitive dopamine transporter in Drosophila melanogaster: a primordial carrier for catecholamines. Mol. Pharmacol. 59, 83–95. (doi: https://doi.org/10.1124/mol.59.1.83) Robinson, G.E., Strambi, C., Strambi, A. and Feldlaufer, M.F., 1991. Comparison of juvenile hormone and ecdysteroid haemolymph titres in adult worker and queen honey bees (Apis mellifera). J. Insect Physiol. 37, 929–935. (doi: 10.1016/0022-1910(91)90008-N) Robinson, G.E., Vargo, E.L., 1997. Juvenile hormone in adult eusocial hymenoptera: gonadotropin and behavioral pacemaker. Arch. Insect Biochem. Physiol. 35, 559–583. (doi: 10.1002/(SICI)1520-6327(1997)35:4<559::AID-ARCH13>3.0.CO;2-9) 25

Röseler, P., 1977. Juvenile hormone control of oögenesis in bumblebee workers, Bombus terrestris. J. Insect Physiol. 23, 985–992. (doi: 10.1016/0022-1910(77)90126-3) Sasagawa, H., Kawahara, Y. Quantitative determination method for a juvenile hormone III titer in honeybee haemolymph by high-performance liquid chromatography. Agric. Biol. Chem. 52, 1295–1297. (doi: 10.1271/bbb1961.52.1295) Sasaki, K., Nagao, T., 2001. Distribution and levels of dopamine and its metabolites in brains of reproductive workers in honeybees. J. Insect Physiol. 47, 1205–1216. (doi: 10.1016/s0022-1910(01)00105-6) Sasaki, K., Yamasaki, K., Tsuchida, K., Nagao, T., 2009. Gonadotropic effects of dopamine in isolated workers of the primitively eusocial wasp, Polistes chinensis. Naturwissenschaften 96, 625–629. (doi: 10.1007/s00114-009-0510-4) Sasaki, K., Matsuyama, S., Harano, K., Nagao, T., 2012. Caste differences in dopamine-related substances and dopamine supply in the brains of honeybees (Apis mellifera L.). Gen. Comp. Endocrinol. 178, 46–53. (doi: 10.1016/j.ygcen.2012.04.006) Sasaki, K., Nagao, T., 2013. Juvenile hormone-dopamine systems for the promotion of flight activity in males of the large carpenter bee Xylocopa appendiculata. Naturwissenschaften 100, 1183–1186. (doi: 10.1007/s00114-013-1116-4) Schulz, D.J., Robinson, G.E., 1999. Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age-related changes in the mushroom bodies. J. Comp. Physiol. A 184, 481–488. (doi: 10.1007/s003590050348) Shpigler, H., Siegel, A.J., Huang, Z., Bloch, G., 2016. No effect of juvenile hormone on 26

task performance in a bumblebee (Bombus terrestris) supports an evolutionary link between endocrine signaling and social complexity. Horm. Behav. 85, 67–75. (doi: 10.1016/j.yhbeh.2016.08.004) Taylor, D.J., Robinson, G.E., Logan, B.J., Laverty, R., Mercer, A.R., 1992. Changes in brain amine levels associated with the morphological and behavioural development of the worker honeybee. J. Comp. Physiol. A 170, 715–721. (doi: 10.1007/BF00198982) Vergoz, V., Lim, J., Duncan, M., Cabanes, G., Oldroyd, B.P., 2012. Effects of natural mating and CO2 narcosis on biogenic amine receptor gene expression in the ovaries and brain of queen honey bees, Apis mellifera. Insect Mol. Biol. 21, 558–67. (doi: 10.1111/j.1365-2583.2012.01159.x) Yoneda, M., 2008. Induction of colony initiation by Japanese native bumble bees using cocoons of the exotic bumblebee Bombus terrestris. Entomol. Sci. 11, 123–126. (doi: 10.1111/j.1479-8298.2007.00258.x)

27

Table 1 Correlations of biogenic amine levels between the brain and meso-metathoracic ganglia (Th.), terminal abdominal ganglion (Ab.) or hemolymph (Hemo.) in mating queens. The bold letters indicate significant correlations.

Dopamine

Octopamine

Tyramine

Serotonin

rs

P

rs

P

rs

rs

P

0.668

<0.01

0.218

0.285

−0.016 0.937

0.099

0.627

0.463

<0.05

0.269

0.198

−0.045 0.828

0.069

0.742

0.702

<0.001 -

-

-

-

-

Tissues P

Brain vs. Th. (n = 25) Brain vs. Ab. (n = 24) Brain vs. Hemo. (n = 25)

28

-

Figure legends

Figure 1. Sampling schedule of mated queens of Bombus ignitus. Three groups of queens were used in the study.

Figure 2. Biogenic amine levels in the brains of queens 1 day after mating (1DAM), diapausing queens (Dp), and colony-founding queens (CF). Levels (mean ± SE) of dopamine (DA) (A), serotonin (5HT) (B), tyramine (TA) (C), octopamine (OA) (D), a metabolite of dopamine (NADA) (E), and a metabolite of serotonin (NA5HT) (F). The numbers in parentheses indicate the number of individuals examined. Different letters above the bar indicate significant differences between the groups (Steel–Dwass test, P <0.05). Statistical values were calculated with a Kruskal–Wallis test to examine the differences among the three experimental groups.

Figure 3. Biogenic amine levels in the meso-metathoracic ganglia in queens 1 day after mating (1DAM), diapausing queens (Dp), and colony-founding queens (CF). Levels (mean ± SE) of dopamine (DA) (A), serotonin (5HT) (B), tyramine (TA) (C), octopamine OA) (D), NADA (E), and NA5HT (F). The numbers in parentheses indicate the number of individuals examined. Different letters above the bar indicate significant differences between the groups (Steel–Dwass test, P <0.05). Statistical values were calculated with a Kruskal–Wallis test to examine the differences among the three experimental groups.

Figure 4. Biogenic amine levels in the terminal abdominal ganglion in queens 1 day 29

after mating (1DAM), diapausing queens (Dp), and colony-founding queens (CF). Levels (mean ± SE) of dopamine (DA) (A), serotonin (5HT) (B), tyramine (TA) (C), octopamine (OA) (D), NADA (E), and NA5HT (F). The numbers in parentheses indicate the number of individuals examined. Different letters above the bar indicate significant differences between the groups (Steel–Dwass test, P <0.05). Statistical values were calculated with a Kruskal–Wallis test to examine the differences among the three experimental groups.

Figure 5. Levels (mean ± SE) of dopamine (DA) (A) and NADA (B) in the hemolymph, lengths of the largest terminal oocytes (mean ± SE) (C), and proportion of individuals at each ovarian stage (D) in queens 1 day after mating (1DAM), diapausing queens (Dp), and colony-founding queens (CF). The numbers in parentheses indicate the number of individuals examined. Different letters above the bar indicate significant differences between the groups (Steel–Dwass test, P <0.05). Statistical values were calculated with a Kruskal–Wallis test to examine the differences among the three experimental groups.

Figure 6. Correlation between brain dopamine levels and lengths of the largest terminal oocytes in isolated virgin queens (A) and isolated workers (B). Data for 13–15-day-old queens (A-1) and 18–20-day-old queens (A-2), 6-day-old workers (B-1) and 10-day-old workers (B-2) are presented. Proportions of individuals at each ovarian stage are also presented (C).

30

31

B

DA-brain H = 15.598 P < 0.001

50

a

Dopamine levels (pmol / brain)

40

5HT-brain

a

30

b

20 10

CF

1DAM

Dp

CF

(10)

(7)

(8)

(10)

(7)

(8)

D

TA-brain

OA-brain 8

H = 3.003 P = 0.223

Octopamine levels (pmol / brain)

Tyramine levels (pmol / brain)

1

Dp

4 3 2 1

b

7

H = 7.645 P < 0.05 ab

6

a

5 4 3 2 1 0

0 1DAM

Dp

CF

1DAM

Dp

CF

(10)

(7)

(8)

(10)

(7)

(8)

F

NADA-brain H = 13.173 P < 0.01

a

a

10 8 6

NA5HT-brain

b

4

H = 13.277 P < 0.01

1

NA5HT levels (pml/brain)

E NADA levels (pmol/brain)

2

1DAM

C

12

3

0

0

14

H = 1.148 P = 0.563

4

Serotonin levels (pmol / brain)

A

a a

0.5

b

2 0 1DAM

Dp

CF

(10)

(7)

(8)

0 1DAM

Dp

CF

(10)

(7)

(8)

Figure 02

DA-thoracic ganglia Dopamine levels (pmol/ganglia)

15

B

5HT-thoracic ganglia 2

H = 12.072 P < 0.01

a

10

ab b

5

Serotonin levels (pmol/ganglia)

A

1.5 1 0.5

0 Dp

CF

(10)

(7)

(8)

TA-thoracic ganglia Tyramine levels (pmol/ganglia)

3

0

D

1

CF

(7)

(8)

H = 0.575 P = 0.75

5 4 3 2 1 0

0 1DAM

Dp

CF

(10)

(7)

(8)

NADA-thoracic ganglia 7

F

4 3 2 1

Dp

CF

(10)

(7)

(8)

0.6

H = 0.55 P = 0.76

0.5

NA5HT levels (pmol/ganglia)

5

1DAM

NA5HT-thoracic ganglia

H = 2.699 P = 0.259

6

NADA levels (pmol/ganglia)

Dp

(10)

6

H = 3.697 P = 0.158

2

E

1DAM

OA-thoracic ganglia Octopamine levels (pmol/ganglia)

C

1DAM

H = 5.394 P = 0.07

0.4 0.3 0.2 0.1 0

0 1DAM

Dp

CF

(10)

(7)

(8)

1DAM

Dp

CF

(10)

(7)

(8)

Figure 03

A

DA-abdominal ganglion H = 3.207 P = 0.201

1.5 1 0.5

0.2 0.1

CF

1DAM

Dp

CF

(9)

(7)

(8)

(9)

(7)

(8)

TA-abdominal ganglion

D

H = 6.090 a P < 0.05

0.8 0.6

a

0.4

a 0.2

1DAM

Dp

CF

(9)

(7)

(8)

NADA-abdominal ganglion 1

F

0.4 0.2 0 1DAM

(9)

Dp

CF

(7)

(8)

0.2

0.1

1DAM

Dp

CF

(9)

(7)

(8)

NA5HT-abdominal ganglion 0.2

NA5HT levels (pmol/ganglion)

0.6

H = 1.469 P = 0.48

0

H = 0.023 P = 0.989

0.8

OA-abdominal ganglion 0.3

Octopamine levels (pmol/ganglion)

Tyramine levels (pmol/ganglion)

0.3

Dp

0

NADA levels (pmol/ganglion)

0.4

1DAM

1

E

H = 0.725 P = 0.696

0

0

C

5HT-abdominal ganglion 0.5

Serotonin levels (pmol/ganglion)

2

Dopamine levels (pmol/ganglion)

B

H = 0.196 P = 0.907

0.1

0 1DAM

Dp

CF

(9)

(7)

(8)

Figure 04

10

Dopamine levels (pmol / µL)

B

DA-hemolymph

30

H = 6.213 P < 0.05

a

8 6

NADA-hemolymph

ab

4

b

2

25

NADA levels (pmol / µL)

A

Dp

CF

(10)

(7)

(8)

5

Terminal oocyte length (mm)

15 10

b

H = 16.187 P < 0.001

4

b

3 2

0

D

1DAM

Dp

CF

(10)

(7)

(8)

Ovarian stage Proportion of individuals

1DAM

Terminal oocyte length

1

ab

5

0

C

20

a

H = 10.736 P < 0.01

1 0.8 Stage 4

0.6

Stage 3

0.4

Stage 2 Stage 1

a

a

1DAM

Dp

CF

1DAM

Dp

CF

(10)

(7)

(8)

(10)

(7)

(8)

0

0.2 0

Figure 05

rs = -0.209 P = 0.419 n = 16

1 2 3 4 5 Terminal oocyte length (mm)

B-1 140 120 100 80 60 40 20 0

rs = 0.587 P = 0.05 n = 12

y = 41.564x + 27.976

0

Isolated virgin queens (18-20 days old)

140 120 100 80 60 40 20 0

rs = 0.287 P = 0.236 n = 18

0

B-2

Isolated workers (6 days old)

C

Amount of dopamine (pmol / brain)

140 120 100 80 60 40 20 0 0

Amount of dopamine (pmol / brain)

A-2

Isolated virgin queens (13-15 days old)

1 2 3 4 Terminal oocyte length

5

Amount of dopamine (pmol / brain)

Amount of dopamine (pmol / brain)

A-1

1 2 3 4 5 Terminal oocyte length (mm)

Isolated workers (10 days old) 140 120 100 80 60 40 20 0

rs = 0.418 P < 0.05 n = 27

y = 5.7609x + 28.32

0

1 2 3 4 5 Terminal oocyte length (mm)

Ovarian stage Proportion of individuals

1 0.8 Stage 4

0.6

Stage 3

0.4

Stage 2 Stage 1

0.2 0 13-15 days 18-20 days

Virgin queens

6 days

10 days

Workers

Figure 06

32

Highlights


Colony founding queens had depressed dopamine levels in the brains.




Brain dopamine levels were correlated with other ganglia and hemolymph in queens.




In virgin queens, dopamine levels were not correlated with ovarian activity.




In workers, dopamine levels were positively correlated with ovarian activity.




Dopamine may play a role in ovarian activity in workers, but not in queens.

33