Seminal fluid enhances sperm viability in the leafcutter ant Atta colombica

The seminal fluid that accompanies sperm in ejaculates has been shown or suggested to affect sperm competition and paternity success of insects by preventing female remating, inducing oviposition, and forming mating plugs. In Atta leafcutter ants, queens have multiple mates but never remate later in life, although they may live and produce fertilized eggs for several decades. The mating biology and life history of these ants therefore suggests that the major function of seminal fluid is to maximize sperm viability during copulation, sperm transfer, and initial sperm storage. We tested this hypothesis by comparing the viability of testis sperm and ejaculated sperm (mixed with seminal fluid) and found a significant positive effect of seminal fluid on sperm viability. We further quantified this positive effect by adding accessory gland secretion (a major component of seminal fluid) in a dilution series, to show that minute quantities of accessory gland secretion achieve significant increases in sperm viability. Sperm stored by queens for 1 year benefited in a similar way from being exposed to accessory gland compounds after dissection in control saline solution. Our results provide the first empirical evidence that seminal fluid is important for the production of viable ejaculates and that the accessory glands of Atta males—despite their small size—are functional and produce a very potent secretion.     

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     

Mating system,optimal number of matings,and sperm transfer in the Argentine ant Iridomyrmex humilis

Summary In ants, because males have a finite sperm supply and females mate only at the beginning of their reproductive lives, it is possible to infer which is the limiting sex from a few parameters: the amount of sperm produced by males, the amount of sperm stored by females, and the numerical sex ratio. In the Argentine ant Iridomyrmex humilis mating takes place in the nest. Laboratory experiments and field data showed that the numerical sex ratio is heavily male-biased (10.1:1) and that the maximum number of sperm a female can store is similar to the number of sperm a male possesses. Thus females are the limiting sex in this species. In a set of mating experiments, one queen was presented with 1–20 males. The highest proportion of successful matings occurred when females were presented with two males. There was a significant negative correlation between the amount of sperm queens stored and the number of males present in the mating arena. This relationship most likely resulted from male interference during the copulation process. When several males were present in the arena, the mating pair was frequently disturbed by other males trying to copulate. Newly mated queens collected from the field stored 172,000 ± 76,000 sperm, a quantity most similar to that measured in laboratory mating experiments with a ratio of 5 or 10 males per queen. Because the operational sex ratio in I. humilis is highly male-biased, male interference may also decrease the amount of sperm queens store in the field. In many ants, fewer sperm stored by queens should decrease their reproductive success because they would run out of sperm earlier in their reproductive lifetimes. However, comparison of the amount of sperm present in young and old I. humilis queens collected in the field suggests that most use only a small proportion of their sperm supply during their lifetimes. Males mate once and discharge all their sperm during a single mating. Females may mate with several males but dissection of these males indicated that in most cases only one of them had empty seminal vesicles thus suggesting that a single male is responsible for most of the sperm transfer. Thus caution should be exercised in inferring multiple inseminations, as is frequently done in eusocial insects, from the observation of multiple copulations. Correspondence to: L. Keller     

Oligogyny by unrelated queens in the carpenter ant, Camponotus ligniperdus

Multilocus DNA fingerprinting and microsatellite analysis were used to determine the number of queens and their mating frequencies in colonies of the carpenter ant, Camponotus ligniperdus (Hymenoptera: Formicidae). Only 1 of 61 analyzed queens was found to be double-mated and the population-wide effective mating frequency was therefore 1.02. In the studied population, 8 of 21 mature field colonies (38%) contained worker, male, or virgin queen genotypes which were not compatible with presumed monogyny and therefore suggested oligogyny, i.e., the cooccurrence of several mutually intolerant queens within one colony. Estimated queen numbers in oligogynous colonies ranged between two and five. According to the results of the genetic analysis, most of the queens coexisting in oligogynous colonies were not closely related. Pleometrosis is very rare and queenless colonies adopt mated queens both in the laboratory and field. Therefore, the most plausible explanation for the origin of oligogynous colonies in C. ligniperdus is the adoption of unrelated queens by orphaned mature colonies. The coexistence of unrelated, but mutually intolerant queens in C. ligniperdus colonies demonstrates that oligogyny should be considered as a phenomenon distinct from polygyny. Received: 18 December 1997 / Accepted after revision: 20 June 1998     

Positive association of queen number and queen-mating frequency Myrmica ants: a challenge to the genetic-variability hypotheses

Detailed knowledge of the mating system in specific social insect populations is essential for testing general evolutionary hypotheses of multiple paternity in eusocial Hymenoptera. We have studied the mating frequency of queens in a polygynous population of the red ant Myrmica sulcinodis. Genetic mother-offspring analysis showed that double mating occurred at a considerable frequency, but that the effective number of queen-mates remained close to one. After quantifying the effects of multiple maternity (polygyny) and multiple paternity (polyandry) on the genetic diversity of workers, we conclude that multiple paternity in M. sulcinodis did not evolve as an adaptation to increase genetic variation within colonies. Contrary to the predictions from `genetic variability' hypotheses, we found a positive correlation between colony-specific queen number and the average number of mates per queen. Such positive association of queen number and frequency of multiple mating was also found after analysing comparative data across six species of Myrmica ants. These results suggest that resticted dispersal of young queens may be a common factor promoting both polygyny and polyandry at the same time, and that moderate degrees of multiple mating may be an unselected consequence of (1) mating at low cost when mating occurs close to the nest and (2) mating in swarms with a highly male biased operational sex ratio. Future comparative tests of genetic-variability hypotheses should therefore not include species with such evolutionary derived mating system characteristics. Received: 30 April 1998 / Accepted after revision: 19 August 1998     

Sperm precedence and sperm storage in multiply mated red flour beetles

In insects, the last male to mate with a female often gains access to a disproportionate number of subsequent fertilizations. This study examined last-male sperm precedence patterns in doubly and triply mated Tribolium castaneum females. Sperm storage processes were investigated by measuring the quantity of sperm stored within the female spermatheca following single, double, and triple matings. Both doubly mated and triply mated females exhibited high last-male sperm precedence for progeny produced during the first 48 h following the last mating, with females in both groups exhibiting parallel declines in sperm precedence 1 and 2 weeks later. The number of sperm stored by females increased by 33% between singly mated and doubly mated females, indicating that the spermatheca is filled to only two-thirds capacity following insemination by the first male. Based on the proportion of stored sperm from first and second matings, we tested predictions about sperm precedence values based on models of random sperm mixing. High initial last-male sperm precedence strongly supports stratification of last-male sperm. By 1–2 weeks after double matings, sperm precedence declined to levels indistinguishable from values expected under random mixing. These results provide insight into mechanisms of sperm storage and utilization in this species. Received: 20 August 1997 / Accepted after revision: 24 May 1998     

Extended family structure in the ant Formica paralugubris: the role of the breeding system

In populations of various ant species, many queens reproduce in the same nest (polygyny), and colony boundaries appear to be absent with individuals able to move freely between nests (unicoloniality). Such societies depart strongly from a simple family structure and pose a potential challenge to kin selection theory, because high queen number coupled with unrestricted gene flow among nests should result in levels of relatedness among nestmates close to zero. This study investigated the breeding system and genetic structure of a highly polygynous and largely unicolonial population of the wood ant Formica paralugubris. A microsatellite analysis revealed that nestmate workers, reproductive queens and reproductive males (the queens' mates) are all equally related to each other, with relatedness estimates centring around 0.14. This suggests that most of the queens and males reproducing in the study population had mated within or close to their natal nest, and that the queens did not disperse far after mating. We developed a theoretical model to investigate how the breeding system affects the relatedness structure of polygynous colonies. By combining the model and our empirical data, it was estimated that about 99.8% of the reproducing queens and males originated from within the nest, or from a nearby nest. This high rate of local mating and the rarity of long-distance dispersal maintain significant relatedness among nestmates, and contrast with the common view that unicoloniality is coupled with unrestricted gene flow among nests. Received: 8 February 1999 / Received in revised form: 15 June 1999 / Accepted: 19 June 1999     

Multiple mating and facultative polygyny in the Panamanian leafcutter ant Acromyrmex echinatior

Queen mating frequency of the facultatively polygynous ant Acromyrmex echinatior was investigated by analysing genetic variation at an (AG)_n repeat microsatellite locus in workers and sexuals of 20 colonies from a single Panamanian population. Thirteen colonies were found to be monogynous, 5 colonies contained multiple queens, whereas the queen number of 2 colonies remained unresolved. Microsatellite genotypes indicated that 12 out of 13 queens were inseminated by multiple males (polyandry). The mean queen mating frequency was 2.53 and the mean genetically effective paternity frequency was 2.23. These values range among the highest found in ants, and the results are in keeping with the high mating frequencies reported for other species of leafcutter ants. Consistent skew in the proportional representation of different patrilines within colonies was found, and this remained constant in two consecutive samples of offspring. Dissections showed that all examined queens from multiple-queen colonies were mated egg-layers. The mean relatedness value among nestmate workers in polygynous colonies was lower than that for monogynous colonies. No diploid males were detected in a sample of 70 genotyped males. Worker production of males was detected in one queenless colony. We discuss our findings in relation to known patterns of multiple maternity and paternity in other eusocial Hymenoptera. Received: 2 September 1998 / Received in revised form: 3 February 1999 / Accepted: 7 February 1999     

Low queen mating frequency in the seed-harvester ant Pogonomyrmex (Ephebomyrmex) pima: implications for the evolution of polyandry

The evolution of polyandry is a central problem in the study of insect mating systems, and both material and genetic benefits have been proposed to offset the presumed costs of multiple mating. Although most eusocial Hymenoptera queens mate with just one or occasionally two males, high levels of polyandry are exhibited by several taxa, including seed-harvester ants of the genus Pogonomyrmex. Previous studies of queen mating frequency in Pogonomyrmex have focused on monogynous (one queen per colony) species in the subgenus Pogonomyrmex. We performed a genetic mother–offspring analysis of mating frequency in Pogonomyrmex (Ephebomyrmex) pima, a queen-dimorphic species with dealate and intermorph queens that differ in colony structure (intermorph colonies contain multiple queens). Our results demonstrate that both dealate and intermorph queens of P. (E.) pima are typically single maters, unlike their congeners analyzed thus far. Polyandry appears to be a derived trait in Pogonomyrmex, but comparative tests between P. (E.) pima queen morphs and across the genus provide no evidence that it evolved as an adaptation to increase genetic diversity within colonies or to obtain more sperm, respectively.     

Inbreeding and local mate competition in the ant Cardiocondyla batesii

The ant species Cardiocondyla batesii is unique in that, in contrast to all other ant species, both sexes are flightless. Female sexuals and wingless, ergatoid males mate in the nest in autumn and young queens disperse on foot to found their own colonies in spring. The close genetic relatedness between queens and their mates (r_qm=0.76±SE 0.12) and the high inbreeding coefficient (F=0.55; 95%CI 0.45–0.65) suggest that 83% of all matings are between brothers and sisters. As expected from local mate competition theory, sex ratios were extremely female biased, with more than 85% of all sexuals produced being young queens. Despite the common occurrence of inbreeding, we could not detect any adult diploid males. Though the probability of not detecting multiple mating was relatively high, at least one-third of all queens in our sample had mated more than once. Multiple mating to some extent counteracts the effects of inbreeding on worker relatedness (r_ww=0.68±SE 0.05) and would also be beneficial through decreasing diploid male load, if sex was determined by a single locus complementary system.Communicated by L. Sundström     

High genetic relatedness among nestmate queens in the polygynous ponerine ant Gnamptogenys striatula in Brazil

Gnamptogenys striatula is a polygynous ant species, in which all workers are potentially able to mate. The reproductive status, relatedness and pedigree relationships among nestmate queens and winged females in a Brazilian population were investigated. We collected all the sexual females of 12 colonies (2–44 queens per colony, plus 2–18 winged females in 3 colonies). Dissections revealed that 98% of the queens were inseminated and that the queens in the most polygynous colonies did not lay equal numbers of eggs. The sexual females and a sample of the population were genotyped using eight microsatellite markers. Relatedness among nestmate queens was among the highest recorded to date (0.65±0.25), and tests of pedigree relationship showed that they were likely to be full-sisters, and sometimes cousins. Mated winged females were always full-sisters, the estimated genetically effective queen numbers were low and tests of pedigree relationship showed that only a few queens in the colony could be the mothers. These results suggest that the high queen-queen relatedness in polygynous colonies of G. striatula is maintained by an unusual mechanism: winged females are mostly produced by only one or a few queens, and these groups of full-sisters are recruited back into their original nest after mating. Received: 26 November 1999 / Revised: 7 September 2000 / Accepted: 7 September 2000     

Sperm limitation and the evolution of extreme polyandry in honeybees (Apis mellifera L.)

Honeybee queens (Apis mellifera) show extreme levels of polyandry, but the evolutionary mechanisms underlying this behaviour are still unclear. The sperm-limitation hypothesis, which assumes that high levels of polyandry are essential to get a lifetime sperm supply for large and long-lived colonies, has been widely disregarded for honeybees because the semen of a single male is, in principle, sufficient to fill the spermatheca of a queen. However, the inefficient post-mating sperm transfer from the queens lateral oviducts into the spermatheca requires multiple matings to ensure an adequate spermatheca filling. Males of the African honeybee subspecies A. m. capensis have fewer sperm than males of the European subspecies A. m. carnica. Thus, given that sperm limitation is a cause for the evolution of multiple mating in A. mellifera, we would expect A. m. capensis queens to have higher mating frequencies than A. m. carnica. Here we show that A. m. capensis queens indeed exhibit significantly higher mating frequencies than queens of A. m. carnica, both in their native ranges and in an experiment on a North Sea island under the same environmental conditions. We conclude that honeybee queens try to achieve a minimum number of matings on their mating flights to ensure a sufficient lifetime sperm supply. It thus seems premature to reject the sperm-limitation hypothesis as a concept explaining the evolution of extreme polyandry in honeybees.Communicated by R.E. Page     

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     

Sociogenetic organization of the red ant Myrmica rubra

Knowledge of the sociogenetic organization determining the kin structure of social insect colonies is the basis for understanding the evolution of insect sociality. Kin structure is determined by the number and relatedness of queens and males reproducing in the colonies, and partitioning of reproduction among them. This study shows extreme flexibility in these traits in the facultatively polygynous red ant Myrmica rubra. Relatedness among worker nestmates varied from 0 to 0.82. The most important reason for this variation was the extensive variation in the queen number among populations. Most populations were moderately or highly polygynous resulting in low relatedness among worker nestmates, but effectively monogynous populations were also found. Polygynous populations also often tend to be polydomous, which is another reason for low relatedness. Coexisting queens were positively related in two populations out of five and relatedness was usually similar among workers in the same colonies. Due to the polydomous colony organization and short life span of queens, it was not possible to conclusively determine the importance of unequal reproduction among coexisting queens, but it did not seem to be important in determining the relatedness among worker nestmates. The estimates of the mating frequency by queens remained ambiguous, which may be due to variation among populations. In some populations relatedness among worker nestmates was high, suggesting monogyny and single mating by queens, but in single-queen laboratory nests relatedness among the worker offspring was lower, suggesting that multiple mating was common. The data on males were sparse, but indicated sperm precedence and no relatedness among males breeding in the same colony. A comparison of social organizations and habitat requirements of M. rubra and closely related M. ruginodis suggested that habitat longevity and patchiness may be important ecological factors promoting polygyny in Myrmica. Received: 15 May 1995/Accepted after revision: 17 October 1995     

Sperm competition as a mechanism of female choice in the field cricket,Gryllus bimaculatus

Summary While traditionally viewed as an extension of intermale competition, mechanisms of sperm competition may be used by multiply mating females for mate choice. In the field cricket G. bimaculatus sperm were shown to mix in the spermatheca. The proportion of offspring sired by the second male increased with spermatophore attachment duration and, therefore, the number of sperm transferred. There was no second male advantage for single matings after an initial double mating. However, the proportion ofoffspring sired by the second male increased in proportion to the number of times he mated such that second males mating three times after an initial double mating had the advantage at fertilization. The data suggested that sperm were utilized in proportion to their numerical representation in the spermatheca. The mechanism of sperm precedence may, therefore, be one of sperm dilution. Female G. bimaculatus may control the degree of sperm competition as a mechanism of mate choice. By accepting large quantities of sperm from chosen males they may determine the paternity of their offspring by diluting out the sperm stored from previous matings.     

Parentage and sex allocation in the facultatively polygynous ant Myrmicatahoensis

Most social groups have the potential for reproductive conflict among group members. Within insect societies, reproduction can be divided among multiple fertile individuals, leading to potential conflicts between these individuals over the parentage of sexual offspring. Colonies of the facultatively polygynous ant Myrmicatahoensis contain from one to several mated queens. In this species, female sexuals were produced almost exclusively by one queen. The parentage of male sexuals was more complex. In accordance with predictions based on worker sex-allocation preferences, male-producing colonies tended to have low levels of genetic relatedness (i.e., high queen numbers). Correspondingly, males were often reared from the eggs of two or more queens in the nest. Further, over half of the males produced appeared to be the progeny of fertile workers, not of queens. Overall investment ratios were substantially more male biased than those predicted by genetic relatedness, suggesting hidden costs associated with the production of female sexuals. These costs are likely to include local resource competition among females, most notably when these individuals are adopted by their maternal nest. Received: 3 March 1998 / Accepted after revision: 20 June 1998     

Levels of polyandry and intracolonial genetic relationships in Apis florea

DNA was extracted from worker and drone pupae of each of five colonies of the dwarf honey bee Apis florea. Polymerase chain reactions (PCR) were conducted on DNA extracts using five sets of primers known to amplify microsatellite loci in A. mellifera. Based on microsatellite allele distributions, queens of the five colonies mated with at least 5–14 drones. This is up to 3 times previous maximum estimates obtained from sperm counts. The discrepancy between sperm count and microsatellite estimates of the number of matings in A. florea suggests that despite direct injection of semen into the spermatheacal duct, either A. florea drones inject only a small proportion of their semen, or queens are able to rapidly expel excess semen after mating. A model of sexual selection (first proposed by Koeniger and Koeniger) is discussed in which males attempt to gain reproductive dominance by increasing ejaculate volume and direct injection of spermatozoa into the spermatheca, while queens attempt to maintain polyandry by retaining only a small fraction of each male's ejaculate. It is shown, at least in this limited sample, that the effective number of matings is lower in A. florea than in A. mellifera.     

Independence, not conflict, characterizes dart-shooting and sperm exchange in a hermaphroditic snail

Although the sexes are united in hermaphrodites, conflict can still occur because the male and female functions have separate interests. We examined the evidence for conflict in the mating system of the terrestrial snail Cantareus aspersus (formerly Helix aspersa) where sharp, calcareous darts are ‘shot’ during courtship. We predicted that the use of the dart would either reflect or create conflict and this would be evident in either the courtship behavior or the transference of sperm. Previous studies demonstrated that the dart functions after sperm transfer to increase sperm survival. Using detailed observations of mating snails, we examined the factors that determine dart shooting order, the behavioral responses after being hit by a dart, the accuracy of dart shooting, and the allocation of sperm resources. We found that each dart was shot independently, and each animal appeared to be interested only in getting off the best possible shot, probably one that penetrates deeply near the genital pore. There is no evidence of mating conflict. Every snail transfers sperm to its partner, and the size of the donation does not depend on the success or failure of either snail’s dart shot. Although the receipt of a dart does not appear to cause harm, it may produce indirect costs due to the partial loss of control over fertilization. We conclude that mating in C. aspersus is a partnership in which independent actors demonstrate unconditional reciprocity during courtship and sperm transfer.     

Strategic mating effort in a simultaneous hermaphrodite

Sexual selection theory for simultaneously hermaphroditic animals predicts an overall preference for inseminating partners that have a relatively higher female fecundity. Previous work on the link between male mating decisions and female fecundity has primarily focused on the effect of the partners’ body size using existing variation in this trait within a study population. On the assumption that the body size is positively correlated with female fecundity, sperm donors should preferentially inseminate relatively larger individuals to obtain a higher fitness gain through their male sex function. However, empirical evidence for such size-dependent mate choice in simultaneous hermaphrodites is equivocal, possibly because of confounding variables. We studied the mating behavior of the simultaneously hermaphroditic flatworm Macrostomum lignano and tested for a strategic mating effort in response to the feeding status of the partner. We experimentally manipulated the feeding status of potential mating partners in order to generate variation in female fecundity among them and tested whether this affected the copulation number and the number of sperm that the focal worm managed to store in the partner’s sperm storage organ. We found that the manipulation of the feeding status had a strong effect on the body size of the potential mating partners and that focal worms copulated more frequently with, and stored more sperm in well-fed partners compared to unfed partners. Our results suggest that M. lignano adjusts its mating effort in response to the feeding status of the mating partner.     

Male remating and female fitness in the wolf spider Pardosa astrigera: the role of male mating history

Although the effects of male mating history on female reproductive output and longevity have been studied in insects, few such studies have been carried out in spiders. In a mating system in which females are monandrous while males are polygynous, females may incur the risk by mating with successful males that have experienced consecutive matings and suffer from the possible depletion of sperm and/or associated ejaculates. Here, we examine the effects of male mating history on male courtship and copulation duration, female reproductive fitness, and female adult longevity of the wolf spider, Pardosa astrigera. Results indicated that male mating frequency had little effect on their subsequent copulation success, and of 35 males tested, about half of the males were able to copulate with five virgin females successively at an interval of 24 h. Male mating history had little effect on their courtship duration. However, male mating history significantly affected male copulation duration, female adult longevity, and reproductive output. Males that mated more frequently copulated longer and more likely failed to cause their mates to produce a clutch, although there was no significant difference in the number of eggs laid and the number of eggs hatched regardless of the first clutch or the second one. Multiple mating of male P. astrigera resulted in significant reduction in female adult longevity. Our results indicate that monandrous females mating with multiple-mated males may incur substantial fitness costs.