Cross-species correlation between queen mating numbers and worker ovary sizes suggests kin conflict may influence ovary size evolution in honeybees

During social evolution, the ovary size of reproductively specialized honey bee queens has dramatically increased while their workers have evolved much smaller ovaries. However, worker division of labor and reproductive competition under queenless conditions are influenced by worker ovary size. Little comparative information on ovary size exists in the different honey bee species. Here, we report ovariole numbers of freshly dissected workers from six Apis species from two locations in Southeast Asia. The average number of worker ovarioles differs significantly among species. It is strongly correlated with the average mating number of queens, irrespective of body size. Apis dorsata, in particular, is characterized by numerous matings and very large worker ovaries. The relation between queen mating number and ovary size across the six species suggests that individual selection via reproductive competition plays a role in worker ovary size evolution. This indicates that genetic diversity, generated by multiple mating, may bear a fitness cost at the colony level.     

Female feeding regime and polyandry in the nuptially feeding nursery web spider, Pisaura mirabilis

We examined the influence of female feeding regime on polyandry in the nuptially feeding nursery web spider (Pisaura mirabilis). In this species, the nuptial gift, a dead prey item wrapped in the male’s silk, is physically separate from the ejaculate. We manipulated female feeding regime (starved or fed) and the presence or absence of a gift with three successive males to test direct-benefits hypotheses (nuptial gift or sperm supply) for the expression of polyandry. The presence of a gift was necessary for copulation, as no male without a gift successfully copulated. Female mating behavior most strongly supports polyandry due to the accumulation of gifted food items (“nuptial gift” direct-benefits hypothesis). Starved females that were presented with a gift accepted significantly more gifts and inseminations than fed females. Most starved females (74%) copulated two or more times, as opposed to only 3% of the fed females. Nearly all of the females that accepted a gift subsequently copulated. The nuptial gift item seems to function as male mating effort and females appear to receive multiple matings as part of a feeding strategy.     

A colony-level response to disease control in a leaf-cutting ant

Parasites and pathogens often impose significant costs on their hosts. This is particularly true for social organisms, where the genetic structure of groups and the accumulation of contaminated waste facilitate disease transmission. In response, hosts have evolved many mechanisms of defence against parasites. Here we present evidence that Atta colombica, a leaf-cutting ant, may combat Escovopsis, a dangerous parasite of Atta's garden fungus, through a colony-level behavioural response. In A. colombica, garden waste is removed from within the colony and transported to the midden - an external waste dump - where it is processed by a group of midden workers. We found that colonies infected with Escovopsis have higher numbers of workers on the midden, where Escovopsis is deposited. Further, midden workers are highly effective in dispersing newly deposited waste away from the dumping site. Thus, the colony-level task allocation strategies of the Atta superorganism may change in response to the threat of disease to a third, essential party.     

Size-correlated division of labour and spatial distribution of workers in the driver ant,<Emphasis Type="Italic"> Dorylus molestus</Emphasis>

Driver ants ( Dorylus spp.) show a high degree of worker polymorphism. Previous reports suggest that large Dorylus workers are specialised for defensive tasks. In this study, we first quantitatively tested whether there is a size-correlated division of defensive labour among workers. Second, we determined whether the spatial distribution of workers outside the nest can be predicted based on such size-specific differences in task allocation. We show that the division of defensive behaviour among different-sized workers is not strict. However, there is a significant correlation between worker size and the tendency to carry out defensive tasks. First, workers of larger size were more likely than smaller workers to participate in colony defence. Second, larger workers were more frequent near the nest containing the reproducing individuals and the brood. Finally, large workers were more common in open sections of the trail than in covered sections, which are likely to be less exposed to predators.     

Monogamy in large bee societies: a stingless paradox

High genetic diversity is important for the functioning of large insect societies. Across the social Hymenoptera (ants, bees, and wasps), species with the largest colonies tend to have a high colony-level genetic diversity resulting from multiple queens (polygyny) or queens that mate with multiple males (polyandry). Here we studied the genetic structure of Trigona spinipes, a stingless bee species with colonies an order of magnitude larger than those of polyandrous honeybees. Genotypes of adult workers and pupae from 43 nests distributed across three Brazilian biomes showed that T. spinipes colonies are usually headed by one singly mated queen. Apart from revealing a notable exception from the general incidence of high genetic diversity in large insect societies, our results reinforce previous findings suggesting the absence of polyandry in stingless bees and provide evidence against the sperm limitation hypothesis for the evolution of polyandry. Stingless bee species with large colonies, such as T. spinipes, thus seem promising study models to unravel alternative mechanisms to increase genetic diversity within colonies or understand the adaptive value of low genetic diversity in large insect societies.     

Small queens and big-headed workers in a monomorphic ponerine ant

Evolution of caste is a central issue in the biology of social insects. Comparative studies on their morphology so far suggest the following three patterns: (1) a positive correlation between queen–worker size dimorphism and the divergence in reproductive ability between castes, (2) a negative correlation among workers between morphological diversity and reproductive ability, and (3) a positive correlation between queen–worker body shape difference and the diversity in worker morphology. We conducted morphological comparisons between castes in Pachycondyla luteipes, workers of which are monomorphic and lack their reproductive ability. Although the size distribution broadly overlapped, mean head width, head length, and scape length were significantly different between queens and workers. Conversely, in eye length, petiole width, and Weber’s length, the size differences were reversed. The allometries (head length/head width, scape length/head width, and Weber’s length/head width) were also significantly different between queens and workers. Morphological examinations showed that the body shape was different between queens and workers, and the head part of workers was disproportionately larger than that of queens. This pattern of queen–worker dimorphism is novel in ants with monomorphic workers and a clear exception to the last pattern. This study suggests that it is possible that the loss of individual-level selection, the lack of reproductive ability, influences morphological modification in ants.     

Underground anemotactic orientation in leaf-cutting ants: perception of airflow and experience-dependent choice of airflow direction during digging

Air exchange between the large nests of Atta vollenweideri leaf-cutting ants and the environment strongly relies on a passive, wind-induced ventilation mechanism. Air moves through nest tunnels and airflow direction depends on the location of the tunnel openings on the nest mound. We hypothesized that ants might use the direction of airflow along nest tunnels as orientation cue in the context of climate control, as digging workers might prefer to broaden or to close tunnels with inflowing or outflowing air in order to regulate nest ventilation. To investigate anemotactic orientation in Atta vollenweideri, we first tested the ants’ ability to perceive air movements by confronting single workers with airflow stimuli in the range 0 to 20 cm/s. Workers responded to airflow velocities ≥ 2 cm/s, and the number of ants reacting to the stimulus increased with increasing airflow speed. Second, we asked whether digging workers use airflow direction as an orientation cue. Workers were exposed to either inflow or outflow of air while digging in the nest and could subsequently choose between two digging sites providing either inflow or outflow of air, respectively. Workers significantly chose the side with the same airflow direction they experienced before. When no airflow was present during initial digging, workers showed no preference for airflow directions. Workers developed preferences for airflow direction only after previous exposure to a given airflow direction. We suggest that experience-modified anemotaxis might help leaf-cutting ants spatially organize their digging activity inside the nest during tasks related to climate control.     

Variable virulence among isolates of Ascosphaera apis: testing the parasite–pathogen hypothesis for the evolution of polyandry in social insects

The queens of many eusocial insect species are polyandrous. The evolution of polyandry from ancestral monoandry is intriguing because polyandry undermines the kin-selected benefits of high intracolonial relatedness that are understood to have been central to the evolution of eusociality. An accumulating body of evidence suggests that polyandry evolved from monoandry in part because genetically diverse colonies better resist infection by pathogens. However, a core assumption of the “parasite–pathogen hypothesis”, that there is variation in virulence among strains of pathogens, remains largely untested in vivo. Here, we demonstrate variation in virulence among isolates of Ascosphaera apis, the causative organism of chalkbrood disease in its honey bee (Apis mellifera) host. More importantly, we show a pathogen–host genotypic interaction for resistance and pathogenicity. Our findings therefore support the parasite-parasite hypothesis as a factor in the evolution of polyandry among eusocial insects.     

Circadian consequences of social organization in the ant species<Emphasis Type="Italic"> Camponotus compressus</Emphasis>

The locomotor activity rhythm of different castes of the ant species Camponotus compressus was monitored individually under laboratory light/dark (LD) cycles, and under continuous darkness (DD). The colony of this ant species comprises two sexual castes, the queens and the males, and three worker castes, namely the major, media, and minor workers. The virgin males and virgin queens display rhythmic activity patterns, but the mated queens were arrhythmic while laying eggs, with the rhythmicity resuming soon after egg-laying. Under the LD regime, major workers showed nocturnal patterns, while about 75% of the media workers displayed nocturnal patterns and about 25% showed diurnal patterns. Under the DD regime, most major workers exhibited circadian rhythm of activity with a single steady state, whereas media workers displayed two types of activity patterns, with activity patterns changing after 6–9 days in DD (turn-arounds). The pre-turn-around of the ants that showed nocturnal activity patterns during LD entrainment was <24 h after release into DD, which then became >24 h, after 6–9 days. On the other hand, the pre-turn-around of those ants that exhibited diurnal patterns during LD entrainment was first >24 h after release into DD, and then became <24 h, after 6–9 days. The activity of the minor workers neither entrained to LD cycles nor showed any sign of free-run in DD. It appears that the circadian clocks of the ant species C. compressus are flexible, and may perhaps depend upon the tasks assigned to them in the colony.     

Multiple paternity in<Emphasis Type="Italic"> Rana dalmatina</Emphasis>, a monogamous territorial breeding anuran

Polyandry and sperm competition in anurans have rarely been documented. We investigated the genetic paternity inferred from allozyme variations in 650 tadpoles from four natural ponds in a territorial breeding anuran, Rana dalmatina. Multiple paternity was demonstrated, although R. dalmatina is regarded as a monogamous species. Polyandrous mating was not a common event, occurring only in 17.9% of clutches, with no significant differences among clutches. The proportions of tadpoles fathered by a second male did not significantly differ among ponds, showing that multipaternity was not restricted to a single site. Such a polyandry may result from synchronous multiple amplexus and should reduce the heterozygote deficit related to the breeding-pond fidelity usually exhibited by most anurans.     

Non-specific association between filamentous bacteria and fungus-growing ants

Fungus-growing ants and their fungal cultivar form a highly evolved mutualism that is negatively affected by the specialized parasitic fungus Escovopsis. Filamentous Pseudonocardia bacteria occurring on the cuticle of attine ants have been proposed to form a mutualistic interaction with these ants in which they are vertically transmitted (i.e. from parent to offspring colonies). Given a strictly vertical transmission of Pseudonocardia, the evolutionary theory predicts a reduced genetic variability of symbionts among ant lineages. The aim of this study was to verify whether actinomycetes, which occur on Acromyrmex octospinosus leaf-cutting ants, meet this expectation by comparing their genotypic variability with restriction fragment length polymorphisms. Multiple actinomycete strains could be isolated from both individual ant workers and colonies (one to seven strains per colony). The colony specificity of actinomycete communities was high: Only 15% of all strains were isolated from more than one colony, and just 5% were present in both populations investigated. Partial sequencing of 16S ribosomal deoxyribonucleic acid of two of the isolated strains assigned both of them to the genus Streptomyces. Actinomycetes could also be isolated from workers of the two non-attine ant species Myrmica rugulosa and Lasius flavus. Sixty-two percent of the strains derived from attine ants and 80% of the strains isolated from non-attine ants inhibited the growth of Escovopsis. Our data suggest that the association between attine ants and their actinomycete symbionts is less specific then previously thought. Soil-dwelling actinomycetes may have been dynamically recruited from the environment (horizontal transmission), probably reflecting an adaptation to a diverse community of microbial pathogens.     

Evidence for intra-colonial genetic variance in resistance to American foulbrood of honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>): further support for the parasite/pathogen hypothesis for the evolution of polyandry

Explanations for the evolution of multiple mating by social insect (particularly honey bee) queens have been frequently sought. An important hypothesis is that multiple mating is adaptive because it increases intracolonial genetic diversity and thereby reduces the likelihood that parasites or pathogens will catastrophically infect a colony. We tested one assumption of this model: that honey bee worker patrilines should differ in disease resistance. We used American foulbrood (caused by the bacterium Paenibacillus larvae) as a model pathogen. We found that patrilines within colonies do indeed vary in their resistance to this disease.     

Clonal structure affects the assembling behavior in the Japanese queenless ant <Emphasis Type="Italic">Pristomyrmex punctatus</Emphasis>

The queenless ant Pristomyrmex punctatus (Hymenoptera: Myrmicinae) has a unique society that differs from those of other typical ants. This species does not have a queen, and the workers lay eggs and produce their clones parthenogenetically. However, a colony of these ants does not always comprise members derived from a single clonal line. In this study, we examined whether P. punctatus changes its “assembling behavior” based on colony genetic structure. We prepared two subcolonies—a larger one comprising 200 individuals and a smaller one comprising 100 individuals; these subcolonies were established from a single stock colony. We investigated whether these subcolonies assemble into a single nest. The genetically monomorphic subcolonies (single clonal line) always fused into a single nest; however, the genetically polymorphic subcolonies (multiple clonal lines) did not tend to form a single colony. The present study is the first to demonstrate that the colony genetic structure significantly affects social viscosity in social insects.     

Irregular brood patterns and worker reproduction in social wasps

The potential for reproductive conflict among colony members exists in all social insect societies. For example, queens and workers may be in conflict over the production of males within colonies. Kin selection theory predicts that in a colony headed by a multiply-mated queen, worker reproduction is prevented by worker policing in the form of differential oophagy. However, few studies have demonstrated that workers actually lay eggs within queenright colonies. The purpose of this study was to determine if workers laid male eggs within unmanipulated queen-right colonies of the polyandrous social wasps Vespula maculifrons and V. squamosa. We focused our analysis on an unusual brood pattern within colonies, multiple egg cells. We were primarily interested in determining if individuals reared in these irregular circumstances were queen or worker offspring. To address this question, we genotyped 318 eggs from eight V. maculifrons and two V. squamosa colonies. No worker‑reproduction was detected in any of the queenright colonies; all of the eggs found in multiple egg cells were consistent with being queen‑produced. However, the frequency of multiple egg cells differed among colonies, suggesting that queens vary in the frequency of errors they make when laying eggs within cells. Finally, we suggest that workers may not be laying eggs within queenright colonies and that worker reproduction may be controlled through mechanisms other than differential oophagy in polyandrous Vespula wasps.     

Does the queen win it all? Queen–worker conflict over male production in the bumblebee,<Emphasis Type="Italic"> Bombus terrestris</Emphasis>

Social insects provide a useful model for studying the evolutionary balance between cooperation and conflict linked to genetic structure. We investigated the outcome of this conflict in the bumblebee, Bombus terrestris, whose annual colony life cycle is characterized by overt competition over male production. We established artificial colonies composed of a queen and unrelated workers by daily exchange of callow workers between colony pairs of distinct genetic make-up. Using microsatellite analysis, this procedure allowed an exact calculation of the proportion of worker-derived males. The development and social behavior of these artificial colonies were similar to those of normal colonies. Despite a high worker reproduction attempt (63.8% of workers had developed ovaries and 38.4% were egg-layers), we found that on average 95% of the males produced during the competition phase (CPh) were queen-derived. However, in four colonies, queen death resulted in a considerable amount of worker-derived male production. The different putative ultimate causes of this efficient control by the queen are discussed, and we suggest a possible scenario of an evolutionary arms race that may occur between these two female castes.     

Alternative mating behaviors of the queen polymorphic ant <Emphasis Type="Italic">Temnothorax longispinosus</Emphasis>

Mating behaviors of ants fall into two categories: female calling, in which a female alate releases pheromones that attract males, and male swarming, in which large male aggregations attract females. Female calling is common in species with queens that return to their natal nest to found colonies dependently after mating, while male swarming is common in species with queens that disperse to found independently. In some species that display both founding strategies, a queen-size polymorphism has evolved in which dependent-founding queens are smaller than independent-founding queens. Dependent founding is likely difficult if gynes (virgin queens) are mating in distant swarms. Therefore, a queen may adopt one or the other mating strategy based on its size and founding behavior. We investigated mating behaviors in the queen-polymorphic ant, Temnothorax longispinosus. Observations in laboratory mating arenas indicated that small gynes exhibited significantly lower flight activity than large gynes. Both forms mated in male swarms, and neither form exhibited female calling. The reduced flight activity of the small morph may facilitate returning to the natal nest after mating, provided the mating swarm is located nearby. Therefore, alternative colony-founding behaviors may be possible without the evolution of female-calling behavior; however, the reduced flight activity of small morphs may require that mating swarms are not distant from the natal nest.     

Biogenic amine levels, reproduction and social dominance in the queenless ant Streblognathus peetersi

Social harmony often relies on ritualised dominance interactions between society members, particularly in queenless ant societies, where colony members do not have developmentally predetermined castes but have to fight for their status in the reproductive and work hierarchy. In this behavioural plasticity, their social organisation resembles more that of vertebrates than that of the “classic” social insects. The present study investigates the neurochemistry of the queenless ant species, Streblognathus peetersi, to better understand the neural basis of the high behavioural plasticity observed in queenless ants. We report measurements of brain biogenic amines [octopamine, dopamine, serotonin] of S. peetersi ants; they reveal a new set of biogenic amine influences on social organisation with no common features with other “primitively organised societies” (bumble bees) and some common features with “highly eusocial” species (honey bees). This similarity to honey bees may either confirm the heritage of queenless species from their probably highly eusocial ancestors or highlight independent patterns of biogenic amine influences on the social organisation of these highly derived species.     

Two pathways ensuring social harmony

Reproductive division of labour is a characteristic trait of social insects. The dominant reproductive individual, often the queen, uses chemical communication and/or behaviour to maintain her social status. Queens of many social insects communicate their fertility status via cuticle-bound substances. As these substances usually possess a low volatility, their range in queen–worker communication is potentially limited. Here, we investigate the range and impact of behavioural and chemical queen signals on workers of the ant Temnothorax longispinosus. We compared the behaviour and ovary development of workers subjected to three different treatments: workers with direct chemical and physical contact to the queen, those solely under the influence of volatile queen substances and those entirely separated from the queen. In addition to short-ranged queen signals preventing ovary development in workers, we discovered a novel secondary pathway influencing worker behaviour. Workers with no physical contact to the queen, but exposed to volatile substances, started to develop their ovaries, but did not change their behaviour compared to workers in direct contact to the queen. In contrast, workers in queen-separated groups showed both increased ovary development and aggressive dominance interactions. We conclude that T. longispinosus queens influence worker ovary development and behaviour via two independent signals, both ensuring social harmony within the colony.     

Age-related changes in biogenic amines in individual brains of the ant Pheidole dentata

The behavioral development of minor workers of the ant Pheidole dentata involves a progression of tasks beginning with brood care and culminating in foraging as individuals age. To understand the role of brain neurochemistry in age-related division of labor, we measured the levels of serotonin, dopamine and octopamine in individual brains of minor workers of different age. Serotonin and dopamine levels were significantly correlated with worker age: both increased as minor workers matured, and serotonin rose significantly in the oldest ants. In addition, the serotonin:dopamine ratio was significantly higher in the oldest workers. Octopamine levels did not change with age, although the ratios of octopamine:serotonin and octopamine:dopamine were significantly higher in the youngest workers. These age-associated changes in biogenic amine levels suggest an involvement of neuromodulators in minor worker behavioral ontogeny and temporal polyethism in P. dentata.     

Forest clearing and sex ratio in forest-dwelling wood ant <Emphasis Type="Italic">Formica aquilonia</Emphasis>

Sex ratios of ants have been shown to vary with food resource levels in several studies, but it is not known whether forest clear-cutting has any effect on sex ratio of aphid-tending forest-dwelling ants. We investigated whether the offspring sex ratio of the forest dwelling ant Formica aquilonia varied as a response to clear-cutting. We found that the proportion of males was smaller in clear-cuts than in adjacent forests. Our results are among the first showing that anthropogenic changes in forest structures may have a potential to modify sex ratios of social insects and other forest-dwelling animals.