Seasonal variation in ornament expression,body size,energetic reserves,immune response,and survival in males of a territorial insect

Abstract 1. Seasonal variation in immune response has rarely been investigated in invertebrates and, therefore, we have studied this using territorial adult males of the damselfly, Hetaerina americana (Fabricius), in several generations, for a year and a half. 2. We investigated and related seasonal variation in red pigmented wing spot size (an ornamental trait) and body size to fat reserves using four immunity components – melanisation ability, phenoloxidase (PO) and nitric oxide (NO) activity, and haemolymph protein concentration – and survival ability after a bacterial immune challenge. 3. There was seasonal variation in spot expression, being more intense in the non‐winter months and less intense in winter months, and, to some extent, a similar pattern was found for NO. Although there was also variation in melanisation, PO activity, protein concentration and fat reserves, this was not consistently related to variation in spot size. Survival was lower in the winter than in non‐winter months. 4. Animals with larger spot and body size had greater values of melanisation, PO and NO activity, and protein concentration. The relation of spot and body size with fat reserves is still not clear. 5. Unlike other studies, ornament was not similarly correlated with all typical immune components (at best, mainly NO). 6. Similar to what occurs in vertebrates, survival is lower during the winter months. In the case of vertebrate studies, however, the same individual suffers these changes. In the American rubyspot, distinct cohorts demonstrated seasonal differences.     

To be or not to be? Mating success and survival trade‐offs when switching between alternative reproductive tactics

Hormones underlie the decision of assuming a territorial or a nonterritorial role, with territorial individuals usually having higher hormonal levels than nonterritorial individuals. As a territorial status is linked to higher mating opportunities, it is unclear why animals do not keep high hormonal levels and one explanation is that this would imply survival costs. We have tested this using males of the territorial damselfly Argia emma in the field. We increased juvenile hormone (JH) levels using methoprene in both territorial and nonterritorial males and predicted that: (i) males will keep (the case of territorial males) or become (the case of nonterritorial males) territorial after hormonal increase, and (ii) there will be an increase in mating success for nonterritorial males only and an impaired survival for both male tactics. Hormonally treated males remained or became territorial but had their survival impaired compared with control groups. Also, hormonally treated, ex‐nonterritorial males increased their mating success compared with the other control, nonterritorial males. The reduced survival can be explained proximally by the energy devoted either to the enhanced aggression showed during territory defence or immune function (as detected previously in damselflies). Although nonterritorial males may increase their mating success by switching to a territorial tactic, they are possibly unable to do it naturally as JH is dietary dependent and usually nonterritorial animals are in poorer condition than territorial animals.     

Immune system activation interacts with territory-holding potential and increases predation of the damselfly Calopteryx splendens by birds

Activation of the immune system in insects has been shown to be costly in the laboratory setting, but experimental studies in the field are lacking. The costs of immunity in the wild may be different to those in the laboratory because animals in the wild are simultaneously subjected to a suite of selective agents. We have measured the costs of immune system activation in a wild population of the territorial damselfly Calopteryx splendens. Immune-challenged males were found to be less likely to be territorial and had lower overall survival rates than control or sham-manipulated males. Because territorial males have a higher mating success than nonterritorial males, this result suggests that immune-challenged males are also likely to suffer reduced mating success. However, the activation of the immune system as such did not increase predation risk; this occurred due to a combination of the former with a reduced territory-holding potential. As such, immune-challenged males not holding a territory were most susceptible to predation by birds. The size of the wing spots, a known sexually selected male trait, predicted territorial behaviour in control and sham-manipulated males, but not in immune-challenged males. Our data show that immune system activation can have several costs acting in unison and that ubiquitous ecological interactions, such as predation, may affect trade-offs between immunity and other life history traits.     

Short-term changes in plasma levels of hormones during establishment and defense of a breeding territory in male song sparrows, Melospiza melodia

When territorial male song sparrows are captured and removed from their territories, previously unmated and nonterritorial males will take over those vacant territories within 12-72 hr. Plasma levels of testosterone are elevated in these replacement males as well as in their neighbors. Since the latter already have territories, it is suggested that the agonistic interactions over territory boundaries, or behavioral stimuli from challenging males, rather than ownership of a territory per se, stimulates secretion of testosterone. To test this hypothesis further, male song sparrows were challenged by experimental simulation of a territorial intrusion. This procedure involved placing a caged male song sparrow in the center of the subject's territory and playing tape recorded conspecific song through an adjacent speaker. Responding males were then captured at intervals after onset of the intrusion. Plasma levels of testosterone were significantly higher in males exposed to experimental territorial intrusion than they were in controls, supporting the hypothesis that behavioral stimuli emanating from an intruding male can act as supplementary information stimulating secretion of testosterone.     

Testis-testicular gland complex of two Tripterygion species (Blennioidei, Teleostei): differences between territorial and non-territorial males

The morphological differences between the testis and testicular gland of territorial and nonterritorial males of Tripterygion tripteronotus and T. delaisi were examined and correlated with differences in reproductive behaviour. In territorial males of both species the testicular gland is much more developed than in non-territorial males. Larger cellular and nuclear sizes in the territorial males indicate that the activity of the gland cells is enhanced. These cells contain SER, numerous lipid droplets and mitochondria with lamellar cristae. Absence of 3β-HSD activity at these sites points to lack of a steroidogenic potency. In both territorial and non-territorial fish, steroid-producing Leydig cells have been demonstrated in the connective tissue betweeen the testis and the testicular gland, and around the collecting sperm duct. In addition, 3β-HSD activity has been found in the scarce interstitial Leydig cells of territorial fish. Morphometric data indicate an enhanced activity of the Leydig cells in territorial fish.     

Sex differences in insect immune function: a consequence of diet choice?

Males often have reduced immune function compared to females but the proximate mechanisms underlying this taxonomically widespread pattern are unclear. Because immune function is resource-dependent and sexes may have different nutritional requirements, we hypothesized that sexual dimorphism in immune function may arise from differential nutrient intake (acquisition hypothesis). To test this hypothesis, we examined patterns of phenoloxidase (PO) activity in relation to nutrient consumption in Queensland fruit flies (Q-flies). In the first experiment, flies were allowed to choose their preferred nutrient intake. Compared with males, female Q-flies had higher PO activity, consumed more calories, and preferred a higher protein:carbohydrate (P:C) diet, suggesting that differential acquisition could explain sex differences. In the second experiment, we restricted flies to one of 12 diets varying in protein and carbohydrate concentrations and mapped PO activity for each sex onto a nutritional landscape. Counter to our hypothesis, females had higher PO activity than males at any given level of nutrient intake. Both carbohydrate and protein intake affected PO activity in females but only protein affected PO activity in males. Our results indicate that sex differences in Q-fly immune function are not solely explained by sex differences in nutrient intake, although nutrition does contribute to the magnitude of these sex differences.     

Interactions between population processes in a cyclic species: parasites reduce autumn territorial behaviour of male red grouse

The causes of population cycles fascinate and perplex ecologist. Most work have focused on single processes, whether extrinsic or intrinsic, more rarely on how different processes might interact to cause or mould the unstable population dynamics. In red grouse (Lagopus lagopus scoticus), two causal mechanisms have been supported: territorial behaviour (changes in autumn aggressiveness) and parasites (parasite induced reduction in fecundity). Here, we report on how these two regulatory processes might interact, by testing whether the parasite suspected to cause the grouse cycles, the nematode Trichostrongylus tenuis, reduces male autumn territorial behaviour. We either treated males with an anthelmintic, to remove parasites (dosed or D-males), or challenged them with infective T. tenuis larvae, to increase parasite intensity (challenged or C-males). We first show that dosing was effective in removing T. tenuis parasites, while parasite intensities increased in challenged birds during the autumn. Because old males initially had more parasites than young males, the treatments generated greater differences in parasite intensity in old than in young males. We also show that various aspects of territorial behaviour (increase in testosterone-dependent comb size in autumn, territorial call rate, likelihood of winning territorial interactions and over-winter survival) were significantly higher in dosed than in challenged males, but in old birds only. Our data thus supported the hypothesis that parasites reduce male aggressiveness during the autumn territorial contests, and could thereby influence recruitment. Our results also highlight that the territorial behaviour of young males, which have fewer parasites, is not as limited by parasites as that of old, previously territorial males. We discuss the implications of these findings for our understanding of the processes regulating red grouse populations and causing their complex, unstable population dynamics.     

Territoriality and testosterone in an equatorial population of rufous-collared sparrows, Zonotrichia capensis

Territorial aggression, displayed by male vertebrates in a reproductive context, is generally thought to be mediated by testosterone. The challenge hypothesis predicts that in socially monogamous species, territorial challenges should induce an increase in plasma testosterone concentrations, which will enhance aggressive behaviour and territory defence. This hypothesis is based on northern latitude birds and needs to be tested in tropical birds before it can be universally accepted. We tested the challenge hypothesis in an equatorial population of rufous-collared sparrows in Papallacta, Ecuador. This population shows an extended breeding period during which males aggressively guard territories. During the early breeding season, males were challenged with conspecific or heterospecific simulated territorial intrusions (STIs) lasting 10 min. Conspecific-challenged males responded more aggressively than heterospecific-challenged males. However, there was no increase in plasma testosterone in response to the conspecific STI. During the breeding season, males were challenged with conspecific STIs lasting 0, 10 or 30 min. Males behaved aggressively regardless of STI duration, and did not differ in plasma testosterone or luteinizing hormone concentrations. During the breeding season, males were implanted with testosterone-filled or empty silastic tubes and subsequently challenged with a conspecific STI. Testosterone implants significantly raised plasma testosterone concentrations, but testosterone-implanted males were not more aggressive than blank-implanted controls. Combined, these findings suggest that testosterone concentrations above breeding baseline are not related to territorial aggression in this population and therefore do not support the challenge hypothesis.     

Castration Lowers Aggression but not Social Dominance in Male Haplochromis burtoni (Cichlidae)

Adult males of the African cichlid fish Haplochromis burtoni are either territorial or nonterritorial. In nature and aquaria, only territorial males breed and they have markedly more developed testes than do nonterritorial males. Territorial males are clearly dominant over nonterritorial males of the same age and size, and also exhibit higher levels of aggression. In this study, territorial males were gonadectomized in order to assess the effects of lowered androgen levels on their aggression and dominance status. Completely castrated males showed a pronounced reduction in both testosterone and 11-ketotestosterone, as well as significantly lower aggression scores than sham-operated males. Partially castrated males (> 0.005 g gonadal tissue remaining at autopsy) exhibited intermediate levels of both androgen levels and aggression. Surprisingly, in dyadic encounters with normal nonterritorial males of the same size, castrated territorial males retained their dominance despite decreased androgen levels and aggression.     

Effect of juvenile hormone on senescence in males with terminal investment

Senescence, a decline in survival and reproductive prospects with age, is controlled by hormones. In insects, juvenile hormone (JH) is involved in senescence with captive individuals, but its effect under natural conditions is unknown. We have addressed this gap by increasing JH levels in young and old wild males of the damselfly Hetaerina americana. We assessed survival in males that were treated with a JH analogue (methoprene), which is known to promote sexual activity, and an immune challenge, which is known to promote terminal investment in reproduction in the studied species. We replicated the same procedure in captivity (to control for environmental variation), where males were deprived of any activity or food. We expected old males to show the lowest survival after being treated with JH and immune‐challenged, because the effect of terminal investment on senescence would be exacerbated by JH. However, this should be the case for wild animals, but not for captive animals, as the effects of JH and immune challenge should lead to an increase in high energetic‐demanding activities only occurring in the wild. Old animals died sooner compared with young animals in both the wild and captivity, confirming that males are subject to senescence. In wild but not captive animals, JH decreased survival in young males and increased it in old males, confirming that JH is sensitive to the environment when shaping animal senescence. Immune challenge had no effect on survival, suggesting no effect of terminal investment on senescence. Additionally, contrary to the expected effects of terminal investment, with an immune challenge, recapture rates increased in young males and decreased in old males. Our results show that male senescence in the wild is mediated by JH and that terminal investment does not cause senescence. One explanation is that animals undergoing senescence and terminal investment modify their feeding behaviour to compensate for their physiological state.     

The Challenge Hypothesis and Seasonal Changes in Aggression and Steroids in Male Northern Fence Lizards (Sceloporus undulatus hyacinthinus)

The challenge hypothesis has been very successful in explaining patterns of testosterone secretion in response to social stimuli in avian species. However, there have been few studies in nonavian vertebrates. We tested the challenge hypothesis in male northern fence lizards (Sceloporus undulatus hyacinthinus). These males are highly territorial and nonparental. Consequently, the challenge hypothesis predicts that plasma testosterone concentrations will be insensitive to aggressive interactions. Testosterone concentrations indeed were not significantly affected by either a short (3–15 min) simulated territorial intrusion (“challenge”) in June or a longer (50–60 min) intrusion in July. Levels of corticosterone were elevated in challenged males in the long, but not the short, intrusion. Challenged males displayed significantly more intense territorial behaviors than did unchallenged control males. The intensity of territorial behavior changed significantly across the active season and was positively related to testosterone concentrations. Thus, while testosterone concentrations do not appear to be involved in rapid changes in aggressive behavior in the fence lizard, they probably are important in larger-scale behavioral changes throughout the season.     

Spatial and temporal population differences in male density and condition in the American rubyspot, Hetaerina americana (Insecta: Calopterygidae)

Increased resource availability should favor higher animal density. It may also affect sex ratio, the male condition, and mating competition over access to females, although the direction of these variables is not straightforward to predict. Using a non-experimental approach, we carried out preliminary research using the territorial American rubyspot (Hetaerina americana) by comparing two spatially separated populations and the same population in two different seasons (each comparison with varying population densities). We first compared the sex ratio by counting males (using two categories, territorial and non-territorial) and females; relative foraging time (as an indicator of how much feeding resources each site provides); wing spot size (a sexual ornament), body size and immune melanization response (these two variables were used to assess male condition); and fighting time and contest number (to assess competition). For the seasonal comparison we used a third population in which we only compared male spot size and two indicators of condition and immune response, phenoloxidase (PO) and nitric oxide (NO) activity. The high-density population had higher values of non-territorial males (but similar sex ratio), relative foraging time, contest time and number, wing spot (but similar body size) and melanization response than the low-density population. According to season, at high density, males had higher values of wing spots, PO, and NO. Our results suggest that in a population where animals have more dietary resources, males reach a better condition despite the competition being more intense.     

Manipulations of the AVT system shift social status and related courtship and aggressive behavior in the bluehead wrasse

Arginine vasotocin (AVT) and its mammalian homologoue arginine vasopressin (AVP) influence male sexual and aggressive behaviors in many species. We tested the effects of AVT and an AVP-V(1a) receptor antagonist on the display of alternative male tactics in a tropical coral reef fish, the bluehead wrasse Thalassoma bifasciatum. We gave AVT injections to territorial and nonterritorial males of the large and colorful phenotype (terminal phase) and an AVP-V(1a) receptor antagonist, Manning compound, to territorial males in the field. AVT increased courtship independent of status, while its effects on territoriality and aggression were dependent upon male status. In territorial males, AVT increased courtship and tended to decrease the number of chases toward initial phase individuals. In nonterritorial males, AVT increased courtship, chases toward initial phase individuals, and territorial behavior while decreasing feeding. These are all behaviors rarely seen in nonterritorial males, so AVT made these males act like territorial TP males. The AVP-V(1a) receptor antagonist had opposite effects. It decreased courtship and territorial defense, making these males act more like nonterritorial males. Manipulations of the AVT system shifted males within a single phenotype from the nonterritorial social status to the territorial social status and vice versa. Since the entire suite of behaviors related to territoriality was affected by AVT system manipulations, our results suggest that the AVT system may play a key role in motivation of behaviors related to mating.     

Territorial damselflies do not show immunological priming in the wild

Adaptive immunity allows vertebrates to gain protection against repeated pathogenic infections. Analogous responses (priming) have been recently uncovered in invertebrates. However, whether such responses are widespread is not known. The present study investigated the presence of immunological priming in males of a species whose phylogenetic position places it in one of the less derived insect orders. It is hypothesized that the efficiency of such a response could be related to animal condition, as assessed by the expression of a sexually selected ornament. Hetaerina americana Fabricius (Odonata: Calopterygidae) males bear a conspicuous ornament (a red wing spot), which is evolutionarily maintained via male territorial competition. Using field‐collected animals, a group of males is challenged with bacteria before exposure to a higher dose of the same or a different bacteria, and survival is compared with that of infected males not previously challenged, as well as control groups. Gram‐positive and Gram‐negative bacteria are used. To explore how long priming may take to work, the second exposure is carried out either after 1 or 5 days. Red spot and body size are entered in the analysis as predictors of survival within and between groups. There is no difference in survival among groups, which suggests no priming effect. Overall, red spot and body size are not consistent in explaining survival.     

Wing Colour Properties do not Reflect Male Condition in the American Rubyspot (Hetaerina americana)

Adult males of the American rubyspot ( Hetaerina americana ) dispute riverine territories where females arrive to mate. On the wing basis, these males bear a red pigmentation spot whose area correlates with territorial disputes and mating rate: males with larger spots are more successful. This is explained by the fact that spot size correlates with fat muscular reserves which fuel flight during territorial intrusions. To further our understanding of sexual selection acting on the spot, here we have examined possible differences in three spot colour properties (red chroma, hue and brightness) in three distinct adult male ages [young, middle-aged (when males are more likely to defend a territory) and old], social status (territorial and non-territorial in middle-aged males), and under two potentially, energetically and costly situations: when faced with an immune challenge [comparing a nylon-implanted male group vs. a non-implanted male group in two ages, teneral (previous to colour formation) and middle-aged] and low diet levels (comparing a male set of middle-aged animals that received food ad libitum vs. a male set that received no food). Our results indicate no change in colour properties across any of these comparisons. Taken together, these and previous results suggest that only spot size but not the spot characteristics we measured here, is sexually selected in males of this species at least in terms of pre-copulatory male–male competition. That some of these colour properties have been related to male condition in other calopterygid damselflies cannot be generalized to the American rubyspot.     

Immunity and reproduction during colony foundation in the dampwood termite, Zootermopsis angusticollis

Abstract.  Termite primary reproductives may be exposed to pathogens when dispersing from their parental nest and establishing a new colony. Immunity and reproduction are investigated during colony foundation by implanting a nylon filament into the abdomen of mated and unmated female and male primary reproductives of the dampwood termite Zootermopsis angusticollis. Primary reproductives are paired in combinations of female/male, female/female and male/male and, using confocal microscopy, immune defence is assessed by measuring the degree of encapsulation of nylon implants during three periods of colony foundation: (I) shortly after pairing; (II) during copulation/oocyte maturation; and (III) during oviposition. There are differences in the encapsulation response of mated and unmated termites that are contingent on the period of colony foundation when termites are challenged. Mated females and males have significantly greater encapsulation responses than their unmated counterparts shortly after pairing, perhaps as a prophylactic measure against exposure to disease. The encapsulation response of mated and unmated males does not differ significantly during periods II and III. The onset of oviposition is significantly delayed in mated females that received implants during periods I and II. Mated females have a significantly reduced encapsulation response during the time of copulation and oocyte maturation, but not during oviposition. Overall, males have a significantly greater ability than females to encapsulate a nylon implant. The findings suggest that reproduction can reduce the immune response in female primary reproductives. The results are discussed in light of trade-offs between immunity and reproduction during the critical life-history phase of colony establishment in termites.     

Androgen control of social status in males of a wild population of stoplight parrotfish, Sparisoma viride (Scaridae)

In the protogynous stoplight parrotfish (Sparisoma viride), large males defend territories that encompass the home-ranges of several mature females. However, high-quality habitat is in short supply, such that smaller, competitively inferior males do not defend territories. We investigated the role of 11-ketotestosterone (11KT) and testosterone (T) in the regulation of territorial behavior in a wild population of a protogynous reef fish, the stoplight parrotfish, at Glover's Reef, Belize. Radioimmunoassay of plasma samples from individuals of known social status revealed that nonterritorial males have lower levels of T and 11KT than territorial males. Nonterritorial males allowed access to vacant territories underwent pronounced increases in T and 11KT. When sampled 1 week after territory acquisition, levels of T and 11KT in new territorial males were significantly higher than the levels in established territorial males, but by 3 weeks after territory acquisition, there was no significant difference. We further investigated the hypothesis that such short-term increases in androgen levels are a response to intense male-male interactions during territory establishment. Simulated territorial intrusion promoted increased plasma levels of both T and 11KT while access to vacant territories without neighboring territorial males did not. These findings suggest that the endocrine system plays a role in fine-tuning the levels of territorial aggression exhibited by male stoplight parrotfish. We discuss these results in light of recent theory in behavioral endocrinology.     

Wing pigmentation, immune ability, fat reserves and territorial status in males of the rubyspot damselfly, Hetaerina americana

An explanation for courting traits is that they convey information about the bearers condition to conspecifics, more specifically immune ability. Here we test a series of immune-based assumptions in the territorial damselfly Hetaerina americana, whose males bear wing pigmentation patterns, which are maintained via male–male competition. H. americana males emerge and take some time to mature sexually, after which, depending on their fat reserves, may start defending territories where females arrive at for copulation. Territorial males are eventually defeated and lose their territories. This loss is a consequence of a reduction in muscular fat reserves. We tested whether: (a) territorial males had more pigmented wings, more intense melanine-based immune response (encapsulation response to a nylon filament implant) and higher fat reserves than non-territorial males; (b) pigmentation is related to immunity and fat reserves; (c) the immune response held constant in two different episodes (3 days between each) in the same male during territorial tenure; and (d) immune response and fat reserves decreased after experimentally simulated fighting event. Our results agree with current views of immune ability and courting traits: (1) territorial males had more wing pigmentation, higher immune responses and fat reserves than non-territorial males; (2) pigmentation was also correlated with immunity and fat reserves; and (3) immune response was similarly intense in the two episodes during territorial tenure. However, this response and fat reserves were considerably lower after fighting compared to that of territorial males and non-territorial males. Our work points out a link between fat reserves and immune ability which agree with previous studies in insects. Given, however, that in this species the use of wing pigmentation via male–male competition is more likely to provide information about current fat reserves than immunity, it is suggested that immune ability is only indirectly selected and may not be the information that pigmentation would convey to conspecifics.     

Parasitism and alternative reproductive tactics in Northern chamois

Alternative reproductive tactics (ARTs), discrete phenotypic variations evolved to maximize fitness, may entail different cost‐benefit trade‐offs. In large mammals, differences in costs associated with ARTs—including energy expenditure and parasite infection—are typically greatest during the breeding season. Nonetheless, physiological and behavioral differences between ARTs can manifest throughout the year, possibly involving costs that may contribute to maintain ARTs within populations. Using the number of nematode larvae per gram of feces (LPG) as a proxy, we explored the temporal changes in lung parasite infection in territorial and nonterritorial male chamois Rupicapra in the Gran Paradiso National Park (Italy), between 2011 and 2012. We aimed to identify which tactic‐specific physiological and behavioral features (including age, hormonal levels, inter‐ and intrasexual interactions, and space use) or climatic factors (temperature and precipitation) best explained yearly variation in parasite infection within and between ARTs. Generalized additive mixed models showed that the fecal larval counts of lung nematodes underwent strong temporal changes in both male types. Differences between ARTs (with higher LPG values in territorial than nonterritorial males) were greatest during the rut and—to a lesser extent—in spring, respectively, at the peak and at the onset of territoriality. The difference in LPG between tactics was largely explained by the greater levels of hormone metabolites in territorial males during the rut. The other variables did not contribute significantly to explain the different shedding of larvae within and between ARTs. Our analysis suggests that different values of LPG between territorial and nonterritorial males are largely a result of tactic‐specific differences in the secretion of hormone metabolites, but only during the rut. To clarify whether rut‐related parasitism contributes to the maintenance of ARTs, tactic‐specific life history trade‐offs, for example, between reproduction and parasite‐related mortality, must be investigated.     

A Mismatch between the Perceived Fighting Signal and Fighting Ability Reveals Survival and Physiological Costs for Bearers

Signals of fighting indicate an animal''s intention to attack and so they serve to prevent costly aggressive encounters. However, according to theory, a signal that is different in design (i.e. a novel signal) but that fails to inform fighting intentions will result in negative fitness consequences for the bearer. In the present study we used males of the territorial damselfly Hetaerina americana, which have a red wing spot during territory defense that has evolved as a signal of fighting ability. By producing a novel signal (covering the red spot with blue ink) in territory owners, we investigated: a) the behavioral responses by conspecific males; b) survival cost and c) three physiological mediators of impaired survival: muscular fat reserves, muscle mass and immune ability. We predicted that males with the novel signal would be attacked more often by conspecifics as the former would fail to convey fighting ability and intentions adequately. This will result in lower survival and physiological condition for the novel signal bearers. We found that, compared to control males (males whose red spot was not changed), experimental males had reduced survival, were less able to hold a territory, and had a reduced muscle mass. It seems that spot modified males were not able to effectively communicate their territory tenancy, which may explain why they lost their defended sites. Our results provide support for theoretical models that a novel signal that fails to informing fighting ability may lead to a fitness cost for bearers.