- Email: [email protected]
Effects of physical and social experiences and octopamine receptor agonist on fighting behavior of male crickets Velarifictorus aspersus (Orthoptera: Gryllidae)
Accepted Manuscript Effects of physical and social experiences and octopamine receptor agonist on fighting behavior of male crickets Velarifictorus aspersus (Orthoptera: Gryllidaeik)
Wei-Nan Kang, Yang Zeng, Dao-Hong Zhu PII: DOI: Reference:
S1226-8615(17)30563-0 doi:10.1016/j.aspen.2018.02.008 ASPEN 1147
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
3 September 2017 24 January 2018 12 February 2018
Please cite this article as: Wei-Nan Kang, Yang Zeng, Dao-Hong Zhu , Effects of physical and social experiences and octopamine receptor agonist on fighting behavior of male crickets Velarifictorus aspersus (Orthoptera: Gryllidaeik). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aspen(2017), doi:10.1016/j.aspen.2018.02.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effects of physical and social experiences and octopamine receptor agonist on fighting behavior of male crickets Velarifictorus aspersus (Orthoptera:
PT
Gryllidaeik)
SC
RI
Wei-Nan Kang, Yang Zeng, Dao-Hong Zhu*
Institute of Evolutionary Ecology and Conservation Biology, Central South University of
MA
NU
Forestry and Technology, Changsha, Hunan 410004, China
AC
CE
PT E
D
* Corresponding author, E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Fighting commonly occurs among animals and is very important for resolving conflicts between conspecific individuals over limited resources. The plasticity of fighting strategies and neurobiological mechanisms underlying fighting behavior of insects are not fully understood.
PT
In the present study, we examined whether physical and social experiences affected the
RI
aggressiveness of males of the cricket Velarifictorus aspersus Walker, and whether an
SC
octopamine (OA) receptor agonist could affected the aggressiveness of males exposed to different experiences. We found that flight and winning a fight significantly enhanced male
NU
aggressiveness, while losing a fight significantly suppressed male aggressiveness, consistent
MA
with the findings of existing studies on other cricket species. We also found that female presence had a stronger enhancing effect on male aggressiveness than flight or winning a fight. These
D
findings demonstrated that physical and social experiences can affect the fighting behavior of
PT E
male V. aspersus. Topical application of a 0.15 M solution of an OA receptor agonist (chlordimeform, CDM) significantly increased male aggression level, suggesting that OA may
CE
play an important role as a neuromodulator in controlling fighting behavior of males of this
AC
species. Despite displaying a significantly higher aggression level (level 5 or 6), CDM-treated losers did not escalate to physical combat, while fights between courting males usually resulted in physical escalation. It is likely that fighting behavior is only partly regulated by OA, and additional regulatory pathways may be involved in achieving physical combat. Key words: fighting behavior; experiences; octopamine; Velarifictorus aspersus
2
ACCEPTED MANUSCRIPT Introduction Fighting commonly occurs among animals and is very important for resolving conflicts between conspecific individuals over limited resources, such as food, territory, and mates (Alexander 1961). In sevaral cricket species, males compete for territorial shelters and females,
PT
and their fighting behavior has been described extensively in the ethological literature
RI
(Alexander, 1961; Adamo and Hoy, 1995; Hofmann and Stevenson, 2000). Crickets can exhibit
SC
a number of different behaviors during agonistic interactions, but their fighting behavior can be characterized as an escalating contest following a ritualized sequence of events (Stevenson et
NU
al., 2000). Studies on the fighting behavior of crickets provide key insights for understanding
MA
ecological significance and physiological mechanism of fighting behavior in insects (Simmons, 1986; Stevenson et al., 2005).
D
Fighting requires a considerable energy, both for the development of weapons and for
PT E
engagement in fiercely physical combat, and may also cause injuries, which ultimately have a negative impact on survivorship and reproduction (Crespi, 1988; Short and Balaban, 1994; Hsu
CE
et al., 2006). Animals often develop great plasticity in fighting strategies during evolution as a
AC
consequence of trade-offs between costs and benefits of the behavior (Zeng et al., 2016). For example, life experiences of male crickets, such as the presence of females, winning a fight, possession of shelters and flight can have a significant impact on their fighting behavior and promote aggressiveness (Adamo and Hoy, 1995; Stevenson et al., 2005; Killian and Allen, 2008; Reaney et al., 2011; Rillich et al., 2011). However, group rearing and losing a fight may suppress aggressiveness of male crickets (Stevenson et al., 2005; Stevenson and Rillich, 2013). The neurobiological mechanisms underlying fighting behavior of insects are not well 3
ACCEPTED MANUSCRIPT understood, although considerable progress has been made during the last decade. Recent studies have provided insight into the neuromodulation of this complex behavior (aggression) in insects, but, as stated by Bubak et al. (2014), these studies have sometimes yielded contradictory or ambiguous results. Using male crickets as a model, researchers have
PT
discovered that biogenic amines, especially octopamine (OA) (the insect analog of
RI
noradrenaline), mediate fighting behavior. In the cricket species Gryllus bimaculatus DeGeer,
SC
depletion of OA and dopamine reduced aggression levels (Stevenson et al., 2000), while injection of chlordimeform (CDM) hydrochloride, an OA receptor agonist, promoted
NU
aggressiveness of males (Stevenson et al., 2005), suggesting that OA may act as a
MA
neuromodulator in regulating fighting behavior of male crickets. The role of OA in the regulation of aggression has also been demonstrated in other insects (e.g., ants: Kamhi et al.,
D
2015; fruit flies: Baier et al., 2002; stalk-eyed flies: Hoyer et al., 2008; Zhou et al., 2008; Bubak
PT E
et al., 2014). Activation of neuronal OA receptors has also been shown to underlie the releasing effect of flight on loser aggressiveness (Hofmann and Stevenson, 2000; Stevenson et al., 2005)
CE
as well as the enhancing effect of winning (Rillich and Stevenson, 2011) and shelter occupation
AC
(Rillich et al., 2011) on cricket aggression. Males of the cricket, Velarifictorus aspersus Walker, live solitarily in burrows, and they usually fight with each other in competition for burrows and mates using their extremely long mandibles (Zeng et al., 2016). Velarifictorus aspersus is wing-dimorphic and adults are either long-winged (LW) and flight capable or short-winged (SW) and flight incapable (Zeng and Zhu, 2012; Zeng et al., 2014). In fighting experiments between LW and SW males, LW males were more successful when fighting for territory, while SW males were more successful when 4
ACCEPTED MANUSCRIPT fighting for a mate, and this behavioral variation is though to influence the life-history strategies of males with different wing phenotypes (Zeng et al., 2016). In order to understand the plasticity of fighting strategies and neurobiological mechanisms underlying fighting behavior of crickets, we tested whether different physical (winning, losing, or flying) and social (presence of females)
PT
experiences had different effects on aggressiveness of the males of V. aspersus. In addition, we
RI
also tested whether an OA receptor agonist influenced the aggressiveness of male crickets
SC
exposed to different experiences. Materials and methods
NU
Experimental individuals
MA
Experimental V. aspersus crickets (Gryllidae) were obtained from an established laboratory colony that originated from a population collected in Hainan Province, China. Nymphs were
D
reared with ad libitum access to food and water in a large container (35 × 23 × 20 cm) under a
PT E
light:dark cycle of 16:8 h at 30 °C, as described by Zeng and Zhu (2012). After molting to adulthood, males were housed separately in a small container (13 × 13 × 8.5 cm) until they
CE
were used in the experiments described below. Earlier observations showed that aggressiveness
AC
of LW and SW males did not differ significantly, when they fought against males of the same morph (Mann-Whitney U test, Z = -0.483, p = 0.629) or without females (Mann-Whitney U test, Z = -0.89, p = 0.374) (Kang, Zeng, and Zhu, unpublished data). Thus, both LW and SW males were used in this study. Effects of experiences on fighting behavior of male crickets Before each experiment, head widths of males were measured with a digital caliper (0.01 mm; Mitutoyo Corporation, Japan). For all experiments, two males of the same wing morph 5
ACCEPTED MANUSCRIPT were matched by head width ( < 0.1 mm difference), to minimize the effect of body size, and placed in a clean glass tube (diameter: 3 cm; height: 18 cm) to rest for 5 min. They were then carefully introduced into a clean plastic container (diameter: 13 cm; height: 12 cm) to fight. All experiments were completed within 30 min, and the highest aggression level achieved was
PT
recorded according to the standard criteria used by Zeng et al. (2016) (Table 1). Fight duration
RI
was calculated from antennal contact until establishment of dominance (one cricket sings and
SC
attacks, while the other cricket escapes). Because males did not actually fight when aggression level was 0, such males were excluded from fight duration recording. To examine the effects of
NU
different experiences on fighting behavior, the following experiments were conducted: (1) As
MA
an experimental control, two males without any experience were simultaneously introduced into the container to fight; (2) to examine the effect of flight on fighting behavior, crickets were
D
suspended in a wind stream to fly for 5 min as described by Zeng et al. (2014), and matched to
PT E
fight immediately thereafter; (3) to test whether the presence of females affected male aggressiveness, a sexually mature virgin female was placed in the container and allowed 5 min
CE
to get acclimate to the new environment. Then, two males were simultaneously introduced into
AC
the container, and allowed to fight with each other; (4) to examine the effect of winning on male aggressiveness, males were first matched to fight to establish dominance, and a second fight was set up between two winners after 1.5 h; (5) to examine the effect of losing on male aggressiveness, males were first matched to fight to establish dominance, and a second fight was set up between two losers after 1.5 h. Aggression level and fight duration were recorded once the fight was over. Determination of CDM functional dosage 6
ACCEPTED MANUSCRIPT CDM was used as an OA receptor agonist in place of CDM hydrochloride, which has been used for the same purpose in previous cricket studies (Stevenson et al. 2005), but is not available in China. CDM (Sigma-Aldrich) was not water-soluble and was thus dissolved in acetone (purity: 99.5 %) to make 0.1, 0.15, and 0.2 M solutions. Males received 1 μL of 0.1, 0.15, or
PT
0.2 M CDM, or 1 μL acetone (for control) on the abdomen from a pipette, and drug-treated
RI
males were matched to fight each other after 1.5 h, according to the protocol used by Stevenson
SC
et al. (2005). Based on the results of this experiment, a functional dosage of 0.15 M CDM was used in subsequent experiments.
NU
Effects of CDM on fighting behavior of males exposed to different physical and social
MA
experiences
In order to investigate the effect of CDM on fighting behavior of males exposed to different
D
experiences, the following experiments were conducted; (1) to test the effect of CDM on
PT E
aggressiveness of males with previous fighting experiences, males were first matched to fight. After a clear establishment of dominance, winner and loser were treated with CDM (1 μL; 0.15
CE
M), and then returned to their own containers for 1.5 h. Thereafter, a second fight was set up
AC
between two CDM-treated winners or two CDM-treated losers. As experimental controls, untreated winners or losers were matched to fight 1.5 h after the first fight; (2) to test the effect of CDM on fighting behavior of flown males, each male was treated with CDM (1 μL; 0.15 M). After 1.5 h, drug-treated males were suspended to fly for 5 min, and matched to fight. The control consisted of untreated males suspended to fly for 5 min and matched to fight; (3) to test the effect of CDM on fighting behavior of courting males, each male was treated with CDM (1 μL; 0.15 M) and after 1.5 h, drug-treated males were placed into a new arena to fight against 7
ACCEPTED MANUSCRIPT each other in the presence of a female. As an experimental control, untreated males were allowed to fight for a female. Aggression level and fight duration were recorded once the fight was over. Statistical methods
PT
Data are presented as median ± interquartile ranges. Differences between two groups were
RI
tested for statistical significance using the Mann-Whitney U test using SPSS 13.0 (IBM, USA).
SC
Multiple groups were compared with the Steel-Dwass multiple comparison test performed in R (version 3.3.3; R Foundation for Statistical Computing).
NU
Results
MA
Effects of physical and social experiences on fighting behavior Fights between two naive males (i.e., with no previous experience) rarely escalated to
D
physical combat, and the observed median aggression level was only 2 (interquartile range: 1–
PT E
4). Winning and flight significantly increased the aggression level (Steel-Dwass test, winners versus naive males: t = 3.911, p < 0.001; flown versus naive males: t = 2.805, p = 0.040), with
CE
median aggression level increasing to 4 (interquartile range: 3–6) and 3.5 (interquartile range:
AC
3–4), respectively. When males were fighting for a mate, almost all fights escalated to physical combat, and the median aggression level was 6 (interquartile range: 5–6), which was significantly higher than the aggression level of other groups (Steel-Dwass test, courting versus naive males: t = 6.639, p < 0.001; courting versus flown males: t = 4.277, p < 0.001; courting males versus winners: t = 2.763, p = 0.045; and courting males versus losers: t = 5.647, p < 0.001). However, only female presence significantly increase the fight duration (Steel-Dwass test, flown versus naive males: t = 0.357, p = 0.997; courting versus naive males: t = 4.417, p 8
ACCEPTED MANUSCRIPT < 0.001; and winners versus naive males: t = 1.506, p = 0.559). In contrast, losing a fight significantly suppressed male aggression and also shortened fight duration (Steel-Dwass test, losers versus naive males, aggression level: t = 4.609, p < 0.001; fight duration: t = 3.402, p = 0.006) (Fig. 1).
PT
Determination of CDM functional dosage
RI
Aggression level and duration of fight between males treated with acetone were similar to
SC
those of untreated males (Steel-Dwass test, aggression level: t = 1.825, p = 0.359; fight duration: t = 1.149, p = 0.780). Application of 0.15 M CDM significantly increased aggression level of
NU
males (Steel-Dwass test, t = 4.629, p < 0.001), while the same effect was not observed in males
MA
treated with 0.1 or 0.2 M CDM (Steel-Dwass test, 0.1 M CDM versus untreated: t = 1.914, p = 0.310; 0.2 M CDM versus untreated: t = 1.348, p = 0.661) (Fig. 2A). In addition, treatment with
D
CDM also increased fight duration, and 0.15 M CDM was the most effective dosage (Steel-
PT E
Dwass test, 0.15 M CDM versus untreated: t = 6.515, p < 0.001; 0.15 M CDM versus acetone: t = 5.100, p < 0.001) (Fig. 2B). Because treatment with acetone did not affect male
CE
aggressiveness, we used untreated males as control group in subsequent experiments.
AC
Effects of CDM on fighting behavior of males exposed to different physical and social experiences
Aggression level and fight duration between two winners treated with CDM tended to be lower compared to untreated winners, although the difference was not significant (MannWhitney U test, p > 0.05) (Fig. 3A, B). However, both of aggression level and fight duration between losers treated with CDM were significantly hight compared to untreated losers (MannWhitney U test, aggression level: Z = -4.522, p < 0.001; fight duration: Z = -3.022, p = 0.003) 9
ACCEPTED MANUSCRIPT (Fig. 3C, D). A significant suppressive effect on aggression level was observed when flown males or courting males were treated with CDM (Mann-Whitney U test, flown males: Z = 3.125, p = 0.002; courting males: Z = -4.356, p < 0.001) (Fig. 4 and Fig. 5). When courting males were treated with CDM, fight duration decreased significantly (Mann-Whitney U test, Z
RI
(Mann-Whitney U test, Z = -1.709, p = 0.087) (Fig. 4 and Fig. 5).
PT
= -1.989, p = 0.047), whereas fight duration of flown males was not affected by CDM treatment
SC
Discussion
In this study, we found that males of V. aspersus were not very aggressive toward each other
NU
when females were absent (median aggression level = 2), but became highly aggressive in
MA
presence of a female (median aggression level = 6), suggesting an important role of females in motivating males to fight. Our result is consistent with existing studies on several other cricket
D
species (Gryllidae), such as G. bimaculatus (Simmons, 1986; Adamo and Hoy, 1995) and G.
PT E
integer Scudder (Kortet and Hedrick, 2007), where males were also observed to fight more frequently and more intensely in presence of a female.
CE
Previous fighting experience influences fight performance in animals, e.g., winners become
AC
hyper-aggressive while losers are not aggressive for a certain period of time after the experience (Stevenson et al., 2005; Hsu et al., 2006; Rillich and Stevenson, 2011, 2014). In this study, we observed that males of V. aspersus became more aggressive after winning, but less aggressive after losing. A short flight significantly enhanced aggressiveness in males of V. aspersus, which is consistent with observations made in other cricket species (Hofmann and Stevenson, 2000). In G. bimaculatus and G. texensis Cade and Otte, short flight bouts were demonstrated to also promote male reproductive behaviors (Dyakonova and Krushinsky, 2008; Guerra and Pollack, 10
ACCEPTED MANUSCRIPT 2009), and the positive effect of flight on aggressiveness was considered to be associated with the enhancement of reproductive efforts. However, in a previous study on V. aspersus, we found that a short time of flight (5 min) did not enhance the development of male accessory glands (Zeng et al., 2014). Because males often spend a great deal of energy on various reproductive
PT
expenditures besides accessory gland development, more studies are needed to test whether
RI
short flight bouts can enhance reproductive behavior in male V. aspersus. In addition, winners
SC
and flown males were less aggressive than courting males, and this finding may have two implications. First, after winning a fight or flying, males may be too exhausted to engage in a
NU
physical combat, which requires considerable energy. Second, these energy-exhausting
MA
experiences may only partially activate regulatory pathways that promote male fighting behavior, while the presence of females may fully activate these regulatory pathways.
D
OA titer increases during aggressive behavior in male G. bimaculatus (Adamo et al., 1995)
PT E
and OA depletion reduces aggression level, while injection of a specific OA receptor agonist (CDM) promotes aggressiveness of male G. bimaculatus (Stevenson et al., 2000, 2005).
CE
Drosophila mutants lacking the enzyme, tyramine-β-hydroxylase (TβH), which catalyzes the
AC
synthesis of OA from tyramine, exhibit reduced aggression, while treatment with CDM and overexpression of TβH increase the aggression levels of socially-reared flies (Zhou et al., 2008). In the ant, Oecophylla smaragdina (Fabricius), OA titer is higher in aggressive major workers than in non-aggressive minor workers, and pharmacological manipulation of OA in major and minor worker brains was shown to reverse the levels of aggression characteristic of each worker size class (Kamhi et al., 2015). These studies strongly suggest that OA plays an important role in the regulation of male fighting behaviors. In the current study, topical application of 1 μL of 11
ACCEPTED MANUSCRIPT 0.15 M CDM significantly increased the aggression level in male V. aspersus, whereas topical application of 0.1 M or 0.2 M CDM did not affect male aggressiveness, suggesting that the release of OA as a neuromodulator may mediate fighting behavior in this species, and its effect may vary depending on OA titer. This finding is consistent with observations made in G.
PT
bimaculatus, although injection of 100 μL of 1 mm CDM was the most effective dosage in
RI
promoting aggressiveness in this species (Stevenson et al., 2005). The difference may be due
SC
to the fact that in our experiment topical application may have decreased the amount of CDM that entered the body of insects.
NU
The effect of CDM topical application on aggressiveness of experienced males varied
MA
depending on the type of experience. Specifically, aggression level increased significantly when CDM-treated losers fought against each other, a phenomenon also observed in other cricket
D
species (Stevenson et al., 2005). However, topical application of CDM to winners, flown males,
PT E
or courting males suppressed their aggressiveness. Excessive CDM treatment was found to decrease aggression in male G. bimaculatus (5 mm) (Stevenson et al., 2005) and male V.
CE
aspersus (0.2 M, this study), and winning and flight increased OA titer in male G. bimaculatus
AC
(Stevenson et al., 2005; Rillich and Stevenson, 2011). It is possible that application of CDM to winners, flown males, or courting males might have resulted in over-excitation of OA neurons, leading to a decrease in aggression. However, we did not quantify OA titer in this study, and further experiments are needed to test this hypothesis. Alternatively, a receptor binding to the agonist may have affected the binding of OA to other subtypes of OA receptors. Because OA plays a role in several functions, such as locomotion and courtship (Chen et al., 2013), CDM may have bound to receptors that control flying or courting behavior when flown or courting 12
ACCEPTED MANUSCRIPT males were treated. Although OA has been extensively demonstrated to be an important regulatory factor of fighting behavior in crickets (Stevenson et al., 2000, 2005; Stevenson and Rillich, 2012), the mechanisms underlying the effects of different experiences on aggressiveness are complicated
PT
and far from being clarified. In male V. aspersus, courting males usually escalated to physical
RI
combat during fights, CDM-treated males did not exhibit physical escalation. It seems likely
SC
that fighting behavior was only partly regulated by OA, and other regulatory pathways may also be involved in controlling escalation to physical combat. For example, inhibition of the
NU
nitric oxide/cyclic guanosine 3',5'-monophosphate pathway in male G. bimaculatus induced
MA
fights to last twice as long and to escalate to significantly higher levels, often involving grappling (level 6) (Stevenson and Rillich, 2015). In addition, activation of the OA pathway
D
may be responsible for the observed enhancing effects of winning a fight or flight on
PT E
aggressiveness (Stevenson et al., 2005; Rillich and Stevenson, 2011), but the dopamine pathway is necessary to recover from the suppressive effect of losing a fight in G. bimaculatus
CE
(Rillich and Stevenson, 2014). To our knowledge, no study has compared OA titers between
AC
males exhibiting low versus high aggressiveness. Therefore, further research is needed to test whether intensity of aggressiveness is positively correlated with OA titer.
13
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the National Nature Science Foundation of China (Grant No. 31070586). The authors thank Prof. Zhiwei Liu (Eastern Illinois University, USA) for revising
PT
the manuscript.
RI
Conflict of interest statement
AC
CE
PT E
D
MA
NU
SC
The authors have no conflict of interest to declare.
14
ACCEPTED MANUSCRIPT References Adamo, S.A., Hoy, R.R., 1995. Agonistic behavior in male and female field crickets, Gryllus bimaculatus, and how behavioral context influences its expression. Anim. Behav. 49, 1491– 1501.
PT
Adamo, S.A., Linn, C.E., Hoy, R.R., 1995. The role of neurohormonal octopamine during ‘fight or flight' behaviour in the field cricket Gryllus bimaculatus. J. Exp. Biol. 198, 1691–
RI
1700.
NU
(Orthoptera: Gryllidae). Behaviour 17, 130–223.
SC
Alexander, R.D., 1961. Aggressiveness, territoriality, and sexual behavior in field crickets
Baier, A., Wittek, B., Brembs, B., 2002. Drosophila as a new model organism for the
MA
neurobiology of aggression? J. Exp. Biol. 205, 1233–1240. Bubak, A.N., Grace, J.L., Watt, M.J., Renner, K.J., Swallow, J.G., 2014. Neurochemistry as a
60, 778–790.
PT E
D
bridge between morphology and behavior: perspectives on aggression in insects. Curr. Zool.
CE
Chen, A., Ng, F., Lebestky, T., Grygoruk, A., Djapri, C., Lawal, H.O., Zaveri, H.A., Mehanzel, F., Najibi, R., Seidman, G., Murphy, N.P., Kelly, R.L., Ackerson, L.C., Maidment,
AC
N.T., Jackson, FR, Krantz, D.E., 2013. Dispensable, redundant, complementary, and cooperative roles of dopamine, octopamine, and serotonin in Drosophila melanogaster. Genetics 193, 159–176. Crespi, B.J., 1988. Adaptation, compromise, and constraint: the development, morphometrics, and behavioral basis of a fighter-flier polymorphism in male Hoplothrips karnyi (Insecta: Thysanoptera). Behav. Ecol. Sociobiol. 23, 93–104. Dyakonova, V., Krushinsky, A., 2008. Previous motor experience enhances courtship behavior 15
ACCEPTED MANUSCRIPT in male cricket Gryllus bimaculatus. J. Insect Behav. 21, 172–180. Guerra, P.A., Pollack, G.S., 2009. Flight behaviour attenuates the trade-off between flight capability and reproduction in a wing polymorphic cricket. Biol. Lett. 5, 229–231. Hofmann, H.A., Stevenson, P.A., 2000. Flight restores fight in crickets. Nature 403, 613.
PT
Hoyer, S.C., Eckart, A., Herrel, A., Zars, T., Fischer, S.A., Hardie, S.L., Heisenberg, M., 2008.
RI
Octopamine in male aggression of Drosophila. Curr. Biol. 18, 159–167.
SC
Hsu, Y., Earley, R.L., Wolf, L.L., 2006. Modulation of aggressive behaviour by fighting experience: mechanisms and contest outcomes. Biol. Rev. Cam. Philos. Soc. 81, 33–74.
NU
Kamhi, J.F., Nunn, K., Robson, S.K.A., Traniello, J.F.A., 2015. Polymorphism and division of
MA
labour in a socially complex ant: neuromodulation of aggression in the Australian weaver ant, Oecophylla smaragdina. Proc. R. Soc. B 282, 79–96.
D
Killian, K.A, Allen, JR, 2008. Mating Resets Male Cricket Aggression. J. Insect Behav. 21,
PT E
535–548.
Kortet, R., Hedrick, A., 2007. A behavioural syndrome in the field cricket Gryllus integer:
CE
intrasexual aggression is correlated with activity in a novel environment. Biol. J. Linn. Soc.
AC
91, 475–482.
Reaney, L.T., Drayton, J.M., Jennions, M.D., 2011. The role of body size and fighting experience in predicting contest behaviour in the black field cricket, Teleogryllus commodus. Behav. Ecol .Sociobiol. 65, 217–225. Rillich, J., Schildberger, K., Stevenson, P.A., 2011. Octopamine and occupancy: an aminergic mechanism for intruder-resident aggression in crickets. Proc. R. Soc. B 278, 1873–1880. Rillich, J., Stevenson, P.A., 2011. Winning fights induces hyperaggression via the action of the 16
ACCEPTED MANUSCRIPT biogenic amine octopamine in crickets. PLoS One 6, e28891 Rillich, J., Stevenson, P.A., 2014. A fighter's comeback: Dopamine is necessary for recovery of aggression after social defeat in crickets. Horm. Behav. 66, 696–704. Short, R.V., Balaban, E., 1994. The differences between the sexes. Cambridge University Press,
PT
Cambridge.
SC
bimaculatus (De Geer). Anim. Behav. 34, 567–579.
RI
Simmons, L.W., 1986. Inter-male competition and mating success in the field cricket, Gryllus
Stevenson, P.A., Dyakonova, V., Rillich, J., Schildberger, K., 2005. Octopamine and
NU
experience-dependent modulation of aggression in crickets. J. Neurosci. 25, 1431–1441.
MA
Stevenson, P.A., Hofmann, H.A., Schoch, K., Schildberger, K., 2000. The fight and flight responses of crickets depleted of biogenic amines. J. Neurobiol. 43, 107–120.
D
Stevenson, P.A., Rillich, J., 2012. The decision to fight or flee-insights into underlying
PT E
mechanism in crickets. Front. Neurosci. 6, 118. Stevenson, P.A., Rillich, J., 2013. Isolation associated aggression-a consequence of recovery
CE
from defeat in a territorial animal. PLoS One 8, e74965.
AC
Stevenson, P.A., Rillich, J., 2015. Adding up the odds-Nitric oxide signaling underlies the decision to flee and post-conflict depression of aggression. Sci. Adv. 1, e1500060. Zeng, Y., Zhu, D.H., Kang, W.N., 2016. Variation in fighting strategies in male wing-dimorphic crickets (Gryllidae). Behav. Ecol. Sociobiol. 70, 429–435. Zeng, Y., Zhu, D.H., Zhao, L.Q., 2014. Critical flight time for switch from flight to reproduction in the wing dimorphic cricket Velarifictorus aspersus. Evol. Biol. 41, 397–403. Zeng, Y., Zhu, D.H., 2012. Trade-off between flight capability and reproduction in male 17
ACCEPTED MANUSCRIPT Velarifictorus aspersus crickets. Ecol. Entomol. 37, 244–251. Zhou, C., Rao, Y., Rau, Y., 2008. A subset of octopaminergic neurons are important for
AC
CE
PT E
D
MA
NU
SC
RI
PT
Drosophila aggression. Nat. Neurosci. 11, 1059–1067.
18
ACCEPTED MANUSCRIPT Table 1 Agonistic behaviors scored in trials with male V. aspersus* Description
Mutual avoidance
Males escape from each other after contact
0
Pre-established dominance
One male attacks, the other runs
1
Antennal fencing
Males rapidly antennate each other’s antennae
2
Mandible spread (one)
One male spreads mandibles and attacks the other
3
Mandible spread (both)
Both opponents spread mandibles
Lunge and butt
Rush at the conspecific and use head to hit the opponent
5
Wrestling
Males interlock mandibles, push and wrestle with each other
6
RI
SC
CE
PT E
D
MA
NU
Modified from Zeng et al. (2016).
AC
*
Score
PT
Behavior
19
4
ACCEPTED MANUSCRIPT Figure captions: Fig. 1 Effects of different experiences on fighting behavior of male Velarifictorus asperses. A: aggression level; B: fight duration. Data are presented as median ± interquartile range, and numbers in brackets indicate the sample size. Different letters indicate significant differences
PT
between groups (Steel-Dwass test, p < 0.05).
RI
Fig. 2 Effect of OA agonist on fighting behavior of naive males. A: aggression level; B: fight
SC
duration. Data are presented as median ± interquartile range, and numbers in brackets indicate the sample size. Different letters indicate significant differences between groups
NU
(Steel-Dwass test, p < 0.05).
MA
Fig. 3 Effect of OA agonist on fighting behavior of males with fighting previous experience. A and B represent fights between winners; C and D represent fights between losers. Data are
D
presented as median ± interquartile range, and the numbers in brackets indicates the sample
PT E
size. Different letters indicate significant differences between groups (Mann-Whitney U test, p < 0.05).
CE
Fig. 4 Effect of OA agonist on fighting behavior of flown males. A: aggression level; B: fight
AC
duration. Data are presented as median ± interquartile range, and the numbers in brackets indicate the sample size. Different letters indicate significant differences between groups (Mann-Whitney U test, p < 0.05). Fig. 5 Effect of OA agonist on fighting behavior of courting males. A: aggression level; B: fight duration. Data are presented as median ± interquartile range, and numbers in brackets indicate the sample size. Different letters indicate significant differences between groups (Mann-Whitney U test, p < 0.05). 20
ACCEPTED MANUSCRIPT Highlights
PT RI SC NU MA D
PT E
CE
Effects of physical and social experiences on aggressiveness of male Velarifictorus asperses were examined. Presence of females, flight, winning a fight enhanced the aggressiveness, but losing a fight suppressed aggressiveness. Aggressiveness was strongest in the presence of a female. Treatment with CDM (OA receptor agonist) resulted in inhibiting the “loser effect” and decreases aggression for winners.
AC
21
Graphics Abstract
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Wei-Nan Kang, Yang Zeng, Dao-Hong Zhu PII: DOI: Reference:
S1226-8615(17)30563-0 doi:10.1016/j.aspen.2018.02.008 ASPEN 1147
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
3 September 2017 24 January 2018 12 February 2018
Please cite this article as: Wei-Nan Kang, Yang Zeng, Dao-Hong Zhu , Effects of physical and social experiences and octopamine receptor agonist on fighting behavior of male crickets Velarifictorus aspersus (Orthoptera: Gryllidaeik). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aspen(2017), doi:10.1016/j.aspen.2018.02.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effects of physical and social experiences and octopamine receptor agonist on fighting behavior of male crickets Velarifictorus aspersus (Orthoptera:
PT
Gryllidaeik)
SC
RI
Wei-Nan Kang, Yang Zeng, Dao-Hong Zhu*
Institute of Evolutionary Ecology and Conservation Biology, Central South University of
MA
NU
Forestry and Technology, Changsha, Hunan 410004, China
AC
CE
PT E
D
* Corresponding author, E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Fighting commonly occurs among animals and is very important for resolving conflicts between conspecific individuals over limited resources. The plasticity of fighting strategies and neurobiological mechanisms underlying fighting behavior of insects are not fully understood.
PT
In the present study, we examined whether physical and social experiences affected the
RI
aggressiveness of males of the cricket Velarifictorus aspersus Walker, and whether an
SC
octopamine (OA) receptor agonist could affected the aggressiveness of males exposed to different experiences. We found that flight and winning a fight significantly enhanced male
NU
aggressiveness, while losing a fight significantly suppressed male aggressiveness, consistent
MA
with the findings of existing studies on other cricket species. We also found that female presence had a stronger enhancing effect on male aggressiveness than flight or winning a fight. These
D
findings demonstrated that physical and social experiences can affect the fighting behavior of
PT E
male V. aspersus. Topical application of a 0.15 M solution of an OA receptor agonist (chlordimeform, CDM) significantly increased male aggression level, suggesting that OA may
CE
play an important role as a neuromodulator in controlling fighting behavior of males of this
AC
species. Despite displaying a significantly higher aggression level (level 5 or 6), CDM-treated losers did not escalate to physical combat, while fights between courting males usually resulted in physical escalation. It is likely that fighting behavior is only partly regulated by OA, and additional regulatory pathways may be involved in achieving physical combat. Key words: fighting behavior; experiences; octopamine; Velarifictorus aspersus
2
ACCEPTED MANUSCRIPT Introduction Fighting commonly occurs among animals and is very important for resolving conflicts between conspecific individuals over limited resources, such as food, territory, and mates (Alexander 1961). In sevaral cricket species, males compete for territorial shelters and females,
PT
and their fighting behavior has been described extensively in the ethological literature
RI
(Alexander, 1961; Adamo and Hoy, 1995; Hofmann and Stevenson, 2000). Crickets can exhibit
SC
a number of different behaviors during agonistic interactions, but their fighting behavior can be characterized as an escalating contest following a ritualized sequence of events (Stevenson et
NU
al., 2000). Studies on the fighting behavior of crickets provide key insights for understanding
MA
ecological significance and physiological mechanism of fighting behavior in insects (Simmons, 1986; Stevenson et al., 2005).
D
Fighting requires a considerable energy, both for the development of weapons and for
PT E
engagement in fiercely physical combat, and may also cause injuries, which ultimately have a negative impact on survivorship and reproduction (Crespi, 1988; Short and Balaban, 1994; Hsu
CE
et al., 2006). Animals often develop great plasticity in fighting strategies during evolution as a
AC
consequence of trade-offs between costs and benefits of the behavior (Zeng et al., 2016). For example, life experiences of male crickets, such as the presence of females, winning a fight, possession of shelters and flight can have a significant impact on their fighting behavior and promote aggressiveness (Adamo and Hoy, 1995; Stevenson et al., 2005; Killian and Allen, 2008; Reaney et al., 2011; Rillich et al., 2011). However, group rearing and losing a fight may suppress aggressiveness of male crickets (Stevenson et al., 2005; Stevenson and Rillich, 2013). The neurobiological mechanisms underlying fighting behavior of insects are not well 3
ACCEPTED MANUSCRIPT understood, although considerable progress has been made during the last decade. Recent studies have provided insight into the neuromodulation of this complex behavior (aggression) in insects, but, as stated by Bubak et al. (2014), these studies have sometimes yielded contradictory or ambiguous results. Using male crickets as a model, researchers have
PT
discovered that biogenic amines, especially octopamine (OA) (the insect analog of
RI
noradrenaline), mediate fighting behavior. In the cricket species Gryllus bimaculatus DeGeer,
SC
depletion of OA and dopamine reduced aggression levels (Stevenson et al., 2000), while injection of chlordimeform (CDM) hydrochloride, an OA receptor agonist, promoted
NU
aggressiveness of males (Stevenson et al., 2005), suggesting that OA may act as a
MA
neuromodulator in regulating fighting behavior of male crickets. The role of OA in the regulation of aggression has also been demonstrated in other insects (e.g., ants: Kamhi et al.,
D
2015; fruit flies: Baier et al., 2002; stalk-eyed flies: Hoyer et al., 2008; Zhou et al., 2008; Bubak
PT E
et al., 2014). Activation of neuronal OA receptors has also been shown to underlie the releasing effect of flight on loser aggressiveness (Hofmann and Stevenson, 2000; Stevenson et al., 2005)
CE
as well as the enhancing effect of winning (Rillich and Stevenson, 2011) and shelter occupation
AC
(Rillich et al., 2011) on cricket aggression. Males of the cricket, Velarifictorus aspersus Walker, live solitarily in burrows, and they usually fight with each other in competition for burrows and mates using their extremely long mandibles (Zeng et al., 2016). Velarifictorus aspersus is wing-dimorphic and adults are either long-winged (LW) and flight capable or short-winged (SW) and flight incapable (Zeng and Zhu, 2012; Zeng et al., 2014). In fighting experiments between LW and SW males, LW males were more successful when fighting for territory, while SW males were more successful when 4
ACCEPTED MANUSCRIPT fighting for a mate, and this behavioral variation is though to influence the life-history strategies of males with different wing phenotypes (Zeng et al., 2016). In order to understand the plasticity of fighting strategies and neurobiological mechanisms underlying fighting behavior of crickets, we tested whether different physical (winning, losing, or flying) and social (presence of females)
PT
experiences had different effects on aggressiveness of the males of V. aspersus. In addition, we
RI
also tested whether an OA receptor agonist influenced the aggressiveness of male crickets
SC
exposed to different experiences. Materials and methods
NU
Experimental individuals
MA
Experimental V. aspersus crickets (Gryllidae) were obtained from an established laboratory colony that originated from a population collected in Hainan Province, China. Nymphs were
D
reared with ad libitum access to food and water in a large container (35 × 23 × 20 cm) under a
PT E
light:dark cycle of 16:8 h at 30 °C, as described by Zeng and Zhu (2012). After molting to adulthood, males were housed separately in a small container (13 × 13 × 8.5 cm) until they
CE
were used in the experiments described below. Earlier observations showed that aggressiveness
AC
of LW and SW males did not differ significantly, when they fought against males of the same morph (Mann-Whitney U test, Z = -0.483, p = 0.629) or without females (Mann-Whitney U test, Z = -0.89, p = 0.374) (Kang, Zeng, and Zhu, unpublished data). Thus, both LW and SW males were used in this study. Effects of experiences on fighting behavior of male crickets Before each experiment, head widths of males were measured with a digital caliper (0.01 mm; Mitutoyo Corporation, Japan). For all experiments, two males of the same wing morph 5
ACCEPTED MANUSCRIPT were matched by head width ( < 0.1 mm difference), to minimize the effect of body size, and placed in a clean glass tube (diameter: 3 cm; height: 18 cm) to rest for 5 min. They were then carefully introduced into a clean plastic container (diameter: 13 cm; height: 12 cm) to fight. All experiments were completed within 30 min, and the highest aggression level achieved was
PT
recorded according to the standard criteria used by Zeng et al. (2016) (Table 1). Fight duration
RI
was calculated from antennal contact until establishment of dominance (one cricket sings and
SC
attacks, while the other cricket escapes). Because males did not actually fight when aggression level was 0, such males were excluded from fight duration recording. To examine the effects of
NU
different experiences on fighting behavior, the following experiments were conducted: (1) As
MA
an experimental control, two males without any experience were simultaneously introduced into the container to fight; (2) to examine the effect of flight on fighting behavior, crickets were
D
suspended in a wind stream to fly for 5 min as described by Zeng et al. (2014), and matched to
PT E
fight immediately thereafter; (3) to test whether the presence of females affected male aggressiveness, a sexually mature virgin female was placed in the container and allowed 5 min
CE
to get acclimate to the new environment. Then, two males were simultaneously introduced into
AC
the container, and allowed to fight with each other; (4) to examine the effect of winning on male aggressiveness, males were first matched to fight to establish dominance, and a second fight was set up between two winners after 1.5 h; (5) to examine the effect of losing on male aggressiveness, males were first matched to fight to establish dominance, and a second fight was set up between two losers after 1.5 h. Aggression level and fight duration were recorded once the fight was over. Determination of CDM functional dosage 6
ACCEPTED MANUSCRIPT CDM was used as an OA receptor agonist in place of CDM hydrochloride, which has been used for the same purpose in previous cricket studies (Stevenson et al. 2005), but is not available in China. CDM (Sigma-Aldrich) was not water-soluble and was thus dissolved in acetone (purity: 99.5 %) to make 0.1, 0.15, and 0.2 M solutions. Males received 1 μL of 0.1, 0.15, or
PT
0.2 M CDM, or 1 μL acetone (for control) on the abdomen from a pipette, and drug-treated
RI
males were matched to fight each other after 1.5 h, according to the protocol used by Stevenson
SC
et al. (2005). Based on the results of this experiment, a functional dosage of 0.15 M CDM was used in subsequent experiments.
NU
Effects of CDM on fighting behavior of males exposed to different physical and social
MA
experiences
In order to investigate the effect of CDM on fighting behavior of males exposed to different
D
experiences, the following experiments were conducted; (1) to test the effect of CDM on
PT E
aggressiveness of males with previous fighting experiences, males were first matched to fight. After a clear establishment of dominance, winner and loser were treated with CDM (1 μL; 0.15
CE
M), and then returned to their own containers for 1.5 h. Thereafter, a second fight was set up
AC
between two CDM-treated winners or two CDM-treated losers. As experimental controls, untreated winners or losers were matched to fight 1.5 h after the first fight; (2) to test the effect of CDM on fighting behavior of flown males, each male was treated with CDM (1 μL; 0.15 M). After 1.5 h, drug-treated males were suspended to fly for 5 min, and matched to fight. The control consisted of untreated males suspended to fly for 5 min and matched to fight; (3) to test the effect of CDM on fighting behavior of courting males, each male was treated with CDM (1 μL; 0.15 M) and after 1.5 h, drug-treated males were placed into a new arena to fight against 7
ACCEPTED MANUSCRIPT each other in the presence of a female. As an experimental control, untreated males were allowed to fight for a female. Aggression level and fight duration were recorded once the fight was over. Statistical methods
PT
Data are presented as median ± interquartile ranges. Differences between two groups were
RI
tested for statistical significance using the Mann-Whitney U test using SPSS 13.0 (IBM, USA).
SC
Multiple groups were compared with the Steel-Dwass multiple comparison test performed in R (version 3.3.3; R Foundation for Statistical Computing).
NU
Results
MA
Effects of physical and social experiences on fighting behavior Fights between two naive males (i.e., with no previous experience) rarely escalated to
D
physical combat, and the observed median aggression level was only 2 (interquartile range: 1–
PT E
4). Winning and flight significantly increased the aggression level (Steel-Dwass test, winners versus naive males: t = 3.911, p < 0.001; flown versus naive males: t = 2.805, p = 0.040), with
CE
median aggression level increasing to 4 (interquartile range: 3–6) and 3.5 (interquartile range:
AC
3–4), respectively. When males were fighting for a mate, almost all fights escalated to physical combat, and the median aggression level was 6 (interquartile range: 5–6), which was significantly higher than the aggression level of other groups (Steel-Dwass test, courting versus naive males: t = 6.639, p < 0.001; courting versus flown males: t = 4.277, p < 0.001; courting males versus winners: t = 2.763, p = 0.045; and courting males versus losers: t = 5.647, p < 0.001). However, only female presence significantly increase the fight duration (Steel-Dwass test, flown versus naive males: t = 0.357, p = 0.997; courting versus naive males: t = 4.417, p 8
ACCEPTED MANUSCRIPT < 0.001; and winners versus naive males: t = 1.506, p = 0.559). In contrast, losing a fight significantly suppressed male aggression and also shortened fight duration (Steel-Dwass test, losers versus naive males, aggression level: t = 4.609, p < 0.001; fight duration: t = 3.402, p = 0.006) (Fig. 1).
PT
Determination of CDM functional dosage
RI
Aggression level and duration of fight between males treated with acetone were similar to
SC
those of untreated males (Steel-Dwass test, aggression level: t = 1.825, p = 0.359; fight duration: t = 1.149, p = 0.780). Application of 0.15 M CDM significantly increased aggression level of
NU
males (Steel-Dwass test, t = 4.629, p < 0.001), while the same effect was not observed in males
MA
treated with 0.1 or 0.2 M CDM (Steel-Dwass test, 0.1 M CDM versus untreated: t = 1.914, p = 0.310; 0.2 M CDM versus untreated: t = 1.348, p = 0.661) (Fig. 2A). In addition, treatment with
D
CDM also increased fight duration, and 0.15 M CDM was the most effective dosage (Steel-
PT E
Dwass test, 0.15 M CDM versus untreated: t = 6.515, p < 0.001; 0.15 M CDM versus acetone: t = 5.100, p < 0.001) (Fig. 2B). Because treatment with acetone did not affect male
CE
aggressiveness, we used untreated males as control group in subsequent experiments.
AC
Effects of CDM on fighting behavior of males exposed to different physical and social experiences
Aggression level and fight duration between two winners treated with CDM tended to be lower compared to untreated winners, although the difference was not significant (MannWhitney U test, p > 0.05) (Fig. 3A, B). However, both of aggression level and fight duration between losers treated with CDM were significantly hight compared to untreated losers (MannWhitney U test, aggression level: Z = -4.522, p < 0.001; fight duration: Z = -3.022, p = 0.003) 9
ACCEPTED MANUSCRIPT (Fig. 3C, D). A significant suppressive effect on aggression level was observed when flown males or courting males were treated with CDM (Mann-Whitney U test, flown males: Z = 3.125, p = 0.002; courting males: Z = -4.356, p < 0.001) (Fig. 4 and Fig. 5). When courting males were treated with CDM, fight duration decreased significantly (Mann-Whitney U test, Z
RI
(Mann-Whitney U test, Z = -1.709, p = 0.087) (Fig. 4 and Fig. 5).
PT
= -1.989, p = 0.047), whereas fight duration of flown males was not affected by CDM treatment
SC
Discussion
In this study, we found that males of V. aspersus were not very aggressive toward each other
NU
when females were absent (median aggression level = 2), but became highly aggressive in
MA
presence of a female (median aggression level = 6), suggesting an important role of females in motivating males to fight. Our result is consistent with existing studies on several other cricket
D
species (Gryllidae), such as G. bimaculatus (Simmons, 1986; Adamo and Hoy, 1995) and G.
PT E
integer Scudder (Kortet and Hedrick, 2007), where males were also observed to fight more frequently and more intensely in presence of a female.
CE
Previous fighting experience influences fight performance in animals, e.g., winners become
AC
hyper-aggressive while losers are not aggressive for a certain period of time after the experience (Stevenson et al., 2005; Hsu et al., 2006; Rillich and Stevenson, 2011, 2014). In this study, we observed that males of V. aspersus became more aggressive after winning, but less aggressive after losing. A short flight significantly enhanced aggressiveness in males of V. aspersus, which is consistent with observations made in other cricket species (Hofmann and Stevenson, 2000). In G. bimaculatus and G. texensis Cade and Otte, short flight bouts were demonstrated to also promote male reproductive behaviors (Dyakonova and Krushinsky, 2008; Guerra and Pollack, 10
ACCEPTED MANUSCRIPT 2009), and the positive effect of flight on aggressiveness was considered to be associated with the enhancement of reproductive efforts. However, in a previous study on V. aspersus, we found that a short time of flight (5 min) did not enhance the development of male accessory glands (Zeng et al., 2014). Because males often spend a great deal of energy on various reproductive
PT
expenditures besides accessory gland development, more studies are needed to test whether
RI
short flight bouts can enhance reproductive behavior in male V. aspersus. In addition, winners
SC
and flown males were less aggressive than courting males, and this finding may have two implications. First, after winning a fight or flying, males may be too exhausted to engage in a
NU
physical combat, which requires considerable energy. Second, these energy-exhausting
MA
experiences may only partially activate regulatory pathways that promote male fighting behavior, while the presence of females may fully activate these regulatory pathways.
D
OA titer increases during aggressive behavior in male G. bimaculatus (Adamo et al., 1995)
PT E
and OA depletion reduces aggression level, while injection of a specific OA receptor agonist (CDM) promotes aggressiveness of male G. bimaculatus (Stevenson et al., 2000, 2005).
CE
Drosophila mutants lacking the enzyme, tyramine-β-hydroxylase (TβH), which catalyzes the
AC
synthesis of OA from tyramine, exhibit reduced aggression, while treatment with CDM and overexpression of TβH increase the aggression levels of socially-reared flies (Zhou et al., 2008). In the ant, Oecophylla smaragdina (Fabricius), OA titer is higher in aggressive major workers than in non-aggressive minor workers, and pharmacological manipulation of OA in major and minor worker brains was shown to reverse the levels of aggression characteristic of each worker size class (Kamhi et al., 2015). These studies strongly suggest that OA plays an important role in the regulation of male fighting behaviors. In the current study, topical application of 1 μL of 11
ACCEPTED MANUSCRIPT 0.15 M CDM significantly increased the aggression level in male V. aspersus, whereas topical application of 0.1 M or 0.2 M CDM did not affect male aggressiveness, suggesting that the release of OA as a neuromodulator may mediate fighting behavior in this species, and its effect may vary depending on OA titer. This finding is consistent with observations made in G.
PT
bimaculatus, although injection of 100 μL of 1 mm CDM was the most effective dosage in
RI
promoting aggressiveness in this species (Stevenson et al., 2005). The difference may be due
SC
to the fact that in our experiment topical application may have decreased the amount of CDM that entered the body of insects.
NU
The effect of CDM topical application on aggressiveness of experienced males varied
MA
depending on the type of experience. Specifically, aggression level increased significantly when CDM-treated losers fought against each other, a phenomenon also observed in other cricket
D
species (Stevenson et al., 2005). However, topical application of CDM to winners, flown males,
PT E
or courting males suppressed their aggressiveness. Excessive CDM treatment was found to decrease aggression in male G. bimaculatus (5 mm) (Stevenson et al., 2005) and male V.
CE
aspersus (0.2 M, this study), and winning and flight increased OA titer in male G. bimaculatus
AC
(Stevenson et al., 2005; Rillich and Stevenson, 2011). It is possible that application of CDM to winners, flown males, or courting males might have resulted in over-excitation of OA neurons, leading to a decrease in aggression. However, we did not quantify OA titer in this study, and further experiments are needed to test this hypothesis. Alternatively, a receptor binding to the agonist may have affected the binding of OA to other subtypes of OA receptors. Because OA plays a role in several functions, such as locomotion and courtship (Chen et al., 2013), CDM may have bound to receptors that control flying or courting behavior when flown or courting 12
ACCEPTED MANUSCRIPT males were treated. Although OA has been extensively demonstrated to be an important regulatory factor of fighting behavior in crickets (Stevenson et al., 2000, 2005; Stevenson and Rillich, 2012), the mechanisms underlying the effects of different experiences on aggressiveness are complicated
PT
and far from being clarified. In male V. aspersus, courting males usually escalated to physical
RI
combat during fights, CDM-treated males did not exhibit physical escalation. It seems likely
SC
that fighting behavior was only partly regulated by OA, and other regulatory pathways may also be involved in controlling escalation to physical combat. For example, inhibition of the
NU
nitric oxide/cyclic guanosine 3',5'-monophosphate pathway in male G. bimaculatus induced
MA
fights to last twice as long and to escalate to significantly higher levels, often involving grappling (level 6) (Stevenson and Rillich, 2015). In addition, activation of the OA pathway
D
may be responsible for the observed enhancing effects of winning a fight or flight on
PT E
aggressiveness (Stevenson et al., 2005; Rillich and Stevenson, 2011), but the dopamine pathway is necessary to recover from the suppressive effect of losing a fight in G. bimaculatus
CE
(Rillich and Stevenson, 2014). To our knowledge, no study has compared OA titers between
AC
males exhibiting low versus high aggressiveness. Therefore, further research is needed to test whether intensity of aggressiveness is positively correlated with OA titer.
13
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the National Nature Science Foundation of China (Grant No. 31070586). The authors thank Prof. Zhiwei Liu (Eastern Illinois University, USA) for revising
PT
the manuscript.
RI
Conflict of interest statement
AC
CE
PT E
D
MA
NU
SC
The authors have no conflict of interest to declare.
14
ACCEPTED MANUSCRIPT References Adamo, S.A., Hoy, R.R., 1995. Agonistic behavior in male and female field crickets, Gryllus bimaculatus, and how behavioral context influences its expression. Anim. Behav. 49, 1491– 1501.
PT
Adamo, S.A., Linn, C.E., Hoy, R.R., 1995. The role of neurohormonal octopamine during ‘fight or flight' behaviour in the field cricket Gryllus bimaculatus. J. Exp. Biol. 198, 1691–
RI
1700.
NU
(Orthoptera: Gryllidae). Behaviour 17, 130–223.
SC
Alexander, R.D., 1961. Aggressiveness, territoriality, and sexual behavior in field crickets
Baier, A., Wittek, B., Brembs, B., 2002. Drosophila as a new model organism for the
MA
neurobiology of aggression? J. Exp. Biol. 205, 1233–1240. Bubak, A.N., Grace, J.L., Watt, M.J., Renner, K.J., Swallow, J.G., 2014. Neurochemistry as a
60, 778–790.
PT E
D
bridge between morphology and behavior: perspectives on aggression in insects. Curr. Zool.
CE
Chen, A., Ng, F., Lebestky, T., Grygoruk, A., Djapri, C., Lawal, H.O., Zaveri, H.A., Mehanzel, F., Najibi, R., Seidman, G., Murphy, N.P., Kelly, R.L., Ackerson, L.C., Maidment,
AC
N.T., Jackson, FR, Krantz, D.E., 2013. Dispensable, redundant, complementary, and cooperative roles of dopamine, octopamine, and serotonin in Drosophila melanogaster. Genetics 193, 159–176. Crespi, B.J., 1988. Adaptation, compromise, and constraint: the development, morphometrics, and behavioral basis of a fighter-flier polymorphism in male Hoplothrips karnyi (Insecta: Thysanoptera). Behav. Ecol. Sociobiol. 23, 93–104. Dyakonova, V., Krushinsky, A., 2008. Previous motor experience enhances courtship behavior 15
ACCEPTED MANUSCRIPT in male cricket Gryllus bimaculatus. J. Insect Behav. 21, 172–180. Guerra, P.A., Pollack, G.S., 2009. Flight behaviour attenuates the trade-off between flight capability and reproduction in a wing polymorphic cricket. Biol. Lett. 5, 229–231. Hofmann, H.A., Stevenson, P.A., 2000. Flight restores fight in crickets. Nature 403, 613.
PT
Hoyer, S.C., Eckart, A., Herrel, A., Zars, T., Fischer, S.A., Hardie, S.L., Heisenberg, M., 2008.
RI
Octopamine in male aggression of Drosophila. Curr. Biol. 18, 159–167.
SC
Hsu, Y., Earley, R.L., Wolf, L.L., 2006. Modulation of aggressive behaviour by fighting experience: mechanisms and contest outcomes. Biol. Rev. Cam. Philos. Soc. 81, 33–74.
NU
Kamhi, J.F., Nunn, K., Robson, S.K.A., Traniello, J.F.A., 2015. Polymorphism and division of
MA
labour in a socially complex ant: neuromodulation of aggression in the Australian weaver ant, Oecophylla smaragdina. Proc. R. Soc. B 282, 79–96.
D
Killian, K.A, Allen, JR, 2008. Mating Resets Male Cricket Aggression. J. Insect Behav. 21,
PT E
535–548.
Kortet, R., Hedrick, A., 2007. A behavioural syndrome in the field cricket Gryllus integer:
CE
intrasexual aggression is correlated with activity in a novel environment. Biol. J. Linn. Soc.
AC
91, 475–482.
Reaney, L.T., Drayton, J.M., Jennions, M.D., 2011. The role of body size and fighting experience in predicting contest behaviour in the black field cricket, Teleogryllus commodus. Behav. Ecol .Sociobiol. 65, 217–225. Rillich, J., Schildberger, K., Stevenson, P.A., 2011. Octopamine and occupancy: an aminergic mechanism for intruder-resident aggression in crickets. Proc. R. Soc. B 278, 1873–1880. Rillich, J., Stevenson, P.A., 2011. Winning fights induces hyperaggression via the action of the 16
ACCEPTED MANUSCRIPT biogenic amine octopamine in crickets. PLoS One 6, e28891 Rillich, J., Stevenson, P.A., 2014. A fighter's comeback: Dopamine is necessary for recovery of aggression after social defeat in crickets. Horm. Behav. 66, 696–704. Short, R.V., Balaban, E., 1994. The differences between the sexes. Cambridge University Press,
PT
Cambridge.
SC
bimaculatus (De Geer). Anim. Behav. 34, 567–579.
RI
Simmons, L.W., 1986. Inter-male competition and mating success in the field cricket, Gryllus
Stevenson, P.A., Dyakonova, V., Rillich, J., Schildberger, K., 2005. Octopamine and
NU
experience-dependent modulation of aggression in crickets. J. Neurosci. 25, 1431–1441.
MA
Stevenson, P.A., Hofmann, H.A., Schoch, K., Schildberger, K., 2000. The fight and flight responses of crickets depleted of biogenic amines. J. Neurobiol. 43, 107–120.
D
Stevenson, P.A., Rillich, J., 2012. The decision to fight or flee-insights into underlying
PT E
mechanism in crickets. Front. Neurosci. 6, 118. Stevenson, P.A., Rillich, J., 2013. Isolation associated aggression-a consequence of recovery
CE
from defeat in a territorial animal. PLoS One 8, e74965.
AC
Stevenson, P.A., Rillich, J., 2015. Adding up the odds-Nitric oxide signaling underlies the decision to flee and post-conflict depression of aggression. Sci. Adv. 1, e1500060. Zeng, Y., Zhu, D.H., Kang, W.N., 2016. Variation in fighting strategies in male wing-dimorphic crickets (Gryllidae). Behav. Ecol. Sociobiol. 70, 429–435. Zeng, Y., Zhu, D.H., Zhao, L.Q., 2014. Critical flight time for switch from flight to reproduction in the wing dimorphic cricket Velarifictorus aspersus. Evol. Biol. 41, 397–403. Zeng, Y., Zhu, D.H., 2012. Trade-off between flight capability and reproduction in male 17
ACCEPTED MANUSCRIPT Velarifictorus aspersus crickets. Ecol. Entomol. 37, 244–251. Zhou, C., Rao, Y., Rau, Y., 2008. A subset of octopaminergic neurons are important for
AC
CE
PT E
D
MA
NU
SC
RI
PT
Drosophila aggression. Nat. Neurosci. 11, 1059–1067.
18
ACCEPTED MANUSCRIPT Table 1 Agonistic behaviors scored in trials with male V. aspersus* Description
Mutual avoidance
Males escape from each other after contact
0
Pre-established dominance
One male attacks, the other runs
1
Antennal fencing
Males rapidly antennate each other’s antennae
2
Mandible spread (one)
One male spreads mandibles and attacks the other
3
Mandible spread (both)
Both opponents spread mandibles
Lunge and butt
Rush at the conspecific and use head to hit the opponent
5
Wrestling
Males interlock mandibles, push and wrestle with each other
6
RI
SC
CE
PT E
D
MA
NU
Modified from Zeng et al. (2016).
AC
*
Score
PT
Behavior
19
4
ACCEPTED MANUSCRIPT Figure captions: Fig. 1 Effects of different experiences on fighting behavior of male Velarifictorus asperses. A: aggression level; B: fight duration. Data are presented as median ± interquartile range, and numbers in brackets indicate the sample size. Different letters indicate significant differences
PT
between groups (Steel-Dwass test, p < 0.05).
RI
Fig. 2 Effect of OA agonist on fighting behavior of naive males. A: aggression level; B: fight
SC
duration. Data are presented as median ± interquartile range, and numbers in brackets indicate the sample size. Different letters indicate significant differences between groups
NU
(Steel-Dwass test, p < 0.05).
MA
Fig. 3 Effect of OA agonist on fighting behavior of males with fighting previous experience. A and B represent fights between winners; C and D represent fights between losers. Data are
D
presented as median ± interquartile range, and the numbers in brackets indicates the sample
PT E
size. Different letters indicate significant differences between groups (Mann-Whitney U test, p < 0.05).
CE
Fig. 4 Effect of OA agonist on fighting behavior of flown males. A: aggression level; B: fight
AC
duration. Data are presented as median ± interquartile range, and the numbers in brackets indicate the sample size. Different letters indicate significant differences between groups (Mann-Whitney U test, p < 0.05). Fig. 5 Effect of OA agonist on fighting behavior of courting males. A: aggression level; B: fight duration. Data are presented as median ± interquartile range, and numbers in brackets indicate the sample size. Different letters indicate significant differences between groups (Mann-Whitney U test, p < 0.05). 20
ACCEPTED MANUSCRIPT Highlights
PT RI SC NU MA D
PT E
CE
Effects of physical and social experiences on aggressiveness of male Velarifictorus asperses were examined. Presence of females, flight, winning a fight enhanced the aggressiveness, but losing a fight suppressed aggressiveness. Aggressiveness was strongest in the presence of a female. Treatment with CDM (OA receptor agonist) resulted in inhibiting the “loser effect” and decreases aggression for winners.
AC
21
Graphics Abstract
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5