Simulation of gamete behaviors and the evolution of anisogamy: reproductive strategies of marine green algae

In marine green algae, isogamous or slightly anisogamous species are taxonomically widespread. They produce positively phototactic gametes in both sexes. We developed a new numerical simulator of gamete behavior using C++ and pseudo-parallelization methods to elucidate potential advantages of phototaxis. Input parameters were set based on experimental data. Each gamete swimming in a virtual rectangular test tank was tracked and the distances between the centers of nearby male and female were measured at each step to detect collisions. Our results shed light on the roles of gamete behavior and the mechanisms of the evolution of anisogamy and more derived forms of sexual dimorphism. We demonstrated that not only gametes with positive phototaxis were favored over those without, particularly in shallow water. This was because they could search for potential mates on the 2-D water surface rather than randomly in three dimensions. Also, phototactic behavior clarified the difference between isogamy and slight anisogamy. Isogamous species produced more zygotes than slightly anisogamous ones only under the phototactic conditions. Our results suggested that sperm limitation might be easily resolved particularly in the slightly anisogamous species. Some more markedly anisogamous species produce the smaller male gametes without any phototactic devices and the larger positively phototactic female gametes. In such species, female gametes attract their partners using a sexual pheromone. This pheromonal attraction system might have played a key role in the evolution of anisogamy, because it could enable markedly anisogamous species achieve 2-D search efficiencies on the water surface. The mating systems appear to be tightly tuned o the environmental conditions of their habitats.     

Effects of gamete behavior and density on fertilization success in marine green algae: insights from three-dimensional numerical simulations

We developed a numerical simulation of mating experiment to study effects of phototactic gamete behavior and density on fertilization success, using the C++ programming language, and pseudo-parallelization methods with input parameters based on experimental data. In our experiments, we found that gametes with positive phototaxis are favored, particularly in shallow water, because they can search for potential mates on the two-dimensional (2-D) water surface rather than randomly in three dimensions. We also found evidence that sperm (male gametes) limitation might not be the dominant selective force in the evolution of isogamous or slightly anisogamous marine green algae because almost all of female gametes can be fertilized on the 2-D water surface meaning they might not be under sperm limited conditions. Gamete density also appears to affect mating success seriously. These findings were produced by some technical progress made recently to rapidly and correctly count the numbers of zygotes formed calculating the locations of huge numbers of male and female gametes in the test tank. Both gamete behavior and density might be determined by environmental conditions of habitat, particularly the depth of water.     

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

 The reproductive strategy of a marine alga with a heteromorphic biphasic life cycle was studied by analyzing various sexual reproductive characters in light of the evolution of anisogamy. Gametophytes of Monostroma angicava were dioecious and their gametes were slightly anisogamous. Volume of gametangium, density of gametangia and area of mature gametangial parts on each gametophyte did not differ from male to female. Therefore, the reproductive biomass investment for gamete production was considered to be the same for each sex. Anisogamy in this alga appeared to be derived from the difference in the number of cell divisions during gametogenesis, because the majority of male gametangia each produced 64 (2⁶) gametes and the female produced 32 (2⁵) gametes. This corresponded with measurements of cell size in male and female gametes. Further, the sex ratio was 1:1 for sexually mature plants sampled at Charatsunai. Therefore, it was suggested that in the field twice as many male gametes are released as female gametes. Liberated gametes of both sexes showed positive phototaxis. The swimming velocity of freshly liberated male gametes was a little higher than that of female gametes. Male gametes had the potential to swim for ca. 72 h and female gametes for ca. 84 h. The difference in gamete motility between the two sexes seemed to be related to cell size. Planozygotes were negatively phototactic and swam more rapidly than gametes of either sex. Received: 5 March 1997 / Revision accepted: 18 July 1997     

Phototaxis and the evolution of isogamy and 'slight anisogamy' in marine green algae: insights from laboratory observations and numerical experiments

The evolution of anisogamy in marine algae was studied through numerical simulations of gamete mating behaviour in three dimensions, using observed traits of marine green algae as input parameters. The importance of phototaxis became apparent from the numerical experiments: all gametes with phototactic systems are favoured over those without, but this advantage is reduced with increasing tank depth or shorter search times. Phototactic gametes were advantaged over non-phototactic gametes if the water was shallower than about 30–40 mm when the time available for gamete encounter was 1000 time steps (5.55 min). If gametes of both sexes are positively phototactic, slightly anisogamous species are at a disadvantage to isogamous species, which invalidates the sperm-limitation theory as a driver for the evolution of slight anisogamy. Conflicting selection forces of search efficiency and zygote fitness may be needed.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 144 , 321–327.     

Sexual fusion of protoplasts in a marine green alga, Bryopsis plumosa

We isolated protoplasts from male and female gametophytes of a strictly dioecious strain of the coenocytic marine green alga Bryopsis plumosa. The protoplasts successfully developed into macrothalli. These in turn produced swimming cells, which appeared similar to biflagellated gametes even when the mixed protoplasts were comprised of protoplasm from male and female gametophytes. We found that swimming cell sizes depended on the male/female protoplasm ratio; macrothalli successfully produced swimming cells with male/female protoplasm ratios of 10:0; 9:1; 7:3; 5:5; 1:9; and 0:10. In male/female protoplasm ratios ranging from equal to strongly female biased (5:5; 3:7; 1:9), swimming cells exhibited normal behaviors of gametes and resultant zygotes, displaying positive and negative phototaxis, respectively. Negatively phototactic swimming cells were quadriflagellated and had two nuclei, apparently as a result of fusion, but never developed into microthalli. Thus, these swimming cells might lack functionality essential for normal gametes. Our findings suggested that natural monoecy observed in this genus did not originate from hybridization of protoplasm between the sexes.     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole     

Changes in photoreceptor currents and their sensitivity to the chemoeffector tryptone during gamete mating in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Chlamydomonas reinhardtii Dangeard generates photoreceptor currents (PCs) upon light excitation. These currents play a key role in the signal transduction chain for photomotility responses. We have previously found that inhibition of PCs by tryptone occurs only in gametes that display chemotaxis toward this agent, and is not observed in chemotactically insensitive vegetative cells. Here we show that the sensitivity to tryptone is characteristic of gametes of both mating types, and examine the influence of gamete mating on PCs and their sensitivity to tryptone. The amplitude of PCs increases after cell fusion, but the sensitivity of these currents to tryptone decreases upon flagellar adhesion and/or an increase in the intracellular cAMP concentration. Net chemotaxis toward tryptone is reduced in young zygotes compared to gametes. We conclude that gamete mating leads to rapid inactivation of a gamete-specific chemosensory system.     

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination.The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote.Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.     

Phototactic responses of Chlamydomonas eugametos gametes before and after fusion

The phototactic behavior of Chlamydomonas eugametos gametes and vis-à-vis pairs was quantitated using a fully automated, computer-controlled microvideo image analysis system. Two different mt- (mating type minus) and one mt+ (mating type plus) strain, together with the two combinations of pairs were studied. One mt- strain of dark-adapted gametes was non-phototactic while the others were positively phototactic at all effective intensities of white light. The mt+ strain exhibited one of the strongest positive responses that has so far been reported in algae (r-values greater than 0.7). After sexual fusion, the mt+ cell powers the swimming vis-à-vis pair. Its phototactic behavior reversed on fusion, with the pairs swimming away from all effective light intensities, irrespective of whether its partner was formerly phototactic or not. However, when adapted to the dark for an hour or more, vis-à-vis pairs swam positively to the light. The ecological consequence could be that pairs settle and develop into zygotes under intermediate light intensities or at light-dark interfaces.     

Epifluorescent microscopic studies on the mechanism of preferential destruction of chloroplast nucleoids of male origin in young zygotes ofChlamydomonas reinhardtii

Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt^–) and female (mt⁺) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of³H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt^–, mt⁺) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin     

Intergeneric pollen – megagametophyte relationships of conifers in vitro

 Germinating pollen from larch (Larix occidentalis), Sitka spruce (Picea sitchensis) and white pine (Pinus monticola) were co-cultured with megagametophytes dissected from cones of other genera (Pseudotsuga menziesii, Larix×eurolepis and Pinus monticola). Pollen was presented to megagametophytes possessing archegonia which were either alive, degenerating or dead. In addition, pollen was presented to fertilized megagametophytes and to megagametophytes that had been cut in half. Megagametophyte penetration by pollen tubes and male gamete release into archegonia were verified by serial sections of glycomethacrylate-embedded specimens. Pollen tubes penetrated through any part of the apex of the megagametophyte. Division of the body cell into the two gametes was regularly observed. Delivery of gametes was confirmed between spruce and larch. Pollen tubes also penetrated fertilized megagametophytes, dead or degenerating archegonia as well as wounded and/or cut surfaces. This demonstrates the inability of the male gametophyte to optimize its mating efforts, since it is unable to differentiate between healthy and unhealthy archegonia. The megagametophyte cells are unable to optimize male selection. They may produce secretions of a generally attractive nature, as pollen is attracted to the apex of the megagametophyte, but archegonia themselves do not produce pollen-specific signals of either a promotive or inhibitory nature. These results open new avenues for the development of novel breeding strategies where natural breeding barriers may be bypassed. Received: 19 March 1998 / Accepted: 29 April 1998     

Photoresponse in the heterotrophic marine dinoflagellate Oxyrrhis marina

Expressed rhodopsins were detected by proteomic analysis in an investigation of potential signal receptors in the cell membrane of the marine heterotrophic dinoflagellate Oxyrrhis marina (CCMP604). We inferred these to be sensory rhodopsins, a type of G-protein-coupled receptor trans-membrane signaling molecule. Because phototactic behavior based on sensory rhodopsins has been reported in other protists, we investigated the photosensory response of O. marina. This dinoflagellate exhibited strongest positive phototaxis at low levels (2-3 μE/m(2)/s) of white light when the cells were previously light adapted and well fed. Positive phototaxis was also found for blue (450 nm), green (525 nm), and red (680 nm) wavelengths. In a further test, O. marina showed significantly greater phototaxis toward concentrated algal food illuminated by blue light to stimulate red chlorophyll-a autofluorescence in the prey, compared with using bleached algae as prey. Concentration of a cytoplasmic downstream messenger molecule, cyclic adenosine monophosphate, a component of the signaling pathway of G-protein-coupled receptor molecules, rapidly increased in O. marina cells after exposure to white light. In addition, treatment with hydroxylamine, a rhodopsin signaling inhibitor, significantly decreased their phototactic response. Our results demonstrate that a heterotrophic marine dinoflagellate can orient to light based on rhodopsins present in the outer cell membrane and may be able to use photosensory response to detect algal prey based on chlorophyll autofluorescence.     

Behavior of flagella and flagellar root systems in the planozygotes and settled zygotes of the green alga Bryopsis maxima Okamura (Ulvophyceae,Chlorophyta) with reference to spatial arrangement of eyespot and cell fusion site

Behaviors of male and female gametes, planozygotes and their microtubular cytoskeletons of a marine green alga Bryopsis maxima Okamura were studied using field emission scanning electron microscopy, high‐speed video microscopy, and anti‐tubulin immunofluorescence microscopy. After fusion of the biflagellate male and female gametes, two sets of basal bodies lay side by side in the planozygote. Four long female microtubular roots extended from the basal bodies to the cell posterior. Four short male roots extended to nearly half the distance to the posterior end. Two flagella, one each from the male and female gametes, become a pair. Specifically, the no. 2 flagellum of the female gamete and one male flagellum point to the right side of the eyespot of the female gamete, which is located at the cell posterior and which is associated with 2s and 2d roots of the female gamete. This spatial relationship of the flagella, microtubular roots, and the eyespot in the planozygote is retained until settlement. During forward swimming, the planozygote swings the flagella backward and moves by flagellar beating. The male and female flagella in the pair usually beat synchronously. The cell withdraws the flagella and becomes round when the planozygote settles to the substratum 20 min after mixing. The axoneme and microtubular roots depolymerize, except for the proximal part and the basal bodies. Subsequently, distinct arrays of cortical microtubules develop in zygotes until 30 min after mixing. These results are discussed with respect to the functional significance of the spatial relationships of flagellar apparatus‐eyespot‐cell fusion sites in the mating gametes and planozygote of green algae.     

Non-random mating in controlled multiple-donor crosses in Gracilaria gracilis (Gracilariaceae,Rhodophyta)

Crosses were performed to identify the sources of variation in zygote production (via cystocarp production) in Gracilaria gracilis, a red haploid-diploid seaweed. First, because male gametes are short-lived (<6?h), the rate of gamete encounters was evaluated in a time-course experiment. Second, the effect of water motion on gamete encounters was assessed by introducing turbulent eddies in the crossing tank and by comparing fertilization rates with and without this added turbulence. Third, variation due to individual performance was explored by performing multiple-donor crosses using 12 males and 12 females from three populations. Paternity of cystocarps produced in these crosses was determined using microsatellite markers. The results show that cystocarp yield increased with exposure time: fertilization occurred in as little as 15?min after the introduction of male branches into the crossing tank and maximum cystocarp production values were observed at 6?h. There were no significant differences in cystocarp production between the two turbulence levels. On the other hand, cystocarp production was highly influenced by male and female parental identities and to a lesser degree by an interaction between the male and female parents. The variation in cystocarp production according to male and female identity was not due to population origin as there was no difference between intra- and inter-population crosses. Thus nonrandom mating occurs in controlled conditions and arises from differential performance in G. gracilis. There was a strong deviation from equality of male performance, implicating post-adhesion events and/or male gamete production as important in generating non-random mating. Consequently, non-random mating may play a role in the evolution of mating patterns in G. gracilis.     

Analysis of gamete and zygote motility in Allomyces

To study the mechanisms of chemotaxis in eukaryotes, the motility patterns of the gametes and zygotes and the chemotactic responses of the male gametes of the lower eukaryote Allomyces macrogynus were examined. Dark-field microscopy of the male gametes showed a smooth swimming pattern interrupted by very brief ‘jerks’ of the cell body that caused a change in swimming direction. Female gametes had a slower swimming velocity than the males and underwent more jerks or turns which accounted for their sluggish motility. The zygotes swam with the fastest velocity and were observed to have a helical swimming pattern involving a continuous turning of the cell body, a behavior absent from the gametes. Introduction of female gametes that produce the chemoattractant sirenin brought about an immediate change in the behavior of the male gametes. They moved in spirals (or helices) towards the source of the chemoattractant (the female gametes), undergoing only a few jerks to reorient the male cells. When very near the female cells, or in high concentrations of added sirenin, many very short motility tracks were observed that finally resulted in contact between the two gamete types. The results indicate that the poor swimming ability of the female gametes facilitates gamete contact, resulting in as many as 30–40 male gametes clustered on a single female cell. Further, male gamete orientation to the sirenin gradient is caused by the chemoattractant suppressing the jerk motion.     

Performance of non-motile male gametes in the sea: analysis of paternity and fertilization success in a natural population of a red seaweed,Gracilaria gracilis

In haploid–diploid red seaweeds, the dispersal of male gametes is presumed limited due to their lack of flagella. It has been suggested that this group suffers from sperm limitation and, consequently, that fertilization is relatively inefficient. Fertilization in most floridean rhodophytes results in the formation a cystocarp, a swelling on the haploid female thallus housing the diploid zygote and its thousands of diploid daughter spores. To study the performance of non-motile male gametes in the sea, we evaluated both female and male fertilization success in a natural population of the red marine alga Gracilaria gracilis. Female fertilization success, estimated by cystocarp yield per unit female thallus, was evaluated with respect to the availability of male gametes. Male fertilization success, estimated by the individual contribution of different males to zygotes, was assessed by paternity analyses on 350 cystocarps produced in one reproductive season using two microsatellite loci. The results show that cystocarp yield is not sperm limited and that the large variation in male fertilization success cannot be solely explained by the distance travelled by the male gamete to find a mate. Taken together, the results suggest that, not only is fertilization efficient, but that male–male competition and/or female choice may play a role in shaping population mating patterns.     

Inheritance pattern of chloroplast DNA is correlated with gamete types based on sex-specific arrangement of the cell fusion site in Caulerpa (Ulvophyceae, Chlorophyta)

Using field emission scanning electron microscopy (FE‐SEM) and fluorescence microscopy, the respective relationships between the arrangement of the gamete cell‐fusion site and the inheritance pattern of chloroplast DNA (cp‐DNA) were studied for Caulerpa brachypus Harvey, C. okamurae Weber‐van Bosse, C. racemosa (Forsskål) J. Agardh var. laete‐virens (Montagne) Weber‐van Bosse, and C. serrulata (Forsskål) J. Agardh var. serrulata f. lata (Weber‐van Bosse) Tseng. The eyespot of the biflagellate gamete was visualized using FE‐SEM. The female gamete, but not the male, has one eyespot on the cell body posterior. In most mating pairs, the female gamete is fused at the anterior left side of the eyespot and the male gamete at a cell surface that is perpendicular to the plane of the flagellar beat when both gametes are mixed. Then, the inheritance pattern of cp‐DNA was observed using fluorescence microscopy after staining with 4′6‐diamidino‐2‐phenylindole. Male and female gametes have one cell nucleus and one chloroplast each. Chloroplasts of the female gamete usually contain 1–11 spherical or rod‐shaped nucleoids. In contrast, nucleoids are not usually detected in the male gamete’s chloroplast. After mixing male and female gametes, the male gamete without nucleoids and female gametes with nucleoids are always associated at the lateral side and become planozygotes. Such a correlation between the arrangement of the cell fusion site and the inheritance pattern of cp‐DNA was found in another member of Caulerpales, Bryopsis maxima Okamura. These results suggest the possibility that the arrangement of the cell fusion site in the gamete is not determined randomly regardless of sex, but is rather correlated with specific mating types. The relation of these results to those for Chlamydomonas is discussed.     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001     

Fluorescence microscopy on dynamic changes of frx B distribution inChlamydomonas reinhardtii

Summary Immunocytology method showed that frx B was primarily located in vesicles adjacent to the pyrenoid inChlamydomonas reinhardtii. In synchronous cells, frx B concentration in these vesicles increased in the light period and decreased in the dark period. Prolonged treatment with 5-fluorodeoxyuridine or aging reduced the detectable cellular frx B content. In these cells, frx B appeared as a single dot attached to the pyrenoid. The frx B content in each mating gamete was initially high. As the mating progressed, frx B-containing vesicles in the male gamete disintegrated while that in the female gamete persisted. The time course of this change coincided with that of the chloroplast DNA nucleoids. Persistant small patches of frx B around the pyrenoid were detected in mature zygotes. Fluorescent membrane potential probe showed that membrane vesicles around the pyrenoid had higher transmembrane potential than other chloroplast membranes. This high membrane potential was sensitive to inhibitors of chlororespiration.     

Mating‐Associated Changes in the Behaviour of Leptothorax gredleri Ant Queens

Throughout their lives, animals adapt their behaviour to environmental fluctuations and to their own requirements. In social insects, behavioural changes are often particularly conspicuous. For example, in many ant species, reproductive sexuals leave their maternal nests and engage in risky mating and dispersal activities. Female sexuals experience, during a short period of time, dramatic changes in terms of behaviour and environmental conditions. But because sexual activities of ants are not easily observed, few studies have quantified in detail how behaviour alters with maturation and mating. We studied how various behavioural traits of Leptothorax gredleri female sexuals, a species in which female sexuals attract males by ‘female calling’, change before and after mating. We tested the hypothesis that behavioural variation reflects the altered requirements of queens to adapt to a particular situation. To this end, we compared geotactic, phototactic and locomotor behaviour across a wide range of life stages from lightly coloured, unmated female sexuals to old, mated queens. The results showed that female sexuals of L. gredleri change conspicuously their geotactic, phototactic and locomotor behavioural traits over their life stages. Three different behavioural states were evident (1) from light to dark female sexuals, individuals have negative phototaxis and reduced locomotor activity; (2) mature female sexuals during the daily period of sexual activity have strong phototaxis, negative geotaxis and an important locomotor activity; and (3) freshly mated and old mated queens avoid light and decrease their locomotor activity. These sharp differences in behaviour between stages match the transition from the relative safety of the nest chamber to the adversary world outside the nest , and back.