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The role of juvenile hormone in competition and cooperation by burying beetles
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Journal of Insect Physiology 52 (2006) 1005–1011 www.elsevier.com/locate/jinsphys
The role of juvenile hormone in competition and cooperation by burying beetles Michelle Pellissier Scott Department of Zoology, University of New Hampshire, Durham, NH 03824, USA Received 28 January 2006; received in revised form 8 April 2006; accepted 10 April 2006
Abstract Few studies have addressed the physiological mechanisms that modulate aggression in insects. In some social insects, there is a correlation of JH and aggression in colony defense and in the establishment of dominance, but only a few studies demonstrate a causal relationship. Burying beetles aggressively defend a breeding resource, a carcass, and juvenile hormone (JH) hemolymph titers increase rapidly upon the discovery of a carcass. In this study, I show that treatment with the JH analog, methoprene, in the absence of a carcass increases the probability of injuries from aggressive interactions, but treatment to one member of a pair of competing Nicrophorus orbicollis females does not increase the probability that she will win control of the resource. In addition, higher JH levels are not associated with greater competitive ability in communally breeding Nicrophorus tomentosus females. Treatment of one female N. tomentosus does not increase her share of the communal brood. Methoprene seems to make a less competitive female more persistent and less willing to concede, which, although maintaining her share of reproduction, results in her exclusion from the brood chamber. r 2006 Elsevier Ltd. All rights reserved. Keywords: Nicrophorus; Burying beetle; Juvenile hormone; Aggression; Dominance hierarchy; Communal breeding
1. Introduction In adult insects, juvenile hormone (JH) most often serves as a gonadotropin to regulate the biosynthesis of vitellogenin and/or its uptake by the developing oocytes (Nijhout, 1994; Gilbert et al., 2000). However, it has been implicated as a factor in the expression of various social behaviors. In honeybees, JH titers increase in response to the absence of foragers to decrease the age of the onset of foraging. Similarly, removal of young bees causes foragers to revert to brood-care behavior and reduces JH titers (Huang and Robinson, 1996). In burying beetles, JH of parental males increases with the absence of a mate, and with an increasing number of begging larvae (Panaitof et al., 2004). JH of both male and female increase in response to the challenge by an intruder (Scott, 2006). Some studies of several social insect species suggest that JH may modulate aggression. The tasks of soldiers and guards in Tel.: +1 603 862 4749; fax: +1 603 862 3784.
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honeybee colonies are associated with higher aggression and higher titers of JH, but both co-vary with increasing age (Huang et al., 1994). Within honeybee colonies, aggression is correlated with JH, but not across colonies, and seems to be modulated through changes in the behavioral threshold sensitivity to alarm pheromones (Robinson, 1987a; Pearce et al., 2001). Queenless worker bumblebees have higher rates of JH synthesis and titers, and of overt aggression and threatening behaviors than those in queenright workers of a similar age (Bloch et al., 1996, 2000a). The role of JH in competition and the establishment of dominance has been studied in several species of Hymenoptera. The establishment of dominant status through aggressive interactions is positively correlated with JH in bumblebees (Bloch et al., 2000a), and high JH titers are correlated with the competition phase of colony development when there is egg laying by workers, oophagy, and overt aggression (Cnaani et al., 2000). In female wasps, dominance status is positively correlated with the volume of the corpora allata (the source of JH), and is significantly
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influenced by treatment with JH or its analog, methoprene (Barth et al., 1975; Ro¨seler et al., 1984); however, once the hierarchy is established, JH treatment has no effect on its maintenance (Ro¨seler, 1985). In this study, I investigate the role of JH on aggression, on the outcome of competition for a breeding resource, and on the outcome of the division of reproduction in communal breeding of two species of burying beetles, Nicrophorus orbicollis and Nicrophorus. tomentosus. Burying beetles require a small carcass (typically 15–50 g) that they bury and prepare as food for their young. In the absence of a carcass, there is seldom any aggression among beetles, but breeding opportunities are probably rare and there is fierce competition for carcasses. If more than one male or female N. orbicollis discover the carcass while it is being buried, there is intrasexual competition and the larger usually wins (Otronen, 1988; Safryn and Scott, 2000). After oviposition, both males and females defend the resource and brood against infanticidal intruders of either sex. On large carcasses, more than one male and/or female N. orbicollis may rear a single brood (Trumbo, 1992), and on all but small carcasses, N. tomentosus often breeds communally (Scott, 1994, 1996). The size of the carcass determines the number of young that can be reared, but communal breeding is favored when the resources of a large carcass exceed the requirements of the young that a single female can produce, and when larger groups of beetles can better rid the carcass of fly larvae (Scott, 1994). The onset of intraspecific aggression, and the defense of carcass and brood, is correlated with JH. Hemolymph titers of JH in N. orbicollis rise very fast upon the discovery of a carcass—it is significantly higher than baseline within 10 min (Trumbo et al., 1995). In males and females, it rises about 4-fold within 12 h and rises in females 10-fold or more by the time larvae are 1–2 days old (Trumbo, 1997, 2002; Panaitof et al., 2004) when parental care is most intense (Fetherston et al., 1990).
of N. orbicollis or midday for N. tomentosus, the carcass was placed in the box only when beetles were on the surface and could be seen to discover it at the same time.
2.2. Effects of JH on aggression and competition Competition in burying beetles is mediated by aggression and the outcome can be either exclusion or cooperation, depending on the size of the resource and the species. First, I examined the effect of JH on aggression in the absence of a resource when normally beetles are not aggressive toward each other. Two N. orbicollis (n ¼ 66), either the same sex or the opposite sex, were placed in a box of soil and no carcass. (See Table 1 for pairings and sample sizes.) They were checked carefully for previous injuries. In most trials, one individual was treated with 200 mg/g beetle of methoprene (JH analog) in acetone (stock: 40 mg/ml) applied topically under the first pair of legs; in 10 trials, both members of a male/female pair were treated with 200 mg/g beetle of methoprene, and in 10 control trials, one of them was treated with 3 ml acetone. N. tomentosus (n ¼ 16) females were treated with 200 mg/g beetle of methoprene or 3 ml acetone, and placed with an untreated male. (Methoprene is commonly used as a JH analog, because it has a longer biological half-life than JH, 14–24 h vs. 1.5 h. Honeybees are typically treated with 200 mg/ female to mimic JH activity (Robinson, 1987b); thus, application of 200 mg/g beetle is very conservative given that breeding burying beetles have JH titers 4–10 times higher than foraging honeybees and are larger, 0.3–0.7 g.) The next day, beetles were located and checked again for injuries. Table 1 The effect of methoprene (JH analog) treatment on aggression in the absence of a carcass No. of pairs without injuries
Nicrophorus orbicollis Control Female treated w/male Female treated w/female Male treated w/male Male treated w/female Both treated
0 4 4 5 1 6
10 6 6 5 15 4
0.043 0.043 0.033 1.0 0.005
Nicrophorus tomentosus Control Female treated w/male
0 6
10 0
0.0001
2. Methods 2.1. Animals and breeding manipulations All N. orbicollis were laboratory-reared from a population derived from beetles captured in Durham, NH. All N. tomentosus were wild, caught at the beginning of their breeding season in Durham, NH. They were maintained in boxes with damp paper towels with like-sex individuals, fed mealworms and beef kidney, and maintained at 20 1C and 14:10 L:D. To breed, beetles were placed in a plastic box (19 14 10 cm3) three-fourth filled with potting soil or peat with a previously frozen mouse (laboratory culls from Duke University Medical Center or the Forsyth Institute, Boston). In cases where the time after discovery of the carcass had to be known exactly, beetles were placed in the box of soil several hours early, and at lights out in the case
P
No. of pairs with injuries
Number of pairs with no injuries, and with one or both beetles injured or dead are shown. Probabilities are the from Fisher’s exact tests (Biom 2.1), comparing the treatment results with controls. A male or female N. orbicollis was treated with 200 mg/g beetle of methoprene or with 3 ml acetone (controls), and placed in a box of soil and no carcass with an untreated male or female. In 10 replicates, both male and female were treated. N. tomentosus females were treated with 200 mg/g beetle of methoprene or with 3 ml acetone (controls), and placed with an untreated male.
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2.3. JH, communal breeding, and reproductive skew Both male and female N. tomentosus readily form cooperative associations on carcasses larger than about 25 g. Although the total brood might be larger than one raised by a single female on the same-size carcass, reproduction is often significantly skewed in favor of the larger (Scott and Williams, 1993; Scott, 1997). To examine JH titers of communally breeding females and compare them to titers of singly breeding females, two females, one 10–20% larger (pronotal width) than the other, and one male were given a 30–35 g carcass. Similarly, a relatively large or relatively small female was given a carcass and a male to breed as a pair. Hemolymph was taken either 6 h later (n ¼ 6 pair, n ¼ 6 large, and n ¼ 5 small), or 12 h later (n ¼ 7 pair, n ¼ 7 large, and n ¼ 8 small). To evaluate the effect of JH on cooperation and reproductive success, two females and one male were given a 30–35 g carcass in a box of soil. One female was 10–20% larger (pronotal width) than the other, and either the larger or smaller female was treated with a high (200 mg/g beetle) or low (50 mg/g beetle) dose of methoprene in acetone applied topically under the first pair of legs; the other female was treated with 2 ml acetone (n ¼ 43; see Table 2
for sample sizes in each treatment). Neither female was treated in controls (n ¼ 38). One of the females, randomly chosen, had previously been fed mealworms injected with rhodamine-B, which dyed the eggs of that female fluorescent pink. The dye has no effect on female fecundity (Scott, 1997). On the fourth day, the brood chamber was exhumed, the presence or absence of each female in the brood chamber was noted, both females were checked for injuries, and all the eggs located. The presence or absence of the dye was confirmed with a Nikon Fluophot microscope (Scott, 1997), and eggs were assigned to the mother. The division of reproductive output was analyzed to see if it differed significantly from the size-specific fecundity (previously assessed, Fig. 1 in Scott, 1997). The predicted vs. observed number of eggs was compared with a G-test of Goodness of Fit with a Williams’ correction. If the probability was 40.05, I concluded there was no reproductive skew. 1000
6 hours 12 hours
800 JH (ng/ml)
The effect of methoprene treatment on aggression and the outcome of competition over a carcass was investigated with N. orbicollis. Two, size-matched (difference p0.12 mm pronotal width), previously inseminated females and no male were placed in a box with a 20–25 g carcass (n ¼ 20). In half of the pairs, one randomly chosen female was treated with 200 mg/g beetle of methoprene, and 3 ml acetone was applied to the other female. In the other pairs (control group), neither female was treated. Females were checked before and two days after for injuries and for ownership of the carcass (i.e. was present with the carcass).
1007
600 400 200 0 small single
small paired
large paired
large single
Fig. 1. Juvenile hormone hemolymph titers 6 and 12 h after the discovery of a carcass for Nicrophorus tomentosus females. Females either bred singly with a male, or communally with a second female (paired) and a male. The size difference between pairs of females was 10–20%. Means and SE are shown for backtransformed data.
Table 2 Reproductive success of pairs of communally breeding N. tomentosus females when one female has been treated with methoprene Small female excluded
Small female not excluded
Controls
6
32
Small female High dose
0
16
Small female Low dose
0
Large female High dose Large female Low dose
P
P
Sig. skew for large female
No skew
Sig. skew for small female
13
22
3
0.16
6
9
1
1.0
10
0.32
2
7
1
1.0
0
11
0.31
4
6
1
1.0
1
5
1.0
2
4
0
1.0
The first three columns show the number of cases where the smaller female was completely excluded from oviposition or not, and the probabilities from Fisher’s exact tests (Biom 2.1) that the treatments differ from controls. The last four columns indicate the number of broods for which reproduction was significantly skewed (i.e. G-test of independence Po0:05) toward either the larger or smaller female, or not skewed, and the probability from a w2 test that the treatment distribution differed from that of the control.
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2.4. JH extraction and radioimmunoassay Hemolymph (5–10 ml, usually 10 ml) was taken from the severed third leg in a calibrated microcapillary tube and added to 0.5 ml chilled acetonitrile. Extraction and RIA followed (Trumbo et al., 1995; Scott and Panaitof, 2004; Panaitof et al., 2004) using the chiral-specific antibody to 10R-JH III, the naturally occurring enantiomer (Hunnicutt et al., 1989). JH III is the only JH produced by these beetles (Trumbo et al., 1995; Scott et al., 2001). JH concentration in the hemolymph was calculated from the standard curve, the volume of hemolymph taken, the extraction efficiency, its dilution in EtOH, and multiplied by 0.5, since the standards were of racemic JH and the antibody is chiral specific. Data were log transformed for analysis, and then back transformed for graphical depiction.
3. Results 3.1. Effects of JH on aggression and competition In both species, treating beetles with methoprene in the absence of a resource significantly increased the number of injuries incurred in most cases (Table 1). The common injuries were missing sections of antennae, legs, or bites taken out of the posterior margin of the elytra. When N. orbicollis females were treated and placed with an untreated male or female, there were significantly more injuries than those suffered by control pairs. The injured beetle was not necessarily the untreated one. There was no strong pattern as to which beetle was injured. There were significantly more injuries when treated males were with untreated males, but not so with untreated females. However, if the female were also treated, there were significantly more injuries than control pairs; of the six injured individuals, five were females. The relative sizes of the individuals did not predict who would be injured. N. tomentosus were not studied in the same detail, but appeared to follow the same pattern. In the absence of a carcass, there were no injuries in control pairs, and all six males of the pairs with treated females were either dead or moribund from injuries. In the presence of a resource, treatment with methoprene did not affect the incidence of injuries. When two sizematched N. orbicollis females competed for a carcass, there were often injuries to one or both females. However, there was no difference in the occurrence of injuries if one female was previously treated or not (seven of ten and seven of ten pairs with injuries, respectively). The effect of methoprene treatment on the outcome of competition of N. orbicollis was somewhat unexpected. Even though methoprene increases aggressiveness, it did not predict the winner of the resource. In only four of ten pairs, the treated females were the winners and the null hypothesis was five of ten (P ¼ 0:68). If the treated female was injured, she was significantly more likely to be the loser
than if the untreated female was injured (six of seven were losers vs. one of two, P ¼ 0:03). 3.2. JH, communal breeding, and reproductive skew I might have expected the larger of two female N. tomentosus to be more competitive and to have higher JH titers, or even that both members of a pair might have elevated JH. However, this was not the case (Fig. 1). The results of a multi-factor ANOVA with 6 or 12 h, single or paired, and larger or smaller as factors found that there were no significant interactions, and JH titers of only single vs. paired was significantly different (P ¼ 0:03). Thus, both small and large females had higher JH titers when single than when paired after both 6 and 12 h. Treatment of either the larger or the smaller female with the high or low dose of methoprene did not change the outcome of how reproduction was shared between the two (Table 2). When neither female was treated, the smaller female was seldom completely excluded from laying any eggs. The frequency of complete exclusion was not significantly different when either female was treated. The frequency with which either the larger or smaller female was able to significantly skew reproduction in her favor when either female was treated was also not different from controls. However, methoprene treatment did significantly increase the probability of injuries, and reduce the probability that the two females would be found peacefully coexisting in the brood chamber on the fourth day (Table 3). There were significantly more injuries to one or both females in three of the four treatment groups than in the controls. When the smaller female was treated with methoprene at either dose, she was always absent (excluded) from the brood chamber when the carcass was exhumed and this is significantly more often than the smaller control female was absent (Table 3). 4. Discussion Treating beetles with the JH analog, methoprene, resulted in a significant increase in injuries from aggressive encounters. Although it was, presumably, the treated beetle that attacked first since there was no resource to compete for, injuries were not always to the untreated individuals. Once attacked, they fought back. Whereas treated N. orbicollis females were always aggressive toward untreated individuals of either sex, treated males were not aggressive toward untreated females. To attack a female, males needed provocation from a treated, aggressive female; injuries when both members of a pair were treated were mostly to the female. Although not tested as extensively, treated N. tomentosus females were even more aggressive than N. orbicollis. In every case, the male was killed or nearly so. Normally, in the absence of a carcass, there is no reason for beetles to be aggressive; it is only adaptive in the presence of a resource. The discovery of a carcass triggers a sharp increase in JH (Trumbo et al.,
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Table 3 Injuries and the presence of either communally breeding N. tomentosus females in the brood chamber when one female has been treated with methoprene No. of pairs with No. of pairs with P some injuries no injuries Controls
Large female with carcass
Large female without carcass
Small female Small female with carcass without carcass
36
2
19
19
P
2
36
Small female High dose
10
6
0.0000
15
1
0
16
0.0003
Small female Low dose
2
8
0.1871
9
1
0
10
0.0035
Large female High dose
4
7
0.0179
9
2
4
7
0.5062
Large female Low dose
5
1
0.0001
6
0
1
5
0.1977
The first three columns show the number of cases with injuries to one or both females, and the probabilities from Fisher’s exact tests (Biom 2.1) that the treatments differ from controls. The last five columns indicate the presence or absence of each female in the brood chamber on the fourth day, and the probabilities from Fisher’s exact tests for the difference for small females between controls and treatments.
1995), so it is not surprising that JH and aggression are correlated; this study strongly suggests that there is a causal relation. The effects of methoprene treatment on aggression in the presence of a carcass are more complicated to interpret. When female N. orbicollis are matched for size, competition is very strong (Safryn and Scott, 2000). Injuries were common when one female was treated or when neither was treated. When a carcass is discovered, JH naturally rises very fast (Trumbo, 1995) and is quickly equivalent in its effectiveness to modulate behavior as the methoprene treatment. Within a few minutes of discovery, both females are essentially in the same motivational state, which is very high as carcasses are rare and unpredictable, and the sole determinant of their lifetime reproductive success. However, the treated female was more likely to be injured when she was the loser, and this suggests that treatment made her more persistent and less willing to concede a contest. The outcome of animal contests is usually determined by resource holding potential (RHP) of either or both contestants (Taylor and Elwood, 2003; Gammell and Hardy, 2003). Treatment may alter the ability of a beetle to assess its own or its opponent’s RHP. Data from N. tomentosus females suggest the same interpretation. In this experiment, females differed in size and the small female, usually subordinate, was allowed to remain in the burial chamber half the time in the control groups. However, when one female was treated, there were significantly more injuries, and the smaller female was excluded from the burial chamber significantly more often when she was treated. This suggests that although she maintained her share of the reproduction, her increased aggressiveness caused the dominant female to exclude her from the brood chamber. The outcome of methoprene treatment on reproductive success was not as predicted. The adaptive hypothesis for this study was that the discovery of a resource stimulates
JH, which prepares the individual for competition. JH then increases aggression and makes the individual a stronger competitor, and this would lead to either a higher probability of winning the carcass in the case of N. orbicollis or a greater share of the brood in the communally breeding N. tomentosus. Although the effects of competition on JH and the increase of aggression supported this hypothesis, JH was not higher when two N. tomentosus females buried a carcass; rather, large N. tomentosus females burying alone had the highest JH. Treated N. orbicollis females were not more likely to be in possession of the carcass, and neither large nor small treated N. tomentosus females increased their share of the brood. Aggression may be the means, but it is not the main determinant of who wins burying beetle competitions. JH appears to increase persistence and possibly, to prolong individual fights, but surprisingly, greater persistence does not improve the odds of winning. Relative size is a significant factor (Safryn and Scott, 2000) and many communication cues (i.e. pheromones and cuticular hydrocarbons) may be involved. In nature, attacks usually follow immediately after contact when sex and status of the opponent are determined. The outcome of most competitions is quickly decided and the loser leaves. However, it is not uncommon for one participant to be seriously injured or killed even when escape is possible (which is not the case in the laboratory). Few studies have addressed the physiological mechanisms modulating aggression in insects. It has been best studied in honeybees. Guard bees are significantly more likely to bite or sting foreign bees than any other caste (Breed et al., 1992). Aggression by guard bees is correlated with JH titers within colonies, i.e. JH titers of aggressive bees are significantly higher that those of non-aggressive bees in the same colony (Pearce et al., 2001). JH is positively correlated with age in honeybees, but guards
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have similar JH titers to foragers and soldiers even though they are 10 days younger, and JH titers of guards are significantly higher than those of wax producers and food storers despite their similar age (Huang et al., 1994). Huang et al. (1994) suggest though that a specific effect of JH on behavior is unlikely as undertakers and foragers, who are not particularly aggressive, have JH levels as high as guards and soldiers. All of these are tasks of older bees, which naturally have higher JH titers. JH plays an equivocal role in the establishment and/or maintenance of colony dominance. In bumblebees, it might seem that JH is associated with dominance behavior, but position in the dominance hierarchy is associated with differences in ovarian development, which correlates with JH titers. With the differences in ovarian development statistically removed, Bloch et al. (2000a) found no support for the hypothesis that JH modulated dominance and aggressive behavior. Treatment with JH I did not increase the probability of becoming dominant in bumblebees (Van Doorn, 1989). In the social wasp Polistes annularis, treatment with JH and JH analogs increased dominance interactions and aggressive interactions, but treatment also increased ovarian development (Barth et al., 1975). As with bumblebees, this may not be an independent effect of JH, but rather, the direct effect of increased ovarian development. But in Polistes gallicus, the effects of ovarian development were statistically removed, and corpora allata volume and dominant status were positively correlated (Ro¨seler et al., 1984). JH has been examined and shown to have no role in aggressive behavior in crickets (Adamo et al., 1994). Biogenic amines appear to be more important in the modulation of aggression in several of the model insect species studied so far. For example, treatment with an octopamine agonist significantly increased aggressive behaviors and the duration of fights in crickets that had recently lost a fight. Aggressiveness was depressed in crickets depleted of octopamine and dopamine, and was restored by an octopamine agonist (Stevenson et al., 2005). Flight also restores aggressiveness in losing crickets; the proposed mechanism is the restricted release of octopamine as a neuromodulator in the central nervous system (Stevenson et al., 2005). Octopamine has also been reported to correlate with dominance and aggressiveness in bumblebees (Bloch et al., 2000b); in fruit flies, octopamine increases aggressiveness and dopamine is negatively correlated (Baier et al., 2002). However, serotonin seems to promote aggression in crustaceans (Kravitz, 2000; Kravitz and Huber, 2003). Thus, the hormonal mechanisms modulating aggression in arthropods seem to be quite varied and more species will need to be investigated before relevant factors or phylogenetic patterns emerge. The exact role of JH in modulating aggressive behavior in burying beetles is unclear. JH could increase aggressiveness indirectly rather than directly. It could act on the behavioral threshold sensitivity to environmental and
social cues, just as it increases the behavior threshold sensitivity to alarm pheromones in honeybees, which in turn modulates aggression (Robinson, 1987a). For example, the difference between N. tomentosus and N. orbicollis in their readiness to breed cooperatively may be that the former has a higher threshold for the combination of carcass size and the presence of same-sex competitors before they are excluded. Social cues are obviously involved in the assessment of the object of aggression. Males and females with high JH are not aggressive toward their mate; both are aggressive only toward consexuals before oviposition and toward both sexes after oviposition (Scott, 2006); in spite of elevated JH N. tomentosus will tolerate consexuals before oviposition but will not let beetles of either sex join after oviposition (Scott, 1994). Aggression could also be triggered by the mismatch of identification cues: partners and co-breeding females are probably identified by co-varying changes in cuticular pheromones caused by changing levels of JH (unpublished data, Mu¨ller et al., 2003). However, in the experiments reported here, JH directly increased aggression in all but treated males to untreated females. In light of the effect of social cues to moderate aggression in nature, it seems that the role of these social cues is to identify a target for aggression when JH has heightened sensitivity to them. Acknowledgments My ideas on burying beetle aggression have benefited from discussions with Fred Nijhout, Dave Borst, Jessica Bolker and Carmen Panaitof. I thank Dave Borst for JH antibody and teaching me JH RIA. The Duke Medical facilities and the Forsyth Institute, Boston, generously supplied me with culls from their laboratory colonies of mice whenever I asked. This work was supported by National Science Foundation IBN 9628832 and Hatch grants from the NH Agriculture Station. This is scientific contribution no. 2290 from the New Hampshire Agriculture Station. References Adamo, S.A., Schildberger, K., Loher, W., 1994. The role of the corpora allata in the adult male cricket (Grillus campestris and Gryllus bimaculatus) in the development and expression of its agonistic behavior. Journal of Insect Physiology 40, 439–446. Baier, A., Wittek, B., Brembs, B., 2002. Drosophila as a new model organism for neurobiology of aggression? Journal of Experimental Biology 205, 1233–1240. Barth, R.H., Lester, L.J., Sroka, P., Kessler, T., Hearn, R., 1975. Juvenile hormone promotes dominance behavior and ovarian development in social wasps (Polistes annularis). Experientia 31, 691–692. Bloch, G., Borst, D.W., Huang, Z.Y., Robinson, G.E., Hefetz, A., 1996. Effects of social conditions on juvenile hormone-mediated reproductive development in Bombus terrestris workers. Physiological Entomology 21, 257–267. Bloch, G., Borst, D.W., Huang, Z.Y., Robinson, G.E., Cnaani, J., Hefetz, A., 2000a. Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris. Journal of Insect Physiology 46, 47–57.
ARTICLE IN PRESS M.P. Scott / Journal of Insect Physiology 52 (2006) 1005–1011 Bloch, G., Simon, T., Robinson, G.E., Hefetz, A., 2000b. Brain biogenic amines and reproductive dominance in bumblebees (Bombus terrestris). Journal of Comparative Physiology A 186, 261–268. Breed, M.D., Smith, T.A., Torres, A., 1992. Role of guard honeybees (Hymenoptera: Apidae) in nestmate discrimination and replacement of removed guards. Annals of the Entomological Society of America 85, 633–637. Cnaani, J., Robinson, G.E., Bloch, G., Borst, D., Hefetz, A., 2000. The effect of queen–worker conflict on caste determination in the bumblebee, Bombus terrestris. Behavioral Ecology and Sociobiology 47, 346–352. Fetherston, I., Scott, M.P., Traniello, J.F.A., 1990. Parental care in burying beetles: male and female roles and the organization of brood care behaviors. Ethology 85, 177–190. Gammell, M.P., Hardy, I.C.W., 2003. Contest duration: sizing up the opposition. Trends in Ecology and Evolution 18, 491–493. Gilbert, L.I., Granger, N.A., Roe, R.M., 2000. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochemistry and Molecular Biology 30, 617–644. Huang, Z.Y., Robinson, G.E., 1996. Regulation of honeybee division of labor by colony age demography. Behavioral Ecology and Sociobiology 39, 147–158. Huang, Z.Y., Robinson, G.E., Borst, D.W., 1994. Physiological correlates of division of labor among similarly aged honeybees. Journal of Comparative Physiology A 174, 731–735. Hunnicutt, D., Toong, Y.C., Borst, D.W., 1989. A chiral-specific antiserum for juvenile hormone. American Zoologist 29, 48a. Kravitz, E.A., 2000. Serotonin and aggression: insights gained from a lobster model system and speculations on the role of amine neurons in a complex behavior. Journal of Comparative Physiology A 186, 221–238. Kravitz, E.A., Huber, R., 2003. Aggression in invertebrates. Current Opinion in Neurobiology 13, 736–743. Mu¨ller, J.K., Eggert, A.-K., Elsner, T., 2003. Nestmate recognition in burying beetles: the ‘‘breeder’s badge’’ as a cue used by females to distinguish their mates from male intruders. Behavioral Ecology 14, 212–220. Nijhout, H.F., 1994. Insect Hormones. Princeton University Press, Princeton, NJ. Otronen, M., 1988. The effect of body size on the outcome of fights in burying beetles (Nicrophorus). Annales Zoologici Fennici 25, 191–201. Panaitof, S.C., Scott, M.P., Borst, D.W., 2004. Plasticity in juvenile hormone in male burying beetles during breeding: physiological consequences of the loss of a mate. Journal of Insect Physiology 50, 715–724. Pearce, A.N., Huang, Z.Y., Breed, M.D., 2001. Juvenile hormone and aggression in honeybees. Journal of Insect Physiology 47, 1243–1247. Robinson, G.E., 1987a. Modulation of alarm pheromone perception in the honeybee: evidence for division of labor based on hormonally regulated response thresholds. Journal of Comparative Physiology B 160, 613–619. Robinson, G.E., 1987b. Regulation of honeybee age polyethism by juvenile hormone. Behavioral Ecology and Sociobiology 20, 329–338.
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Ro¨seler, P.F., 1985. Endocrine basis of dominance and reproduction in polistine paper wasps. In: Ho¨lldobler, B., Lindauer, M. (Eds.), Experimental Behavioral Ecology and Sociobiology. Sinauer, Sunderland, MA, pp. 259–272. Ro¨seler, P.F., Ro¨seler, I., Strambi, A., Augier, R., 1984. Influence of insect hormones on the establishment of dominance hierarchies among foundresses of the paper wasp, Polistes gallicus. Behavioral Ecology and Sociobiology 15, 133–142. Safryn, S.A., Scott, M.P., 2000. Sizing up the competition: do burying beetles weigh or measure their opponents? Journal of Insect Behavior 13, 291–297. Scott, M.P., 1994. Competition with flies promotes communal breeding in the burying beetle, Nicrophorus tomentosus. Behavioral Ecology and Sociobiology 34, 367–374. Scott, M.P., 1996. Communal breeding in burying beetles. American Scientist 84, 376–382. Scott, M.P., 1997. Reproductive dominance and differential ovicide in the communally breeding burying beetle, Nicrophorus tomentosus. Behavioral Ecology and Sociobiology 40, 313–320. Scott, M.P., 2006. Resource defence and juvenile hormone: the ‘‘challenge hypothesis’’ extended to insects. Hormones and Behavior 49, 276–281. Scott, M.P., Panaitof, S.C., 2004. Social stimuli affect juvenile hormone during breeding in burying beetles (Silphidae: Nicrophorus). Hormones and Behavior 45, 159–167. Scott, M.P., Williams, S.M., 1993. Comparative reproductive success of communally breeding burying beetles as assessed by PCR with randomly amplified polymorphic DNA. Proceedings of the National Academy of Science 90, 2242–2245. Scott, M.P., Trumbo, S.T., Neese, P.A., Bailey, W.D., Roe, R.M., 2001. Changes in biosynthesis and degradation of juvenile hormone during breeding by burying beetles: a reproductive or social role? Journal of Insect Physiology 47, 295–302. Stevenson, P.A., Dyakonova, V., Rillich, J., Schildberger, K., 2005. Octopamine and experience-dependent modulation of aggression in crickets. Journal of Neuroscience 25, 1431–1441. Taylor, P.W., Elwood, R.W., 2003. The mismeasure of animal contests. Animal Behaviour 65, 1195–1202. Trumbo, S.T., 1992. Monogamy to communal breeding: exploitation of a broad resource base by burying beetles (Nicrophorus). Ecological Entomology 17, 289–298. Trumbo, S.T., 1997. Juvenile hormone-mediated reproduction in burying beetles: from behavior to physiology. Archives of Insect Biochemistry and Physiology 35, 479–490. Trumbo, S.T., 2002. Hormonal regulation of parental care in insects. In: Pfaff, D.W., et al. (Eds.), Hormones, Brain and Behavior, vol. 3. Academic Press, New York, pp. 115–139. Trumbo, S.T., Borst, D.W., Robinson, G.E., 1995. Rapid elevation of juvenile hormone titer during behavioral assessment of the breeding resource by the burying beetle, Nicrophorus orbicollis. Journal of Insect Physiology 41, 535–543. Van Doorn, A., 1989. Factors influencing dominance behaviour in queenless bumblebee workers (Bombus terrestris). Physiological Entomology 14, 211–221.
Journal of Insect Physiology 52 (2006) 1005–1011 www.elsevier.com/locate/jinsphys
The role of juvenile hormone in competition and cooperation by burying beetles Michelle Pellissier Scott Department of Zoology, University of New Hampshire, Durham, NH 03824, USA Received 28 January 2006; received in revised form 8 April 2006; accepted 10 April 2006
Abstract Few studies have addressed the physiological mechanisms that modulate aggression in insects. In some social insects, there is a correlation of JH and aggression in colony defense and in the establishment of dominance, but only a few studies demonstrate a causal relationship. Burying beetles aggressively defend a breeding resource, a carcass, and juvenile hormone (JH) hemolymph titers increase rapidly upon the discovery of a carcass. In this study, I show that treatment with the JH analog, methoprene, in the absence of a carcass increases the probability of injuries from aggressive interactions, but treatment to one member of a pair of competing Nicrophorus orbicollis females does not increase the probability that she will win control of the resource. In addition, higher JH levels are not associated with greater competitive ability in communally breeding Nicrophorus tomentosus females. Treatment of one female N. tomentosus does not increase her share of the communal brood. Methoprene seems to make a less competitive female more persistent and less willing to concede, which, although maintaining her share of reproduction, results in her exclusion from the brood chamber. r 2006 Elsevier Ltd. All rights reserved. Keywords: Nicrophorus; Burying beetle; Juvenile hormone; Aggression; Dominance hierarchy; Communal breeding
1. Introduction In adult insects, juvenile hormone (JH) most often serves as a gonadotropin to regulate the biosynthesis of vitellogenin and/or its uptake by the developing oocytes (Nijhout, 1994; Gilbert et al., 2000). However, it has been implicated as a factor in the expression of various social behaviors. In honeybees, JH titers increase in response to the absence of foragers to decrease the age of the onset of foraging. Similarly, removal of young bees causes foragers to revert to brood-care behavior and reduces JH titers (Huang and Robinson, 1996). In burying beetles, JH of parental males increases with the absence of a mate, and with an increasing number of begging larvae (Panaitof et al., 2004). JH of both male and female increase in response to the challenge by an intruder (Scott, 2006). Some studies of several social insect species suggest that JH may modulate aggression. The tasks of soldiers and guards in Tel.: +1 603 862 4749; fax: +1 603 862 3784.
E-mail address: [email protected]. 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.006
honeybee colonies are associated with higher aggression and higher titers of JH, but both co-vary with increasing age (Huang et al., 1994). Within honeybee colonies, aggression is correlated with JH, but not across colonies, and seems to be modulated through changes in the behavioral threshold sensitivity to alarm pheromones (Robinson, 1987a; Pearce et al., 2001). Queenless worker bumblebees have higher rates of JH synthesis and titers, and of overt aggression and threatening behaviors than those in queenright workers of a similar age (Bloch et al., 1996, 2000a). The role of JH in competition and the establishment of dominance has been studied in several species of Hymenoptera. The establishment of dominant status through aggressive interactions is positively correlated with JH in bumblebees (Bloch et al., 2000a), and high JH titers are correlated with the competition phase of colony development when there is egg laying by workers, oophagy, and overt aggression (Cnaani et al., 2000). In female wasps, dominance status is positively correlated with the volume of the corpora allata (the source of JH), and is significantly
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influenced by treatment with JH or its analog, methoprene (Barth et al., 1975; Ro¨seler et al., 1984); however, once the hierarchy is established, JH treatment has no effect on its maintenance (Ro¨seler, 1985). In this study, I investigate the role of JH on aggression, on the outcome of competition for a breeding resource, and on the outcome of the division of reproduction in communal breeding of two species of burying beetles, Nicrophorus orbicollis and Nicrophorus. tomentosus. Burying beetles require a small carcass (typically 15–50 g) that they bury and prepare as food for their young. In the absence of a carcass, there is seldom any aggression among beetles, but breeding opportunities are probably rare and there is fierce competition for carcasses. If more than one male or female N. orbicollis discover the carcass while it is being buried, there is intrasexual competition and the larger usually wins (Otronen, 1988; Safryn and Scott, 2000). After oviposition, both males and females defend the resource and brood against infanticidal intruders of either sex. On large carcasses, more than one male and/or female N. orbicollis may rear a single brood (Trumbo, 1992), and on all but small carcasses, N. tomentosus often breeds communally (Scott, 1994, 1996). The size of the carcass determines the number of young that can be reared, but communal breeding is favored when the resources of a large carcass exceed the requirements of the young that a single female can produce, and when larger groups of beetles can better rid the carcass of fly larvae (Scott, 1994). The onset of intraspecific aggression, and the defense of carcass and brood, is correlated with JH. Hemolymph titers of JH in N. orbicollis rise very fast upon the discovery of a carcass—it is significantly higher than baseline within 10 min (Trumbo et al., 1995). In males and females, it rises about 4-fold within 12 h and rises in females 10-fold or more by the time larvae are 1–2 days old (Trumbo, 1997, 2002; Panaitof et al., 2004) when parental care is most intense (Fetherston et al., 1990).
of N. orbicollis or midday for N. tomentosus, the carcass was placed in the box only when beetles were on the surface and could be seen to discover it at the same time.
2.2. Effects of JH on aggression and competition Competition in burying beetles is mediated by aggression and the outcome can be either exclusion or cooperation, depending on the size of the resource and the species. First, I examined the effect of JH on aggression in the absence of a resource when normally beetles are not aggressive toward each other. Two N. orbicollis (n ¼ 66), either the same sex or the opposite sex, were placed in a box of soil and no carcass. (See Table 1 for pairings and sample sizes.) They were checked carefully for previous injuries. In most trials, one individual was treated with 200 mg/g beetle of methoprene (JH analog) in acetone (stock: 40 mg/ml) applied topically under the first pair of legs; in 10 trials, both members of a male/female pair were treated with 200 mg/g beetle of methoprene, and in 10 control trials, one of them was treated with 3 ml acetone. N. tomentosus (n ¼ 16) females were treated with 200 mg/g beetle of methoprene or 3 ml acetone, and placed with an untreated male. (Methoprene is commonly used as a JH analog, because it has a longer biological half-life than JH, 14–24 h vs. 1.5 h. Honeybees are typically treated with 200 mg/ female to mimic JH activity (Robinson, 1987b); thus, application of 200 mg/g beetle is very conservative given that breeding burying beetles have JH titers 4–10 times higher than foraging honeybees and are larger, 0.3–0.7 g.) The next day, beetles were located and checked again for injuries. Table 1 The effect of methoprene (JH analog) treatment on aggression in the absence of a carcass No. of pairs without injuries
Nicrophorus orbicollis Control Female treated w/male Female treated w/female Male treated w/male Male treated w/female Both treated
0 4 4 5 1 6
10 6 6 5 15 4
0.043 0.043 0.033 1.0 0.005
Nicrophorus tomentosus Control Female treated w/male
0 6
10 0
0.0001
2. Methods 2.1. Animals and breeding manipulations All N. orbicollis were laboratory-reared from a population derived from beetles captured in Durham, NH. All N. tomentosus were wild, caught at the beginning of their breeding season in Durham, NH. They were maintained in boxes with damp paper towels with like-sex individuals, fed mealworms and beef kidney, and maintained at 20 1C and 14:10 L:D. To breed, beetles were placed in a plastic box (19 14 10 cm3) three-fourth filled with potting soil or peat with a previously frozen mouse (laboratory culls from Duke University Medical Center or the Forsyth Institute, Boston). In cases where the time after discovery of the carcass had to be known exactly, beetles were placed in the box of soil several hours early, and at lights out in the case
P
No. of pairs with injuries
Number of pairs with no injuries, and with one or both beetles injured or dead are shown. Probabilities are the from Fisher’s exact tests (Biom 2.1), comparing the treatment results with controls. A male or female N. orbicollis was treated with 200 mg/g beetle of methoprene or with 3 ml acetone (controls), and placed in a box of soil and no carcass with an untreated male or female. In 10 replicates, both male and female were treated. N. tomentosus females were treated with 200 mg/g beetle of methoprene or with 3 ml acetone (controls), and placed with an untreated male.
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2.3. JH, communal breeding, and reproductive skew Both male and female N. tomentosus readily form cooperative associations on carcasses larger than about 25 g. Although the total brood might be larger than one raised by a single female on the same-size carcass, reproduction is often significantly skewed in favor of the larger (Scott and Williams, 1993; Scott, 1997). To examine JH titers of communally breeding females and compare them to titers of singly breeding females, two females, one 10–20% larger (pronotal width) than the other, and one male were given a 30–35 g carcass. Similarly, a relatively large or relatively small female was given a carcass and a male to breed as a pair. Hemolymph was taken either 6 h later (n ¼ 6 pair, n ¼ 6 large, and n ¼ 5 small), or 12 h later (n ¼ 7 pair, n ¼ 7 large, and n ¼ 8 small). To evaluate the effect of JH on cooperation and reproductive success, two females and one male were given a 30–35 g carcass in a box of soil. One female was 10–20% larger (pronotal width) than the other, and either the larger or smaller female was treated with a high (200 mg/g beetle) or low (50 mg/g beetle) dose of methoprene in acetone applied topically under the first pair of legs; the other female was treated with 2 ml acetone (n ¼ 43; see Table 2
for sample sizes in each treatment). Neither female was treated in controls (n ¼ 38). One of the females, randomly chosen, had previously been fed mealworms injected with rhodamine-B, which dyed the eggs of that female fluorescent pink. The dye has no effect on female fecundity (Scott, 1997). On the fourth day, the brood chamber was exhumed, the presence or absence of each female in the brood chamber was noted, both females were checked for injuries, and all the eggs located. The presence or absence of the dye was confirmed with a Nikon Fluophot microscope (Scott, 1997), and eggs were assigned to the mother. The division of reproductive output was analyzed to see if it differed significantly from the size-specific fecundity (previously assessed, Fig. 1 in Scott, 1997). The predicted vs. observed number of eggs was compared with a G-test of Goodness of Fit with a Williams’ correction. If the probability was 40.05, I concluded there was no reproductive skew. 1000
6 hours 12 hours
800 JH (ng/ml)
The effect of methoprene treatment on aggression and the outcome of competition over a carcass was investigated with N. orbicollis. Two, size-matched (difference p0.12 mm pronotal width), previously inseminated females and no male were placed in a box with a 20–25 g carcass (n ¼ 20). In half of the pairs, one randomly chosen female was treated with 200 mg/g beetle of methoprene, and 3 ml acetone was applied to the other female. In the other pairs (control group), neither female was treated. Females were checked before and two days after for injuries and for ownership of the carcass (i.e. was present with the carcass).
1007
600 400 200 0 small single
small paired
large paired
large single
Fig. 1. Juvenile hormone hemolymph titers 6 and 12 h after the discovery of a carcass for Nicrophorus tomentosus females. Females either bred singly with a male, or communally with a second female (paired) and a male. The size difference between pairs of females was 10–20%. Means and SE are shown for backtransformed data.
Table 2 Reproductive success of pairs of communally breeding N. tomentosus females when one female has been treated with methoprene Small female excluded
Small female not excluded
Controls
6
32
Small female High dose
0
16
Small female Low dose
0
Large female High dose Large female Low dose
P
P
Sig. skew for large female
No skew
Sig. skew for small female
13
22
3
0.16
6
9
1
1.0
10
0.32
2
7
1
1.0
0
11
0.31
4
6
1
1.0
1
5
1.0
2
4
0
1.0
The first three columns show the number of cases where the smaller female was completely excluded from oviposition or not, and the probabilities from Fisher’s exact tests (Biom 2.1) that the treatments differ from controls. The last four columns indicate the number of broods for which reproduction was significantly skewed (i.e. G-test of independence Po0:05) toward either the larger or smaller female, or not skewed, and the probability from a w2 test that the treatment distribution differed from that of the control.
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2.4. JH extraction and radioimmunoassay Hemolymph (5–10 ml, usually 10 ml) was taken from the severed third leg in a calibrated microcapillary tube and added to 0.5 ml chilled acetonitrile. Extraction and RIA followed (Trumbo et al., 1995; Scott and Panaitof, 2004; Panaitof et al., 2004) using the chiral-specific antibody to 10R-JH III, the naturally occurring enantiomer (Hunnicutt et al., 1989). JH III is the only JH produced by these beetles (Trumbo et al., 1995; Scott et al., 2001). JH concentration in the hemolymph was calculated from the standard curve, the volume of hemolymph taken, the extraction efficiency, its dilution in EtOH, and multiplied by 0.5, since the standards were of racemic JH and the antibody is chiral specific. Data were log transformed for analysis, and then back transformed for graphical depiction.
3. Results 3.1. Effects of JH on aggression and competition In both species, treating beetles with methoprene in the absence of a resource significantly increased the number of injuries incurred in most cases (Table 1). The common injuries were missing sections of antennae, legs, or bites taken out of the posterior margin of the elytra. When N. orbicollis females were treated and placed with an untreated male or female, there were significantly more injuries than those suffered by control pairs. The injured beetle was not necessarily the untreated one. There was no strong pattern as to which beetle was injured. There were significantly more injuries when treated males were with untreated males, but not so with untreated females. However, if the female were also treated, there were significantly more injuries than control pairs; of the six injured individuals, five were females. The relative sizes of the individuals did not predict who would be injured. N. tomentosus were not studied in the same detail, but appeared to follow the same pattern. In the absence of a carcass, there were no injuries in control pairs, and all six males of the pairs with treated females were either dead or moribund from injuries. In the presence of a resource, treatment with methoprene did not affect the incidence of injuries. When two sizematched N. orbicollis females competed for a carcass, there were often injuries to one or both females. However, there was no difference in the occurrence of injuries if one female was previously treated or not (seven of ten and seven of ten pairs with injuries, respectively). The effect of methoprene treatment on the outcome of competition of N. orbicollis was somewhat unexpected. Even though methoprene increases aggressiveness, it did not predict the winner of the resource. In only four of ten pairs, the treated females were the winners and the null hypothesis was five of ten (P ¼ 0:68). If the treated female was injured, she was significantly more likely to be the loser
than if the untreated female was injured (six of seven were losers vs. one of two, P ¼ 0:03). 3.2. JH, communal breeding, and reproductive skew I might have expected the larger of two female N. tomentosus to be more competitive and to have higher JH titers, or even that both members of a pair might have elevated JH. However, this was not the case (Fig. 1). The results of a multi-factor ANOVA with 6 or 12 h, single or paired, and larger or smaller as factors found that there were no significant interactions, and JH titers of only single vs. paired was significantly different (P ¼ 0:03). Thus, both small and large females had higher JH titers when single than when paired after both 6 and 12 h. Treatment of either the larger or the smaller female with the high or low dose of methoprene did not change the outcome of how reproduction was shared between the two (Table 2). When neither female was treated, the smaller female was seldom completely excluded from laying any eggs. The frequency of complete exclusion was not significantly different when either female was treated. The frequency with which either the larger or smaller female was able to significantly skew reproduction in her favor when either female was treated was also not different from controls. However, methoprene treatment did significantly increase the probability of injuries, and reduce the probability that the two females would be found peacefully coexisting in the brood chamber on the fourth day (Table 3). There were significantly more injuries to one or both females in three of the four treatment groups than in the controls. When the smaller female was treated with methoprene at either dose, she was always absent (excluded) from the brood chamber when the carcass was exhumed and this is significantly more often than the smaller control female was absent (Table 3). 4. Discussion Treating beetles with the JH analog, methoprene, resulted in a significant increase in injuries from aggressive encounters. Although it was, presumably, the treated beetle that attacked first since there was no resource to compete for, injuries were not always to the untreated individuals. Once attacked, they fought back. Whereas treated N. orbicollis females were always aggressive toward untreated individuals of either sex, treated males were not aggressive toward untreated females. To attack a female, males needed provocation from a treated, aggressive female; injuries when both members of a pair were treated were mostly to the female. Although not tested as extensively, treated N. tomentosus females were even more aggressive than N. orbicollis. In every case, the male was killed or nearly so. Normally, in the absence of a carcass, there is no reason for beetles to be aggressive; it is only adaptive in the presence of a resource. The discovery of a carcass triggers a sharp increase in JH (Trumbo et al.,
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Table 3 Injuries and the presence of either communally breeding N. tomentosus females in the brood chamber when one female has been treated with methoprene No. of pairs with No. of pairs with P some injuries no injuries Controls
Large female with carcass
Large female without carcass
Small female Small female with carcass without carcass
36
2
19
19
P
2
36
Small female High dose
10
6
0.0000
15
1
0
16
0.0003
Small female Low dose
2
8
0.1871
9
1
0
10
0.0035
Large female High dose
4
7
0.0179
9
2
4
7
0.5062
Large female Low dose
5
1
0.0001
6
0
1
5
0.1977
The first three columns show the number of cases with injuries to one or both females, and the probabilities from Fisher’s exact tests (Biom 2.1) that the treatments differ from controls. The last five columns indicate the presence or absence of each female in the brood chamber on the fourth day, and the probabilities from Fisher’s exact tests for the difference for small females between controls and treatments.
1995), so it is not surprising that JH and aggression are correlated; this study strongly suggests that there is a causal relation. The effects of methoprene treatment on aggression in the presence of a carcass are more complicated to interpret. When female N. orbicollis are matched for size, competition is very strong (Safryn and Scott, 2000). Injuries were common when one female was treated or when neither was treated. When a carcass is discovered, JH naturally rises very fast (Trumbo, 1995) and is quickly equivalent in its effectiveness to modulate behavior as the methoprene treatment. Within a few minutes of discovery, both females are essentially in the same motivational state, which is very high as carcasses are rare and unpredictable, and the sole determinant of their lifetime reproductive success. However, the treated female was more likely to be injured when she was the loser, and this suggests that treatment made her more persistent and less willing to concede a contest. The outcome of animal contests is usually determined by resource holding potential (RHP) of either or both contestants (Taylor and Elwood, 2003; Gammell and Hardy, 2003). Treatment may alter the ability of a beetle to assess its own or its opponent’s RHP. Data from N. tomentosus females suggest the same interpretation. In this experiment, females differed in size and the small female, usually subordinate, was allowed to remain in the burial chamber half the time in the control groups. However, when one female was treated, there were significantly more injuries, and the smaller female was excluded from the burial chamber significantly more often when she was treated. This suggests that although she maintained her share of the reproduction, her increased aggressiveness caused the dominant female to exclude her from the brood chamber. The outcome of methoprene treatment on reproductive success was not as predicted. The adaptive hypothesis for this study was that the discovery of a resource stimulates
JH, which prepares the individual for competition. JH then increases aggression and makes the individual a stronger competitor, and this would lead to either a higher probability of winning the carcass in the case of N. orbicollis or a greater share of the brood in the communally breeding N. tomentosus. Although the effects of competition on JH and the increase of aggression supported this hypothesis, JH was not higher when two N. tomentosus females buried a carcass; rather, large N. tomentosus females burying alone had the highest JH. Treated N. orbicollis females were not more likely to be in possession of the carcass, and neither large nor small treated N. tomentosus females increased their share of the brood. Aggression may be the means, but it is not the main determinant of who wins burying beetle competitions. JH appears to increase persistence and possibly, to prolong individual fights, but surprisingly, greater persistence does not improve the odds of winning. Relative size is a significant factor (Safryn and Scott, 2000) and many communication cues (i.e. pheromones and cuticular hydrocarbons) may be involved. In nature, attacks usually follow immediately after contact when sex and status of the opponent are determined. The outcome of most competitions is quickly decided and the loser leaves. However, it is not uncommon for one participant to be seriously injured or killed even when escape is possible (which is not the case in the laboratory). Few studies have addressed the physiological mechanisms modulating aggression in insects. It has been best studied in honeybees. Guard bees are significantly more likely to bite or sting foreign bees than any other caste (Breed et al., 1992). Aggression by guard bees is correlated with JH titers within colonies, i.e. JH titers of aggressive bees are significantly higher that those of non-aggressive bees in the same colony (Pearce et al., 2001). JH is positively correlated with age in honeybees, but guards
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have similar JH titers to foragers and soldiers even though they are 10 days younger, and JH titers of guards are significantly higher than those of wax producers and food storers despite their similar age (Huang et al., 1994). Huang et al. (1994) suggest though that a specific effect of JH on behavior is unlikely as undertakers and foragers, who are not particularly aggressive, have JH levels as high as guards and soldiers. All of these are tasks of older bees, which naturally have higher JH titers. JH plays an equivocal role in the establishment and/or maintenance of colony dominance. In bumblebees, it might seem that JH is associated with dominance behavior, but position in the dominance hierarchy is associated with differences in ovarian development, which correlates with JH titers. With the differences in ovarian development statistically removed, Bloch et al. (2000a) found no support for the hypothesis that JH modulated dominance and aggressive behavior. Treatment with JH I did not increase the probability of becoming dominant in bumblebees (Van Doorn, 1989). In the social wasp Polistes annularis, treatment with JH and JH analogs increased dominance interactions and aggressive interactions, but treatment also increased ovarian development (Barth et al., 1975). As with bumblebees, this may not be an independent effect of JH, but rather, the direct effect of increased ovarian development. But in Polistes gallicus, the effects of ovarian development were statistically removed, and corpora allata volume and dominant status were positively correlated (Ro¨seler et al., 1984). JH has been examined and shown to have no role in aggressive behavior in crickets (Adamo et al., 1994). Biogenic amines appear to be more important in the modulation of aggression in several of the model insect species studied so far. For example, treatment with an octopamine agonist significantly increased aggressive behaviors and the duration of fights in crickets that had recently lost a fight. Aggressiveness was depressed in crickets depleted of octopamine and dopamine, and was restored by an octopamine agonist (Stevenson et al., 2005). Flight also restores aggressiveness in losing crickets; the proposed mechanism is the restricted release of octopamine as a neuromodulator in the central nervous system (Stevenson et al., 2005). Octopamine has also been reported to correlate with dominance and aggressiveness in bumblebees (Bloch et al., 2000b); in fruit flies, octopamine increases aggressiveness and dopamine is negatively correlated (Baier et al., 2002). However, serotonin seems to promote aggression in crustaceans (Kravitz, 2000; Kravitz and Huber, 2003). Thus, the hormonal mechanisms modulating aggression in arthropods seem to be quite varied and more species will need to be investigated before relevant factors or phylogenetic patterns emerge. The exact role of JH in modulating aggressive behavior in burying beetles is unclear. JH could increase aggressiveness indirectly rather than directly. It could act on the behavioral threshold sensitivity to environmental and
social cues, just as it increases the behavior threshold sensitivity to alarm pheromones in honeybees, which in turn modulates aggression (Robinson, 1987a). For example, the difference between N. tomentosus and N. orbicollis in their readiness to breed cooperatively may be that the former has a higher threshold for the combination of carcass size and the presence of same-sex competitors before they are excluded. Social cues are obviously involved in the assessment of the object of aggression. Males and females with high JH are not aggressive toward their mate; both are aggressive only toward consexuals before oviposition and toward both sexes after oviposition (Scott, 2006); in spite of elevated JH N. tomentosus will tolerate consexuals before oviposition but will not let beetles of either sex join after oviposition (Scott, 1994). Aggression could also be triggered by the mismatch of identification cues: partners and co-breeding females are probably identified by co-varying changes in cuticular pheromones caused by changing levels of JH (unpublished data, Mu¨ller et al., 2003). However, in the experiments reported here, JH directly increased aggression in all but treated males to untreated females. In light of the effect of social cues to moderate aggression in nature, it seems that the role of these social cues is to identify a target for aggression when JH has heightened sensitivity to them. Acknowledgments My ideas on burying beetle aggression have benefited from discussions with Fred Nijhout, Dave Borst, Jessica Bolker and Carmen Panaitof. I thank Dave Borst for JH antibody and teaching me JH RIA. The Duke Medical facilities and the Forsyth Institute, Boston, generously supplied me with culls from their laboratory colonies of mice whenever I asked. This work was supported by National Science Foundation IBN 9628832 and Hatch grants from the NH Agriculture Station. This is scientific contribution no. 2290 from the New Hampshire Agriculture Station. References Adamo, S.A., Schildberger, K., Loher, W., 1994. The role of the corpora allata in the adult male cricket (Grillus campestris and Gryllus bimaculatus) in the development and expression of its agonistic behavior. Journal of Insect Physiology 40, 439–446. Baier, A., Wittek, B., Brembs, B., 2002. Drosophila as a new model organism for neurobiology of aggression? Journal of Experimental Biology 205, 1233–1240. Barth, R.H., Lester, L.J., Sroka, P., Kessler, T., Hearn, R., 1975. Juvenile hormone promotes dominance behavior and ovarian development in social wasps (Polistes annularis). Experientia 31, 691–692. Bloch, G., Borst, D.W., Huang, Z.Y., Robinson, G.E., Hefetz, A., 1996. Effects of social conditions on juvenile hormone-mediated reproductive development in Bombus terrestris workers. Physiological Entomology 21, 257–267. Bloch, G., Borst, D.W., Huang, Z.Y., Robinson, G.E., Cnaani, J., Hefetz, A., 2000a. Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris. Journal of Insect Physiology 46, 47–57.
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