Black yeast symbionts compromise the efficiency of antibiotic defenses in fungus-growing ants

Multiplayer symbioses are common in nature, but our understanding of the ecological dynamics occurring in complex symbioses is limited. The tripartite mutualism between fungus-growing ants, their fungal cultivars, and antibiotic-producing bacteria exemplifies symbiotic complexity. Here we reveal how black yeasts, newly described symbionts of the ant-microbe system, compromise the efficiency of bacteria-derived antibiotic defense in fungus-growing ants. We found that symbiotic black yeasts acquire nutrients from the ants' bacterial mutualist, and suppress bacterial growth. Experimental manipulation of ant colonies and their symbionts shows that ants infected with black yeasts are significantly less effective at defending their fungus garden from Escovopsis, a prevalent and specialized pathogen. The reduction of mutualistic bacterial biomass on ants, likely caused by black yeast symbionts, apparently reduces the quantity of antibiotics available to inhibit the garden pathogen. Success of the ant-fungal mutualism is directly dependent on fungus garden health. Thus our finding that black yeasts compromise the ants' ability to deal with the garden parasite indicates that it is an integral component of the symbiosis. This is further evidence that a full understanding of symbiotic associations requires examining the direct and indirect interactions of symbionts in their ecological community context.     

Patterns of paternity skew in Formica ants

Reproductive skew among cooperatively breeding animals has recently attracted considerable interest. In social insects reproductive skew has been studied in females but not in males. However, cooperative breeding of males occurs when two males mate with the same queen and father offspring. Here we present the first analysis of comparative data on paternity skew in ants. We show that, across seven species of Formica ants, the average skew in paternity among worker offspring of doubly mated queens is negatively correlated with the population-wide frequency of multiple (mostly double) mating. We also demonstrate that this trend is relatively robust in additional analyses taking phylogenetic relationships between species into account. The observed trend is opposite to the one normally found in non-social insects with second-male precedence through sperm displacement, but agrees with predictions based on queen-male conflict over sperm allocation as a consequence of facultative, worker controlled, sex allocation – an interpretation which assumes first-male precedence. However, alternative (but not mutually exclusive) explanations are possible and further studies will be needed to discriminate between these alternatives. Received: 16 May 1997 / Accepted after revision: 26 September 1997     

Mating frequency, colony size, polyethism and sex ratio in fungus-growing ants (Attini)

The mating frequency of queens was estimated for eight attine ant species, Myrmicocrypta ednaella, Apterostigma mayri, Cyphomyrmex costatus, C. rimosus (four lower attines), Trachymyrmex isthmicus, Serico-myrmex amabalis, Acromyrmex octospinosus and Atta colombica (four higher attines), and correlated to colony size, worker polyethism, and sex ratio. Mating frequency was calculated from within-colony relatedness estimated by CAP-PCR DNA fingerprinting. Most queens of lower attines and T. isthmicus mated with only one male, while those of the three higher attines mated with multiple males. Mating frequency was positively correlated with colony size. Polyethism among workers was dependent on worker age in lower attines but on body size in higher attines, suggesting some correlation between mating frequency (i.e., within-colony gene diversity) and caste complexity. The sex ratio was biased toward females in species where the mating frequency equaled one, but toward males in species where the mating frequency was greater than two. Changing in nest site from ground surface to deep underground may have facilitated the evolution of large colony size in Attini, and this may have resulted in the evolution of polyandry (a queen mates with multiple males). With the evolution of polyandry in higher attines, Atta and Acromyrmex in particular have generated high genetic diversity within their colonies and complex social structures. Received: 26 October 1999 / Revised: 25 May 2000 / Accepted: 24 June 2000     

Patterns of courtship behavior and ejaculate characteristics in male red-winged blackbirds

Sperm competition in birds is likely to have important effects on the behavior and physiology of reproduction in both sexes. For males, such competition should select for large sperm reserves and behavioral adjustment of copulation when reserves are low. We investigated both these possibilities in free-living red-winged blackbirds (Agelaius phoeniceus), a highly polygynous species with apparently strong sperm competition. We found that the recent copulatory behavior of males did not affect the propensity to copulate with a model female. Ejaculates collected from individual males at 1-h intervals showed no evidence of sperm depletion, yet repeated ejaculates collected less than 10 min apart did. Male ejaculate size was significantly larger if it was the first one of the day (i.e., after an overnight rest). The average ejaculate size was 12.5 (±12.5 SD) million sperm. Males captured during the breeding season had an average of 111.7 (±52.8) million sperm stored in their seminal glomera. Because males average a peak copulation rate of six per female per day, in one day a male might utilize all the sperm in his seminal glomera if more than two females on his territory are fertilizable. We hypothesize that polygyny and sperm competition in this species have combined to select for rapid replenishment of the seminal glomera throughout the day, in contrast to other species that have been studied. Testis size and sperm reserves of male red-winged blackbirds are intermediate between monogamous species and species with intense sperm competition. Several possible explanations for this are discussed. Received: 21 October 1997 / Accepted after revision: 15 February 1998     

Male competition in Cardiocondyla ants

The two types of males in the ant genus Cardiocondyla differ remarkably in morphology and behavior. Ergatoid males are wingless fighters whose spermatogenesis continues throughout their entire adult lives and which therefore have an “unlimited” sperm supply. They attempt to kill all eclosing ergatoid rivals and thus to increase their share in copulations with the virgin queens reared in their nests. Winged males, on the other hand, are docile and emigrate from the nests a few days after eclosion, probably to mate with queens from other colonies. By this time, their testes have fully degenerated and all sperm is stored in the seminal vesicles. Before emigration, winged males may mate with virgin queens in their maternal nests, but they are nevertheless rarely attacked by ergatoid males. In the laboratory, the life expectancy of ergatoid males is only slightly higher than that of winged males, but because of the emigration of the latter the difference is likely to be more pronounced in the field. Both male morphs are capable of inseminating more than 35 virgin queens. However, winged males older than 14 days mate less often than ergatoid males of similar age, probably due to sperm depletion in later life. The spermathecae of queens inseminated by ergatoid males contained significantly more sperm than those of queens which mated with winged males. We discuss the evolution of intranidal mating and male polymorphism in ants. Received: 8 August 1997 / Accepted after revision: 6 October 1997     

Policing in queenless ponerine ants

Potential reproductive conflicts are common in insect societies. One process that can reduce or suppress conflict is policing. We review worker and "queen" policing in queenless ponerine ants. Queenless ants are an important model system for the study of intracolony reproductive conflict. Policing is widespread in queenless ants because workers are totipotent, so that additional potential conflicts occur in comparison to species where workers cannot mate, and these additional conflicts are frequently reduced by policing. Policing is more diverse than suggested by the examples known in other social insects. In almost all species of social Hymenoptera it can include preventing workers from reproducing by killing worker-laid eggs, but in queenless ants it can additionally include immobilisation or mutilation of workers attempting to reproduce by replacing the gamergate (i.e. mated worker with a queen-like role) or by becoming an extra gamergate. Policing by both workers and by the gamergate are important. Policing can be facultative. Depending on the age of the gamergate, workers can prevent her replacement by immobilising challenging workers or they can favour replacement by immobilising the gamergate. The initial definition of policing was inspired by species in which workers retain ovaries but cannot mate. We broaden the definition to include species, such as queenless ants, where females are totipotent, thereby including not only conflict over male production but also over gamergate replacement and gamergate number. Finally, we compare policing with punishment and dominance hierarchy. Policing is not always punishment and it does not necessarily entail dominance relationships.     

Genetic evidence of multiple parentage in broods of cliff swallows

Summary Parental exclusion analyses based on allozyme data were performed on 105 families of cliff swallows (Hirundo pyrrhonota) from southwestern Nebraska, USA. The protein products of seve polymorphic loci were resolved from blood, and at least one parental exclusion occurred at six of these loci. One or both putative parents were excluded for 35 nestlings from 22 different families. The mean number of non-kin nestlings in these families was 1.59. Non-kin nestlings were found in families with brood sizes ranging from two to five. A greater percentage of families in an 1100-nest colony had non-kin offspring than in two smaller colonics, although the difference was not statistically significant. Application of genetic models to these data and the observed distribution of parental exclusions suggested that multiple parentage in cliff swallows results more often from intraspecific brood parasitism than from forced extra-pair copulations. Based on the calculated probabilities of detecting non-kin, we estimate that 23.7% of all nestlings in our population are not the offspring of one or both of their putative parents. We estimate that about 43% of all cliff swallow nests in Nebraska contain at least one offspring resulting from intraspecific brood parasitism, and that about 6% of nests might contain offspring resulting from extra-pair copulations.     

Worker senescence and the sociobiology of aging in ants

Senescence, the decline in physiological and behavioral function with increasing age, has been the focus of significant theoretical and empirical research in a broad array of animal taxa. Preeminent among invertebrate social models of aging are ants, a diverse and ecologically dominant clade of eusocial insects characterized by reproductive and sterile phenotypes. In this review, we critically examine selection for worker life span in ants and discuss the relationship between functional senescence, longevity, task performance, and colony fitness. We did not find strong or consistent support for the hypothesis that demographic senescence in ants is programmed, or its corollary prediction that workers that do not experience extrinsic mortality die at an age approximating their life span in nature. We present seven hypotheses concerning how selection could favor extended worker life span through its positive relationship to colony size and predict that large colony size, under some conditions, should confer multiple and significant fitness advantages. Fitness benefits derived from long worker life span could be mediated by increased resource acquisition, efficient division of labor, accuracy of collective decision-making, enhanced allomaternal care and colony defense, lower infection risk, and decreased energetic costs of workforce maintenance. We suggest future avenues of research to examine the evolution of worker life span and its relationship to colony fitness and conclude that an innovative fusion of sociobiology, senescence theory, and mechanistic studies of aging can improve our understanding of the adaptive nature of worker life span in ants.     

Colonial recognition of fungus in the fungus-growing ant Acromyrmex subterraneus subterraneus (Hymenoptera: Formicidae)

Summary. Leaf cutting ants live in symbiosis with a basidiomycete fungus that is exploited as a source of nutrients for the ant larvae. Tests of fungus transport demonstrated that Acromyrmex subterraneus subterraneus workers discriminate concolonial fungus from alien fungus, and rejected the latter. Larvae and pupae of the ant were used as controls. Chemical analysis of the fungus revealed a great similarity between its hydrocarbon profile and that found on the ant brood. Experiments with lures showed that chemical extracts from the fungus are responsible for this discrimination process. Moreover, the presence of brood inside the fungus seemed to be important for discrimination of the fungus by workers. Resident workers accepted concolonial broodless fungus less than concolonial fungus inoculated with brood odor. Fungus seems to acquire colonial odor passively, simply by contact with the brood. The impact of fungus volume present in the nest on closure of the colony is discussed. We show here for the first time the importance of a symbiotic vegetal organism in colonial recognition in social insects. Received 14 April 2000; accepted 29 September 2000     

Genetic parentage in the indigo bunting: a study using DNA fingerprinting

Summary Parentage of nestlings in a North Carolina population of indigo buntings (Passerina cyanea) was analyzed using DNA fingerprinting. Three minisatellite DNA probes (wild type M13, Jeffreys' 33.15 and 33.6) were used to analyze nuclear DNA isolated from muscle biopsies of 63 nestlings, their parents, and other local adults. Each probe detected approximately 15 scorable fragments per individual, with 18%–39% overlap between probes. The proportion of bands shared (using all fragments over all three probes) between presumably unrelated adults averaged 0.23. Of the 63 offspring analyzed, 35 had at least one fragment not present in either putative parent. The distribution of offspring with novel fragments was distinctly bimodal. The lower mode (offspring with 0, 1, or 2 novel fragments, N=41) fit a Poisson distribution, a pattern expected if mutation (estimated rate per fragment= 0.01) were the source of the novel fragments. The remaining 22 offspring had more novel fragments than could be explained by mutation alone (minimum of four independent fragments across all three probes, =8.2). A low band-sharing proportion with the resident male ( =0.24) and high band-sharing with the resident female ( =0.60) implicated extra-pair fertilizations as the source of all 22. Thus in this sample, 35% of all nestlings came from extra-pair fertilizations and none from intra-specific brood parasitism. Of 25 broods sampled, 12 (48%) had at least one excluded offspring. In 3 broods all of the offspring excluded the resident male. Band-sharing proportions between excluded nestlings within a brood could not distinguish between single and multiple extra-pair paternity. Although young males tended to be excluded less often than older males, wing length and weight were not associated with the frequency of exclusion. Weight and wing length of females also were not associated with involvement in EPCs. Six of the 22 excluded offspring (in 3 broods) shared a large proportion of bands and had fewer than four novel fragments when compared to the fingerprints of a neighboring territorial male, implicating those males as actual fathers. The parentage of the remaining 16 offspring (in 9 broods) could not be clearly assigned because (1) one or more neighbors were not sampled or (2) difficulties in scoring across gels prevented confident alignment of fingerprint bands, and insufficient DNA was obtained from muscle samples to allow reanalysis of potential actual fathers on the same gel as excluded nestlings.     

An experimental study of competition between fire ants and Argentine ants in their native range

An understanding of why introduced species achieve ecological success in novel environments often requires information about the factors that limit the abundance of these taxa in their native ranges. Although numerous recent studies have evaluated the importance of natural enemies in this context, relatively few have examined how ecological success may result from differences in the magnitude of interference competition between communities in the native and introduced ranges of nonnative species. Here we examine how native-range competitive environments may relate to invasion success for two important invasive species, the red imported fire ant (Solenopsis invicta) and the Argentine ant (Linepithema humile), in a region of native-range sympatry. At two study sites in northern Argentina, we used stable-isotope analysis, a variety of observational approaches, and two different reciprocal removal experiments to test (1) whether S. invicta competes asymmetrically with L. humile (as suggested by the 20th century pattern of replacement in the southeastern United States) and (2) the extent to which these two species achieve behavioral and numerical dominance. Stable-isotope analysis and activity surveys indicated that S. invicta and L. humile are both omnivores and forage during broadly overlapping portions of the diel cycle. Short-term removal experiments at baits revealed no competitive asymmetry between S. invicta and L. humile. Longer-term colony removal experiments illustrated that S. invicta and L. humile experience an approximately equal competitive release upon removal of the other. Our results indicate that neither S. invicta nor L. humile achieves the same degree of behavioral or ecological dominance where they co-occur in native populations as they do in areas where either is common in their introduced range. These results strongly suggest that interspecific competition is an important limiting factor for both S. invicta and L. humile in South America.     

Trail-following behaviour in two Aphaenogaster ants

In the course of our studies on the chemical ecology of the widely distributed Mediterranean ant Aphaenogaster senilis, we found that trail following is triggered by extracts of the poison gland and Dufour’s gland. To assess the specificity of the trail pheromone, we examined whether a cross-reaction exists between trails of A. senilis and the closely related species A. iberica. Specificity seemed to differ amongst these two species, because workers of A. senilis did not follow trails of A. iberica, whereas the latter followed trails made by both species. Chemical analyses of the glandular contents reveal that Dufour’s glands of both species contain mainly alkanes and alkenes exhibiting species-specific profiles. However, differences in the poison gland content of the two species were dramatic, with A. senilis showing high amounts of alkaloids that were completely absent in A. iberica.     

The evolution of uniparental transmission of fungal symbionts in fungus-growing termites (Macrotermitinae)

Mutualistic associations between different organisms are theoretically expected when the interests of independently reproducing units are aligned to form a single reproductive unit. This alignment does not come about easily, because models show that hosts and symbionts can be in conflict over the transmission of symbionts. Selection will favour hosts that are able to limit genetic variation of symbionts, for example by enforcing uniparental vertical transmission, while symbionts will be selected to disperse independently of the host. A crucial factor determining the evolution and elaboration of symbiotic relationships is therefore who controls the transmission of symbionts. In the fungus-growing termites (Macrotermintinae) horizontal transmission seems to be the rule as the termites normally acquire their cultivated fungus (Termitomyces) from the environment. In spite of this general pattern, uniparental, vertical transmission has evolved in two unrelated Macrotermitinae genera, where only one sex of the two primary reproductives carries asexual spores from the fungal comb of its parent colony to inoculate the new fungus comb. Remarkably, symbiont transmission is exclusively paternal in Macrotermes bellicosus, whereas symbionts are maternally inherited in all Microtermes species studied so far. Thus, in Macrotermitinae horizontal transmission is the ancestral state with two independent origins to uniparental, vertical transmission. This is in contrast to fungus-growing ants where uniparental, vertical transmission is the rule. Causes and consequences of this difference are further discussed. Despite this fundamental difference both groups evolved a similar symbiosis that is probably the key for their ecological success: the fungus-growing ants in the neotropics and the fungus-growing termites in the paleotropics.     

Genetic evidence for mixed parentage in nests of the emu (Dromaius novaehollandiae)

Parentage in emus (Dromaius novaehollandiae) was examined by microsatellite analysis using four independent loci. Of 106 chicks sampled in one breeding season from 18 nests, 54 (51%) were not fathered by the nesting male, 12 (11%) were not from the observed mate of the sitting male, and 9 (8%) represented intra-specific brood parasitism, having no alleles in common with either nest parent. Some males (11%) fathered all chicks in their nests, but the majority showed high levels of cuckoldry. Those males commencing incubation earliest in the season tended to have the highest levels of paternity in their own nests. These results reveal a high frequency of extra-pair fertilisations and resultant cuckoldry in a predominantly socially monogamous bird and support recent reports which have described the emu mating system as a complexity of polyandrous, promiscuous and monogamous behaviour. Parentage assignment of chicks resulting from extra-pair fertilisations revealed an evenly scattered pattern of paternity that did not show any particular male dominance in reproductive success. These results lead to a reassessment of behavioural observations of emus, the consequences of parentage distribution, and theories about mating systems and sexual selection. The frequency of extra-pair copulations and intra-specific brood parasitism suggests patterns of descent that differ greatly from those implied by social monogamy. Received: 27 November 1999 / Received in revised form: 8 January 2000 / Accepted: 8 February 2000     

How ants determine the number of potential recruits

In this paper we present a model that determines the number of potential recruits of Lasius niger, when feeding on a liquid sugar source. The core of the model are two rules: (i) there is a number of workers (facultative foragers) that become potential recruits if starved and (ii) facultative foragers are more likely to become starved than nonforagers because they are more likely to donate food in a trophallaxis (sugar-exchange) event. We develop and explore an analytical model based on these rules, deriving the number of potential recruits after an arbitrary period of starvation. We develop a simplified recruitment model and observe that the predictions of the model are in rough agreement with the empirical data.     

Improvement in collective performance with experience in ants

We show that entire ant colonies can improve their collective performance progressively when they repeat the same process. Colonies of Leptothorax albipennis can reduce their total emigration times over successive emigrations. We show that this improvement is based on experience and some memory-like process, rather than a coincidental developmental change or an increased general level of arousal. We demonstrate that the benefits of experience can be lost (i.e. forgotten) if the interval between successive emigrations is too long. We also show that the benefits of experience are more likely to be retained over a longer period if the collective performance has been repeated several times. This is a new demonstration of a process akin to learning in ants and we briefly discuss how it may involve not only improvements in individual performance but also improvements in the ways in which the ants interact with one another.Communicated by L. Sundström     

Induced responses to herbivory in the Neotropical ant-plant association between Azteca ants and Cecropia trees: response of ants to potential inducing cues

Plant defense against herbivores often involves constitutive and inducible mechanisms of resistance. Obligate ant-plants, which provide food and housing for ants, are thought to primarily rely on ants for defense against herbivores. This form of plant defense has largely been viewed as static. We have been investigating the dynamic nature of Azteca ants as an inducible defense of Cecropia trees. Ants rapidly recruit to and patrol sites of foliar damage. We propose that Azteca ants can be viewed as an inducible defense for Cecropia trees because of their sensitivity to cues associated with herbivory, their rapid and aggressive recruiting ability, and their reclaimable and redeployable nature as a plant defense. In this study, we examine ant behavior following plant damage, and the potential cues that indude ant recruitment. We found that ants present on leaves when the plant is damaged leave the damaged leaf and recruit other ants to it, presumably by laying recruitment trails. Volatile leaf cues associated with herbivory were important in eliciting an induced response in two experiments. However, we found that cues associated with a congeneric plant elicited a much stronger ant response than conspecific cues. Although the type of leaf damage (gaping wounds versus leaf edge wounds) did not affect the level of ant recruitment, the extent of damage did. Leaves with one hole punched showed a 50% increase in ants, while leaves with five holes punched in them elicited a 100% increase in ant numbers. In sum, it appears that multiple plant-related cues associated with herbivory are involved in induction of ant recruitment in the Cecropia-Azteca system. We discuss the generality of ant responses to herbivory in obligate ant-plant systems, and in facultative ant-plant associations, which may be more common. Received: 23 March 1998 / Accepted after revision: 5 July 1998     

Reliability of social media data in monitoring the global pet trade in ants

The global pet trade is a major risk to biodiversity and humans and has become increasingly globalized, diversified, digitalized, and extremely difficult to control. With billions of internet users posting online daily, social media could be a powerful surveillance tool. But it is unknown how reliably social media can track the global pet trade. We tested whether Instagram data predicted the geographic distribution of pet stores and the taxonomic composition of traded species in the emerging pet trade in ants (Hymenoptera, Formicidae). We visited 138 online stores selling ants as pets worldwide and recorded the species traded. We scraped ∼38,000 Instagram posts from ∼6300 users referencing ants as pets and analyzed comments on post and geolocation (available for ∼1800 users). We tested whether the number of Instagram users predicted the number of ant sellers per country and whether the species referenced as pets on Instagram matched the species offered in online stores, with a particular focus on invasive species. The location of Instagram users referencing ants as pets predicted the location of ant sellers across the globe (R² = 0.87). Instagram data detected 439 of the 631 ant species traded in online stores (70%), including 59 of the 68 invasive species traded (87%). The number of Instagram users referencing a species was a good predictor of the number of sellers offering the species (R² = 0.77). Overall, Instagram data provided affordable and reliable data for monitoring the emerging pet trade in ants. Easier access to these data would facilitate monitoring of the global pet trade and help implement relevant regulations in a timely manner.