A technique to artificially inseminate leafcutter ants

Artificial insemination (AI), the instrumental transfer of semen from the male to female reproductive organs, offers excellent opportunities to study mating system adaptations as it allows paternity to be experimentally manipulated. AI techniques have been developed for many animals, but rarely for ants, where they would be particularly useful as most species do not mate under controlled lab conditions. Here, we describe an AI technique for Atta leafcutter ants involving (1) the collection of ejaculates via induction of natural ejaculation, (2) storage in glass capillaries, and (3) transfer to queens using a modified AI equipment as used for honeybees. Queens were fixed and anesthetized in a queen holder, after which the sting chamber was opened with two steel hooks, the tip of the semen-containing glass capillary was inserted into the bursa copulatrix and the semen slowly expelled. Sperm was successfully stored in the spermatheca of queens, and some queens produced a small colony as a result. We could furthermore confirm the earlier observations that Atta semen is directly transferred to the spermatheca rather than to the bursa copulatrix as in most other eusocial insects. The technique that we present here can offer novel opportunities to study mating events such as sperm transfer, sperm competition, and cryptic female choice in ants. At present, the number of queens that produce colonies after AI remains low. However, this number will likely increase, as our results indicate that rearing conditions after AI influence colony founding success of artificially inseminated Atta queens.     

Genetic structure of colonies and a male aggregation in the stingless bee Scaptotrigona postica,as revealed by microsatellite analysis

Summary: Stingless bee queens have for long been assumed to mate once on a nuptial flight, early in life. To evaluate critically monandry in one stingless bee, Scaptotrigona postica, worker offspring (adults or brood) were genetically analysed with microsatellite loci, five of which were developed specifically for the species. Marker loci were highly variable; unbiased estimates of heterozygosity were > 0.5. "Foreign" workers, either those having drifted from other colonies (circa 2%) or those of a replacement queen, were identified with the genetic markers and removed from further analysis. Worker genotypes were consistent with some queens having mated once and others having mated with up to six different males. Scaptotrigona postica queens are therefore facultatively polyandrous. Effective mating frequencies, m_e, were generally lower than the number of patrilines observed. Relatedness estimates of nestmates from individual colonies concurred with those derived from direct counts of the number of patrilines and their proportional representation. Putative genotypes of a colony's queen and her mates were deduced from those of her workers. Queens were generally not related to their mates. For one polyandrous queen, her six mates were related to each other, possibly because of numerically biased representation of males from different colonies at mating sites. However, males at an aggregation outside a colony came from numerous colonies.     

Interspecific hybridisations in natural populations of ants by example of a regional fauna (Hymenoptera, Formicidae)

Summary: The identification of hybrids by high-precision stereomicroscopy and chorological data is described in detail by example of the ant species Formica bruni and pressilabris. In a synopsis of heterogenous data, the overall hybrid frequency in the ant fauna of Central Europe is estimated. 17 of the 164 ant species of this region are demonstrated and further 2 species strongly suspected to hybridise. The low-frequency hybridisers, showing overall hybrid ratios < 3%, were native elements of the Central European fauna before the onset of human cultivation and experienced direct interspecific contact for longer periods of their natural history. They developed more effective mechanisms of reproductive isolation beginning at the prezygotic level. Extremely high (12-31%) local hybridisation ratios occurred in species that invaded the area after anthropogenic changes in landscape structure. The segregated distribution of invaders and autochthonous species in the precultural period apparently did not impose the need to evolve more effective mechanisms for reproductive isolation. Prezygotic mechanisms in particular are deficient. In local situations, 19% of Lasius jensi matings and 44% of Leptothorax albipennis matings leading to successful nest foundations were matings with heterospecific partners. Signs for a dissolution of interspecific phenotypic differences are not detectable in most of the species. The dispersal of hybrid genotypes is apparently inhibited. Factors that probably stabilise the genomic integrity of hybridizing parent species are: (a) inability to produce hybrid queens (in L. jensi 2 umbratus), (b) aneuploidy of F₁ females with inability to perform a balanced meiosis (in hybrids of L. albipennis with 3 other species), and (c) selection against hybrids in the epigenetic environment of alleles (Leptothorax nylanderi 2 slavonicus). Extreme ratios of heterospecific matings would mean a dangerous drain of genetic material. A mechanism to reduce these losses is postulated, consisting of a 'cleptogamy' (theft of heterospecific sperm) by queens that missed a conspecific male, an establishment of a functioning colony of F₁ hybrid workers, a depression of the rearing or fertility of hybrid queens, and a maximum production of own sons by the queen. This mechanism could explain the persistence of rare species (Lasius jensi or Leptothorax albipennis) in an environment of more populous heterospecific hybridisation partners.     

Semi-claustral colony founding in the seed-harvester ant Pogonomyrmex californicus: a comparative analysis of colony founding strategies

The evolution of queens that rear their first brood solely using body reserves, i.e. fully claustral, is viewed as a major advance for higher ants because it eliminated the need for queens to leave the nest to forage. In an apparently unusual secondary modification, the seed-harvester ant Pogonomyrmex californicus displays obligate queen foraging, i.e. queens must forage to garner the resources necessary to survive and successfully rear their first brood. I examined the potential benefits of queen foraging by comparing ecological and physiological traits between P. californicus and several congeners in which the queen can rear brood using only body reserves. The primary advantage of foraging appears to lie in providing the queens of P. californicus with the energy to raise significantly more brood than possible by congeners that use only body reserves; the workers reared in the first brood were also heavier in mass than that predicted by their head width. Other correlates of queen foraging in P. californicus relative to tested congeners included a significantly lower total fat content for alate queens, a small queen body size, and a low queen to worker body mass ratio. Queens also forage in several other well-studied species of Pogonomyrmex, suggesting the possibility that queen foraging may be more common than previously thought in higher ants.     

An experimental study of colony-founding in pine saplings by queens of the arboreal ant, Crematogaster ashmeadi

Summary: Newly mated queens of the arboreal ant Crematogaster ashmeadi initiate colonies in old beetle galleries in the dead branches of longleaf pine trees. In a study by Hahn (1996), a number of tree characteristics were correlated with the number of newly-mated queens in those trees, with branch length the best indicator of queen presence. Three of these characteristics, tree height, dead branch length, and the number of dead branches were tested in an experiment to see which, if any, the queens were using to choose a tree. Both tree height and the number of dead branches significantly influenced queen choice: shorter trees (4-5 m) had more queens than tall ones (7-9 m), and trees with 8 branches had significantly more queens than trees with 2 branches. Branch length had no effect on the number of queens. These findings suggest that newly mated Crematogaster ashmeadi queens search for founding trees on the basis of the height of a sapling and its number of dead branches. Modes of searching are discussed.     

Clustered male production by workers in the stingless bee Melipona subnitida Ducke (Apidae, Meliponinae)

Summary: In stingless bees brood cells are sequentially filled with liquid larval food (mass-provisioning), upon which the queen lays an egg. Thereafter the cell is closed by a worker. This study showed that during these processes workers of Melipona subnitida regularly laid eggs that served as food for the queen. Occasionally cells were oviposited in and immediately closed by a worker. These cells always rendered males. Some of these reproductive workers were seen to lay a trophic egg as well. Cells which were exclusively oviposited in by the physogastric queen gave rise to workers and queens only. In one colony it could be verified that three workers alone, which differed in age by one day, laid 15 male-producing eggs within a period of two successive weeks. Among them the number of ovipositions was positively related to the order in which workers eclosed - the oldest worker laying most eggs - and inversely related to the number of times they closed cells oviposited in exclusively by the queen. Apparently the physogastric queen was not able to stop certain workers from reproducing. We therefore conclude that some workers in M. subnitida temporarily dominated their queens in egg-laying.     

Queen polymorphism and queen-morph related facultative polygyny in the ant, <Emphasis Type="Italic">Myrmecina graminicola</Emphasis> (Hymenoptera,Formicidae)

Summary: Polymorphism of the functional queens in Myrmecina graminicola is analyzed. Both gynomorphs (G-§§ G) and a wide range of intermorphs (I-§§ I) occur, which all are usually mated and egg-laying. Colonies having a gynomorphic queen are always monogynous, whereas about 57% of all colonies with intermorphic queens are polygynous, having two or more coexisting functional queens. The female sexual offspring of individual gynomorphic queens either consists of gynomorphs only, or exclusively of intermorphs. Intermorphic queens may have exclusively intermorphic female sexual progeny, or simultaneously both gynomorphs and intermorphs. Single colonies in laboratory culture produce the same kind of female progeny over several subsequent breeding cycles (artificially compressed "years" of 9-10 months). No environmental influence on queen morph determination could be detected. A genetically mediated queen polymorphism, as in Harpagoxenus sublaevis and Leptothorax sp. A, is suggested. Colony sizes vary considerably, with polygynous I-queen colonies being largest (57.2 - 34.3 s.d. workers), followed by G-queen colonies (44.6 - 22.7 s.d.) and monogynous I-queen colonies (34.4 - 23.7 s.d.), suggesting occasional budding of polygynous colonies.     

A morphological comparison of alates between monogynous and polygynous colonies of Myrmica kotokui in northernmost Japan

Summary: Social structure and sexual size of two populations of Myrmica kotokui were compared. Most of the colonies were polygynous in one population and monogynous in the other. Mean body size of the queens was larger in the monogynous population than in the polygynous population. Although the polygynous population contained many large queens, their wing length was significantly shorter than that of queens in the monogynous population. Some females in the polygynous population were intermorphic between typical workers and queens, while the females of the monogynous population were clearly divided into worker and queen castes without any intermorphic females. The body size of the males showed a large variation and the mean was not significantly different between the two populations.     

Sperm extract and inositol 1,4,5-triphosphate induce cytosolic calcium rise in the central cell of Torenia fournieri

Microinjection of soluble sperm extract and Calcium Green-1 10 kDa-dextran conjugate (CG-1) into the mature central cell of Torenia fournieri induced a significant rise in cytosolic free calcium concentration ([Ca²⁺]_i). The rise reached a maximum at 20 min after injection and then steadily declined. Nevertheless, a relatively high level of [Ca²⁺]_i was maintained even 40 min after injection. Microinjection of sperm extract of maize into Torenia central cells, however, did not trigger any increase in [Ca²⁺]_i, suggesting the possibility of distinct triggers in different species. We also injected caged inositol 1,4,5-triphosphate (InsP₃) and caged cyclic ADP-ribose (cADPR) into Torenia central cells to compare the pattern of Ca²⁺ rise induced by the sperm extract. The results showed that [Ca²⁺]_i elevation triggered by the release of InsP₃ after photolysis appears much faster than that induced by sperm extract. The increase in [Ca²⁺]_i reached a maximum at 70-80 s and dropped to the resting level within 300 s after photolysis. Microinjection of cADPR, however, did not induce any changes in [Ca²⁺]_i. The results indicate that sperm extract might contain factors triggering the release of Ca²⁺ in the central cell.     

Temporal dynamics of facultative social parasitism in bumble bees (Hymenoptera: Apidae)

Parasitical behaviour is thought to play an important, yet so far largely unknown role in the evolution and maintenance of insect sociality. In this study, the influence of interspecific, facultative social parasitism upon bumble bee, Bombus, nesting biology was explored. Queens of B. affinis were introduced into 38 various-sized laboratory colonies of B. terricola. Foreign queens were very successful at killing the resident queen and usurping pre-worker nests (77% wins), although usurpation success decreased to 30% in colonies that contained workers. In addition, B. affinis queens were unable to suppress ovarian development in B. terricola workers that emerged prior to nest usurpation, resulting in the eventual death or expulsion of the foreign queen. In contrast, ovarian development was supporessed in workers that eclosed in the presence of a foreign queen, and in workers that were less than 5 h old when confined in small experimental boxes with a B. affinis queen. Foreign queens that usurped a nest prior to worker emergence were assisted by B. terricola workers, and achieved similar reproductive success when compared to a cohort of 22 B. affinis queens that initiated their own nests in the laboratory. These results suggest that there are periods in the ontogeny of Bombus nest defence, recognition and dominance during which bees are sensitive to the effects of nest parasitism. In bumble bees, and in other social insects, individuals of some species may exploit these weaknesses for their own reproductive benefit.     

Paternity and maternity frequencies in Apis mellifera sicula

Summary: Honey bee queens have been shown to mate with a high number of males, but the evolutionary advantage of this high degree of polyandry is still unclear. Mating data from a number of different Apis species and subspecies are needed to help explain polyandry in honey bees. Pupae of four colonies of Apis mellifera sicula from Sicily were genotyped on three polymorphic microsatellite loci. The genotypes of the queens and fathering drones from these colonies were deduced from the genotypes of the pupae. We found no evidence for polygyny, at least we can exclude more than one functional queen, even super-sister queens, if maternity contributions are equal. The four queens mated with at least 5 to 12 (mean: 9.3 - 3.0 SE) drones. We estimate the error in our determination of the mating frequency that is caused by limited genetic resolution of the marker loci to be less than 1 mating given that Hardy-Weinberg assumptions are satisfied. However, the drones the single queens mated with may be a non-random sample of the whole population, so that detection error may be more severe. The average pedigree relatedness among workers within the colonies was estimated to be 0.341. These results are within the range of those found in other A. mellifera subspecies and Apis species except A. dorsata. We speculate that mating frequency may be positively correlated with drone density.     

Queen mating frequency and maternity of males in the stingless bee Trigona carbonaria Smith

Summary. Aspects of the sociobiology of the native Australian stingless bee Trigona carbonaria (Hymenoptera, Meliponini) were investigated using highly variable microsatellite markers. The queen mating frequency was estimated by examining genotypes of samples of workers from five colonies across three to five microsatellite loci. In each case, results were consistent with the workers being progeny of a queen mated with a single male. Microsatellite analysis of haploid males from two of the colonies suggested they predominantly arose from queen-laid eggs. As workers are more related to sons-of-workers than to sons-of-queens in monandrous colonies, this is somewhat surprising, and suggests that there may be colony-level costs associated with worker reproduction, or that queens are able to regulate worker reproduction via the ritualized cell provisioning and oviposition process. Males from another T. carbonaria colony were found to be diploid.     

Sperm use economy of honeybee (Apis mellifera) queens

The queens of eusocial ants, bees, and wasps only mate during a very brief period early in life to acquire and store a lifetime supply of sperm. As sperm cannot be replenished, queens have to be highly economic when using stored sperm to fertilize eggs, especially in species with large and long‐lived colonies. However, queen fertility has not been studied in detail, so that we have little understanding of how economic sperm use is in different species, and whether queens are able to influence their sperm use. This is surprising given that sperm use is a key factor of eusocial life, as it determines the fecundity and longevity of queens and therefore colony fitness. We quantified the number of sperm that honeybee (Apis mellifera) queens use to fertilize eggs. We examined sperm use in naturally mated queens of different ages and in queens artificially inseminated with different volumes of semen. We found that queens are remarkably efficient and only use a median of 2 sperm per egg fertilization, with decreasing sperm use in older queens. The number of sperm in storage was always a significant predictor for the number of sperm used per fertilization, indicating that queens use a constant ratio of spermathecal fluid relative to total spermathecal volume of 2.364 × 10^?6 to fertilize eggs. This allowed us to calculate a lifetime fecundity for honeybee queens of around 1,500,000 fertilized eggs. Our data provide the first empirical evidence that honeybee queens do not manipulate sperm use, and fertilization failures in worker‐destined eggs are therefore honest signals that workers can use to time queen replacement, which is crucial for colony performance and fitness.     

Origin and rearing season of honeybee queens affect some of their physiological and reproductive characteristics

Queen honeybees of Apis mellifera ligustica and Apis mellifera syriaca were raised to investigate physiological and reproductive characteristics and to determine the most suitable time for queen rearing under semi‐arid conditions in Jordan. The queen rearing season as well as the origin of the queens affected the queens’ weight, acceptance, preoviposition period, volume of the spermatheca, and quantity and quality of sperm in the spermatheca. Italian bees were heavier than Syrian bees at emergence. The introduced queen acceptance rate appeared to be a genetic influence of the queen: A. m. ligustica virgin queens were accepted at a higher rate than were A. m. syriaca queens. There were large seasonal variations in the acceptance rate. Experimental bee colonies accepted their virgin queens during spring with good honey flows at a higher rate compared to the other rearing periods. The greatest mating success was achieved in May and the smallest was during July and August. The preoviposition period was shorter in the Syrian than in the Italian queens, and was longer during summer for both honeybee subspecies. The volume of the spermatheca was smaller in Syrian bees and the spermatheca had lower numbers of spermatozoa compared with Italian bees. Thus, under semi‐arid Mediterranean region conditions, it is highly recommended to raise virgin queens in the spring months only to obtain their highest quality.     

Colony Failure Linked to Low Sperm Viability in Honey Bee (Apis mellifera) Queens and an Exploration of Potential Causative Factors

Queen health is closely linked to colony performance in honey bees as a single queen is normally responsible for all egg laying and brood production within the colony. In the U. S. in recent years, queens have been failing at a high rate; with 50% or greater of queens replaced in colonies within 6 months when historically a queen might live one to two years. This high rate of queen failure coincides with the high mortality rates of colonies in the US, some years with >50% of colonies dying. In the current study, surveys of sperm viability in US queens were made to determine if sperm viability plays a role in queen or colony failure. Wide variation was observed in sperm viability from four sets of queens removed from colonies that beekeepers rated as in good health (n = 12; average viability = 92%), were replacing as part of normal management (n = 28; 57%), or where rated as failing (n = 18 and 19; 54% and 55%). Two additional paired set of queens showed a statistically significant difference in viability between colonies rated by the beekeeper as failing or in good health from the same apiaries. Queens removed from colonies rated in good health averaged high viability (ca. 85%) while those rated as failing or in poor health had significantly lower viability (ca. 50%). Thus low sperm viability was indicative of, or linked to, colony performance. To explore the source of low sperm viability, six commercial queen breeders were surveyed and wide variation in viability (range 60–90%) was documented between breeders. This variability could originate from the drones the queens mate with or temperature extremes that queens are exposed to during shipment. The role of shipping temperature as a possible explanation for low sperm viability was explored. We documented that during shipment queens are exposed to temperature spikes (<8 and > 40°C) and these spikes can kill 50% or more of the sperm stored in queen spermathecae in live queens. Clearly low sperm viability is linked to colony performance and laboratory and field data provide evidence that temperature extremes are a potential causative factor.     

K L a Evaluation during the course of fermentation using data reconciliation techniques

The oxygen mass transfer coefficient often serves to compare the efficiency of bioreactors and their mixing devices as well as being an important scale-up factor. In submerged fermentation, four methods are available to estimate the overall oxygen mass transfer coefficient (K_La): the dynamic method, the stationary method based on a previous determination of the oxygen uptake rate (Q_O2X), the gaseous oxygen balance and the carbon dioxide balance. Each method provides a distinct estimation of the value of K_La. Data reconciliation was used to obtain a more probable value of K_La during the production of Saccharomyces cerevisiae, performed in 22.5-l fed-batch bioreactor. The estimate of K_La is obtained by minimising an objective function that includes measurement terms and oxygen conservation models, each being weighted according to their level of confidence. Weighting factors of measurement terms were taken as their respective inverse variance whereas weighting factors of oxygen conservation models were obtained using Monte Carlo simulations. Results show that more coherent and precise estimations of K_La are obtained.     

Sperm influences female hibernation success, survival and fitness in the bumble-bee Bombus terrestris

We present evidence that in the absence of the transfer of male gland compounds in the ejaculate as well as of behavioural male traits, such as mate guarding or harming of females, sperm itself affects female life-history traits such as hibernation success, female longevity and female fitness. Using the bumble-bee Bombus terrestris, we artificially inseminated queens (females) with sperm from one or several males and show that sire groups (groups of brother males) vary in their effects on queen hibernation survival, longevity and fitness. In addition, multiply inseminated queens always had a lower performance as compared to singly inseminated queens. Apart from these main effects, sire groups (in situations of multiple insemination) affected queen longevity and fitness not independently of each other, i.e. certain sire group combinations were more harmful to queens than others. So far, the cause(s) of these effects remain(s) elusive. Harmful male traits as detected here are not necessarily expected to evolve in social insects because males depend on females for a successful completion of a colony cycle and thus have strong convergent interests with their mates.     

Sperm transfer in the antCarebara vidua F. Smith (Hymenoptera: Formicidae)

Summary Sperm transfer in the myrmicine antCarebara vidua was studied by making histological sections of newly mated queens. Queens are multiply inseminated, and longitudinal sections through the sperm- filled vagina show that the bundle of sperm from each male is enclosed in a layer of accessory gland secretion terminating in a plug of secretion in the female's vaginal orifice. This spermatophore breaks down soon after mating. The huge quantity of sperm packed into the spermatheca (ca. 16 mm³) suggests that the queen lays a large number of eggs in her lifetime, confirmed by the average of 1672 ovarioles in the two ovaries.     

The greater seedling high-light tolerance of <Emphasis Type="Italic">Quercus robur</Emphasis> over <Emphasis Type="Italic">Fagus sylvatica </Emphasis>is linked to a greater physiological plasticity

The responses of Quercus robur (oak) and Fagus sylvatica (beech) seedlings to four different light environments (full, 50%, 40% and 15% sunlight) and to a rapid increase in irradiance were explored during the summer, after 2 years of growth in a forest nursery at Nancy (France). Significant differences between the two species were found for most variables. Phenotypic plasticity for morphological variables (root-shoot ratio, leaf size, leaf weight ratio) was higher in beech than in oak, while the reverse was true for anatomical (stomatal density, epidermis thickness, exchange surface area of the palisade parenchyma) and physiological (maximum photosynthetic rate, stomatal conductance, Rubisco activity) variables. Predawn photochemical efficiency (F_v/F_m) was higher in oak than in beech in all light environments except in 15% sunlight. F_v/F_m was significantly lower in 100% sunlight than in the other light environments in beech but not in oak. Maximum photosynthetic rates (A_max) increased with increasing light availability in the two species but they were always higher in oak than in beech. Oak exhibited higher Rubisco activity than beech in full sunlight. The transfer of shade-adapted seedlings to the open caused a decrease of F_v/F_m, which was larger for beech than for oak. Transferred oak but not beech plants recovered gradually to the control F_v/F_m values. The decreased chlorophyll content and the increased non-photochemical quenching observed in high-light beech seedlings were not enough to avoid photoinhibition. The results suggest that a greater tolerance of strong irradiance is linked to an enhanced physiological plasticity (variables related to photosynthesis), while shade tolerance relies on an enhanced plasticity in light-harvesting variables (crown morphology and chlorophyll content).     

Oxygen transfer characteristics of a centrifugal, packed-bed reactor during viscous xanthan fermentation

The oxygen transfer properties of a novel, centrifugal, packed-bed reactor (CPBR) during viscous xanthan fermentation were determined with respect to the effects of the arrangement of the centrifugal, packed bed (CPB) and the recirculation loop (RL). Characterized by the maximum volumetric transfer coefficient (k_La) in xanthan broth, the aeration efficiency of CPBR was compared to those in stirred-tank reactors (STR) equipped with disc turbines (DT) or marine propellers (MP), and to that in a water-in-oil emulsion (WIO). As expected, STR-WIO showed the highest k_La (0.038 s^-1 at 2%) among all systems studied due to reduced broth viscosity; however, practical difficulties exist in product recovery. It was found that, at 3.5% xanthan the k_La in CPBR (0.018 s^-1) was higher than that of STR (0.005 s^-1) and close to that of STR-WIO (0.020 s^-1), indicating improved oxygen transfer at such a xanthan concentration. The exterior baffles along the rotating fibrous matrix offer additional agitation in the viscous broth. A gas-continuous arrangement, in which the CPB was kept above the broth, was able to elevate k_La to 0.023 s^-1, higher than that of STR-WIO. The external RL operated by a peristaltic pump was found to play an important role in CPBR aeration by providing better gas-liquid contact. With the improved oxygen transfer efficiency in CPBR at high xanthan concentrations, the CPBR system is practically the preferred system for xanthan fermentation. The characteristic roles of CPB arrangement and the RL should be considered primarily during scale-up operation.