Sexual Larvae Research Articles

Evidence of queen-rearing suppression by mature queens in the little fire ant, Wasmannia auropunctata.

The little fire ant (LFA), Wasmannia auropunctata, is a serious invasive pest first reported on Hawaii Island in 1999, and has since spread and established itself across the island. LFA is considered one of the worst 100 invasive species and has significant ecological, agricultural, and public health impacts in invaded areas, which include much of the tropical New World. Although localized eradication efforts have proven successful, they are intensive and difficult to implement. Furthermore, LFA's high invasive-ability resists these control efforts in areas where the species is established and can re-infest treated areas. This research set out to determine whether LFA queens have a suppressant effect on new queen production in nests, as a first step in identifying a potential queen pheromone for LFA. A queen pheromone could offer a means to shutdown LFA reproductive capability, potentially by suppressing the production of new queens or inducing the execution of queens or queen-destined larvae. When queenless experimental nests and polygyne experimental nests were compared, six out of eight queenless nests successfully reared both new alate queens (2.25 queens/nest) and drones (3.63 drones/nest) to adulthood, whereas only three of eight polygyne nests reared sexual larvae that failed to develop to adulthood or even the pupal stage. These results suggest that dealate mature LFA queens suppress the production of new alate queens in LFA nests, and is the first evidence that LFA may utilize a queen pheromone.

Myrmecophily and Myrmecophagy of Attacobius lavape (Araneae: Corinnidae) on Solenopsis saevissima (Hymenoptera: Myrmicinae)

Attacobius lavape, a small spider from the Corinnidae family, has been recently described living inside a fire ant colony of Solenopsis saevissima species in the municipality of Morrinhos, south region of the Goiás State, Brazil. Yet several aspects of this spider relationship with the host ant remain unknown. In this way, we performed an extension study to determine its local (Morrinhos) and regional (latitudinal transect) occurrence. We also investigated if the spider uses the host ant as a feeding source. For this, we established arenas with a known number of young and adult ant individuals plus one spider and observed the feeding rate for some determined time. Regarding local distribution, differently from most socially parasitic myrmecophiles, A. lavape showed high local infestation, being found in 47% of the colonies in the sites where the spider occurred, and high transmission, being found in 42% of the 12 collection sites. Regionally, among the 11 study sites, this species only occurred in the municipality of Morrinhos, but its distribution still needs to be verified in the north region. Attacobius lavape consumed eggs, larvae and pupae, confirming that the myrmecophily was explained by myrmecophagy. The spiders consumed eggs (not estimated), 4.45 ± 2.14 larvae and/or 3 ± 0.87 pupae per day. Considering that the mean abundance was approximately seven spiders per colony (extent 1-23), we foresee an impact of 35 larvae and/or 21 pupae consumed per day in each hosting colony. The possibility of consuming sexual eggs, larvae and pupae classifies A. lavape as a potential agent of biological control of S. saevissima.

An asexual strain of Lysiphlebus fabarum (Hymenoptera: Braconidae) gains fitness in superparasitised hosts, but a sexual strain pays costs

1. The fitness impacts of two levels of superparasitism were compared in a sexual and an asexual strain of Lysiphlebus fabarum (Marshall) developing in black bean aphids.2. Asexual females were larger than sexuals, had higher eggs loads, and better adult emergence in two of three treatments, but parasitised fewer available aphids in 24 h (80% fewer when foraging alone).3. Superparasitism resulted in wasps that were larger than those emerging from singly parasitised aphids, and asexual females had larger ovaries, without delayed development. In contrast, sexual wasps took about 2 days longer to develop in superparasitised hosts, and females had smaller ovaries with lower egg loads.4. The growth of host aphids bearing asexual larvae was not reduced relative to healthy aphids, except when heavily superparasitised, whereas sexual larvae reduced aphid growth in all treatments.5. Elimination of supernumerary larvae was virtually complete at 4.5 days after parasitism by sexual females, but no elimination occurred among asexual larvae. The evolution of diminished aggression in asexual larvae may be facilitated by greater genetic similarity, without necessarily leading to gregarious development, assuming the extended life of supernumeraries somehow improves survivor fitness.6. Sons of virgin sexual females developed faster than sons of mated females, implicating parental effects that slowed development of the latter.7. It is concluded that asexual females of this strain use superparasitism to improve host quality, and thus the fitness of their daughters, whereas females of the sexual strain prioritise offspring number.

Caste regulation in the ant Monomorium pharaonis (L.) with emphasis on the role of queens

The role of queens in caste regulation of pharaoh ant colonies has been puzzling biologists for decades. Worker-fertility-inhibiting pheromones are lacking, as workers of this polygynous species are completely sterile. Yet, fertile queens possess inhibiting powers over the production of new gynes and males. Here we test whether queen-produced cuticular compounds or factors related to eggs affect sexual brood survival. Additionally, the influence of dead queens and virgin queens is investigated. Hereto, subcolonies consisting of workers, mixed worker brood and sexual larvae are treated with eggs, living queens, dead queens, virgin queens or queen extracts. The extracts are prepared with (phosphate-buffered saline), hexane or acetone and applied on either glass dummies (chemical cue only) or queen dummies (visual and chemical cue). Subsequently, the number of surviving larvae and their sex ratio are recorded. Only treatments containing living queens show a significant drop in eclosing sexual larvae. In all other treatments the survival of sexual larvae is unaffected. Several hypotheses are discussed involving competition for ‘royal’ food between queens and sexual larvae, food deprival of sexual larvae by workers or cannibalism of sexual brood by queens and/or workers. Although brood cannibalism seems the most valid explanation, as it allows recycling the energy used for brood production throughout the colony, our data could not sufficiently support this hypothesis. Therefore, additional research on different aspects of caste regulation is required to fully understand its mechanism in pharaoh ants.

Queen Red Imported Fire Ants (Solenopsis invictaBuren) Induce Colony Sororicide to Reduce Competition

Queens of red imported fire ants, Solenopsis invicta Buren, regulate colony dynamics by inducing workers to execute sexual larvae; proteins in the venom glands might be involved. Bioassays of the expressed, isolated queen venom proteins, Sol i 2q and Sol i 4q, did not show an increased rate of execution of sexual larvae compared to the control; however, Sol i 4q had a significantly lower execution rate than Sol i 2q and the control. Ants in the Sol i 4q treatments kept their brood primarily in the foraging chamber, closer to the added protein, and did not move them into the pumice nests; however, no difference in execution of sexual larvae between the colony and brood main locations (nest or tube/chamber) were observed. Identification of the crystal structure of the venom protein of red imported fire ant workers, Sol i 2w, revealed a hydrophobic cavity that binds to a ligand with an overall structure similar to LUSH, a pheromone-binding protein in Drosophila. If the proteins of the queen are structurally similar to the proteins of the worker, the possible cavity in Sol i 2q and Sol i 4q potentially binds to the execution pheromone, masking the protein as the pheromonal source instead of the ligand contained within. These hydrophilic queen venom proteins might act as a transport mechanism for the hydrophobic pheromones in the venom. Identification of the source of the pheromone and transport venom proteins is important in understanding the execution of sexual larvae in this pest species.

Mixed asexual and sexual reproduction in the Indo-Pacific reef coral Pocillopora damicornis.

Pocillopora damicornis is one of the best studied reef-building corals, yet it's somewhat unique reproductive strategy remains poorly understood. Genetic studies indicate that P. damicornis larvae are produced almost exclusively parthenogenetically, and yet population genetic surveys suggest frequent sexual reproduction. Using microsatellite data from over 580 larvae from 13 colonies, we demonstrate that P. damicornis displays a mixed reproductive strategy where sexual and asexual larvae are produced simultaneously within the same colony. The majority of larvae were parthenogenetic (94%), but most colonies (10 of the 13) produced a subset of their larvae sexually. Logistic regression indicates that the proportion of sexual larvae varied significantly with colony size, cycle day, and calendar day. In particular, the decrease in sexual larvae with colony size suggests that the mixed reproductive strategy changes across the life of the coral. This unique shift in reproductive strategy leads to increasingly asexual replications of successful genotypes, which (in contrast to exclusive parthenogens) have already contributed to the recombinant gene pool.

The population genetic structure of the waratah anemone (Actinia tenebrosa) around New Zealand

The genetic structure of populations is often shaped by the reproductive system and larval properties of the species. The waratah anemone (Actinia tenebrosa) reproduces through both asexual clones, which have very short-distance dispersal, and sexual larvae, which are believed to disperse much greater distances. The impact of this mixed strategy on the New Zealand population structure of Actinia tenebrosa was investigated using microsatellite markers. The analysis incorporated 24 sampling locations from around New Zealand and one Australian location, using four microsatellite markers, n = 420. We observed low connectivity and high genetic differentiation between all locations sampled, with a distinct pattern of isolation by distance. The most distinct grouping of locations sampled was the north-east of the North Island from Cape Reinga to East Cape, which was identified by SAMOVA and STRUCTURE analyses as being moderately diverged from the remainder of the country. A tentative correlation was observed between genetic clustering and biogeographic regionalisation, where the distribution of a number of genetic clusters matched previously defined biogeographic regions. Within each location sampled, large numbers of clones were present and a latitudinal cline was observed in the relative contribution of asexually and sexually generated recruits, with an increase in asexual recruits on the South Island's east coast.

Genetic Traces of Recent Long-Distance Dispersal in a Predominantly Self-Recruiting Coral

BackgroundUnderstanding of the magnitude and direction of the exchange of individuals among geographically separated subpopulations that comprise a metapopulation (connectivity) can lead to an improved ability to forecast how fast coral reef organisms are likely to recover from disturbance events that cause extensive mortality. Reef corals that brood their larvae internally and release mature larvae are believed to show little exchange of larvae over ecological times scales and are therefore expected to recover extremely slowly from large-scale perturbations.Methodology/Principal FindingsUsing analysis of ten DNA microsatellite loci, we show that although Great Barrier Reef (GBR) populations of the brooding coral, Seriatopora hystrix, are mostly self-seeded and some populations are highly isolated, a considerable amount of sexual larvae (up to ∼4%) has been exchanged among several reefs 10 s to 100 s km apart over the past few generations. Our results further indicate that S. hystrix is capable of producing asexual propagules with similar long-distance dispersal abilities (∼1.4% of the sampled colonies had a multilocus genotype that also occurred at another sampling location), which may aid in recovery from environmental disturbances.Conclusions/SignificancePatterns of connectivity in this and probably other GBR corals are complex and need to be resolved in greater detail through genetic characterisation of different cohorts and linkage of genetic data with fine-scale hydrodynamic models.

Mendelian microsatellite loci for the Caribbean coral Acropora palmata

The genus Acropora constitutes the most species-rich clade of hermatypic corals, and its members are important reef builders throughout their broad tropical range. In the Caribbean, acrop- orid populations have declined over the last 2 decades due to disease, hurricanes, predation, and bleaching episodes, and some are now subjects of conservation efforts. Genetic estimates of popula- tion connectivity and clonal structure should be part of these efforts, but such studies have been ham- pered by low levels of mitochondrial DNA variation in corals, and an apparent dearth of variable sin- gle-copy nuclear markers. Developing microsatellite markers in Acropora has proven especially difficult. We used Southern blotting to reveal that, indeed, some microsatellite motifs (AAC, AAG) are rare in the genome of the Caribbean species Acropora palmata. However, repeats with the motif AAT are both abundant and variable. We developed 8 polymorphic microsatellite markers for A. palmata, and performed crosses to confirm co-dominant inheritance patterns. Five of the 8 mark- ers tested show simple Mendelian inheritance (mean observed heterozygosity = 0.84, mean number of alleles per locus = 8.6). Along with outcrossed sexual larvae, individual egg donors also produced some triploid and selfed larvae that developed normally and survived for 80 h, when the experiment was terminated. The markers reveal variation among 3 Florida populations of A. palmata and among clones within 1 of these populations. Seven of the markers amplify DNA from A. cervicornis and 8f rom the hybrid A. prolifera. These markers should prove to be valuable tools for developing conservation strategies for Caribbean acroporid species.

A queen pheromone induces workers to kill sexual larvae in colonies of the red imported fire ant (Solenopsis invicta).

We conducted five bioassays to study how queens control the execution of sexual larvae by workers in colonies of the red imported fire ant, Solenopsis invicta. In each assay, subset colonies were made from many large polygyne colonies, and the 20 sexual larvae they contained were monitored over time. Sexual larvae mostly survived in queenless colonies, but were mostly killed in colonies with a single dealated queen, regardless of whether or not the queen was fertilized. The larvae were also killed when fresh corpses of queens were added to queenless colonies. Whereas acetone extracts of queens did not produce a significant increase in killings, extracts in buffered saline induced workers to execute most sexual larvae, indicating successful extraction of an execution pheromone. We identified the probable storage location of the chemical as the poison sac, and found both fresh (1 day) and old (21 day) extracts of poison sacs to be equally effective in inducing executions. The pheromone is stable at room temperature, perhaps because venom alkaloids also present in the extracts keep the pheromone from degrading. It is apparently either proteinaceous or associated with a proteinaceous molecule, a novel finding, as no queen pheromone of a proteinaceous nature has been previously demonstrated in ants.

A duration constant for worker-to-larva trophallaxis in fire ants

The allocation of liquid food by workers to larvae, a central process in ant biology, could be regulated by the frequency of trophallaxis, its duration, or both. In 4th-instar fire ant larvae, the duration of trophallaxis, bolus size, and the rate at which boluses were swallowed were all constant, indicating that the volume of food ingested during each worker-larva trophallaxis was both small and constant. Neither larval size over a 20-fold volume range nor larval starvation had a significant effect on duration of trophallaxis (mean = 11s, SD = 2s), bolus swallowing rate (mean = 2/s, SD = 0.5/s), or bolus volume (mean = 0.0675 nl, SD = 0.0002 nl, based on the assumption that the stomodaeum's epithelial layer is not expandable). Larval body orientation and larval location within the brood pile also had no effect on duration. Durations of trophallaxis by workers of different sizes were similar. Durations of trophallaxis for 1st-, 2nd-, and 3rd-instar worker larvae were also constant but greater than that for 4th-instar worker larvae. Fourth-instar minim larvae (from founding colonies) and 4th-instar worker larvae (from mature colonies) were fed for the same duration by workers but for different durations by founding queens. Founding queens fed minim larvae longer than they fed worker larvae. The durations of feedings to 4th-instar sexual larvae were more variable than those to worker larvae. Altogether, these findings indicated that 4th-instar worker larvae ingested a small, nearly constant volume of food (mean = 1.50 nl, SD = 0.005 nl) during each trophallactic event. Consequently, the long-term allocation of liquid food by workers to these larvae is regulated by the frequency of trophallaxis. Several other ant species showed a similar brevity and constancy in the duration of worker-larva trophallaxis. This brevity of worker-larva trophallaxis is in contrast to the duration of worker-worker trophallaxis. Although the duration of worker-larva trophallaxis appears to be determined by the worker, the data are not totally consistent with this interpretation.

Slave addition increases sexual production of the facultative slave-making ant Formica subnuda

Formica subnuda is a facultative slave-making ant, and colonies without slaves are often found. We studied the effect of slave workers on sexual production of F. subnuda by experimentally increasing the proportion of slaves. We added c. 4000 worker pupae of the ant F. podzolica to 15 F. subnuda colonies and kept 15 colonies as controls. The following year we excavated all colonies, counted the proportion of slaves, the total number of workers (colony size) and the number of sexual offspring. The proportion of slaves was significantly higher in the slave-added colonies than in the control colonies. The total production of sexual offspring increased 57% in the treatment colonies in comparison to the controls. When colony size was adjusted to the number of sexual offspring, the treatment colonies produced significantly more sexual offspring than the controls. Slave addition did not alter sex ratios. We suggest that two alternative mechanisms, not mutually exclusive, caused the increase of sexual production in F. subnuda colonies: (1) Most of the added pupae were consumed and stored as fat body in workers at the end of the experimental year; the following spring the excess fat was metabolized and fed to the developing sexual larvae, or (2) a proportion of the added pupae hatched to become slaves; the following spring these slaves foraged actively for protein-rich food for the developing sexual offspring.

Variation in reproductive characteristics of colonies of the freshwater bryozoan Cristatella mucedo

SUMMARY1. We studied relationships between different forms of reproduction and local variation in the reproductive state of colonies of a common freshwater bryozoan Cristatella mucedo Cuvier (Bryozoa: Phylactolaemata). Four sampling locations in central Finland, including both lotic and lentic habitats, were sampled by scuba diving. The traits studied were occurrence of sexual larvae, colony size, number of resting stages (statoblasts) and number of zooids.2. While 76.9% of the sampled colonies carried statoblasts at the time of sampling, only 4.5% of the colonies were brooding sexual larvae. Most of the colonies were relatively small with a mean colony size of 16.6 mm.3. In colonies brooding larvae, the number of statoblasts was positively correlated with the number of larvae. We did not detect a colony size threshold for sexual reproduction or formation of statoblasts. Colonies carrying statoblasts had a lower number of zooids per unit dry weight.4. We found significant variation in reproductive traits of colonies among the sampling locations, and among lotic and lentic habitats. The observed phenotypic differences may reflect broadly distributed, phenotypically plastic clones. Differences in clonal composition of local populations cannot, however, be ruled out and factors that may provide clonal diversity are discussed.

The influence of queens of the ant Paratrechina flavipes on daughter and non‐daughter workers, for the suppression of sexual production

AbstractThe influence that the queen of Paratrechina flavipes (Smith) (Hymenoptera: Formicidae) has upon workers was investigated in field and laboratory nests. There was no significant difference in sexual production, either of gynes or males, between field queenright and queenless nests of the ant. A replicated laboratory experiment revealed that workers lay fewer eggs and rear fewer sexual larvae in the presence of their mother queen, while this reduction is not observed in workers with influence of non‐mother queens. The results seem to support the queen's signal hypothesis by Keller & Nonacs (1993). The contradiction is explained by workers rearing sexuals in queenless satellite nests. There exists a probably complex regulation of the transfer of larvae between satellite and mother nests.

Caste regulation in the pharaoh's ant Monomorium pharaonis: recognition and cannibalism of sexual brood by workers

Abstract In colonies of the pharaoh's ant Monomorium pharaonis (L), new males and queens can be produced at any time by the removal of the inhibitory effect of the presence of existing fertile queens. When queens are absent, workers rear sexual larvae and will also accept sexual larvae introduced from other nests. However, in the presence of fertile queens workers cannibalize sexual brood, and will not accept male or queen larvae or pre‐pupae from other nests, although worker brood is always accepted and reared. Worker larvae are covered in bifurcated hairs, whereas sexual larvae are essentially hairless. Workers may use these morphological cues to distinguish between sexual and worker brood stages.

Social control of egg‐laying rate in queens of the fire ant, Solenopsis invicta*

ABSTRACT Social control of egg‐laying rate in queens of the fire ant (Solenopsis invicta Buren) was studied by experimental manipulation of the number of larvae, pupae and workers in colonies, and the age and size of larvae and workers. Workers and pupae do not stimulate oviposition by queens. The number of fourth instar larvae, on the other hand, bears a positive log‐log relationship to the queen's egg‐laying rate. Such larvae are needed both to stimulate and maintain oviposition. Their withdrawal results, within 48 h, in a decline in queen oviposition almost to zero. Their addition to broodless nests results in peak laying in about 4 days. Larvae in the first three stadia and early in the fourth stadium have a much lower effect upon queen fecundity. Sexual larvae are only c. 5% as stimulating on a weight basis, but equivalent on an individual basis. Several associated measures are positively correlated to egg‐laying rate: weight of the queen, the number of her vitellogenic follicles per ovariole, total vitellogenic follicles, the time she spends feeding and (usually) the number of workers in the retinue that cares for her. The egg volume is negatively correlated with laying rate, so that queens lay more eggs for the same expenditure of material as laying rate increases. As body size of workers increases, they become less effective in transmitting the larval stimulation to the queen, but worker age has no effect on this ability. For a given number of larvae, queens in small, naturally growing colonies lay fewer, larger eggs than do queens in experimental colonies, but their fecundity increases more rapidly in relation to number of larvae. When larvae are fed vital‐dyed food in one experimental colony, and then transferred to an undyed colony, the dye is rapidly transferred to worker crops, and into the queen's eggs, indicating bulk movement of material from larvae to workers to the queen and eggs. Large larvae are more effective at this than small larvae. Fourth instar larvae may be a digestive and metabolic caste that processes protein for egg production by the queen. If that is the case, the queen and fourth instar larvae are linked in a positive feedback loop. Either the logarithmic relation of fecundity to larval numbers or physical limits of the queen may set the maximum egg‐laying rate, and thus determine maximum colony size. The data do not allow a clear choice between these alternatives.

The control of Myrmica rubra workers by queens of their own and other Myrmica species and the interaction between queenless groups of workers

ABSTRACT. Queens of two species of the ant genus Myrmica bonded to workers of the species M. rubra L. as the latter emerge from the pupal skin can use these workers nearly 6 months later to arrest gyne formation in sex‐competent larvae of the same species. Queens of M. ruginodis Nylander var. microgyria (Brian & Brian, 1949) are as good at this as the natural M. rubra, but those of M. sabuleti Meinert (of a race close to M. scabrinodis) are not. Though the M. sabuleti queens induce normal aggression against sexualizing larvae, they are unable to prevent some or all of the workers feeding larvae as though they were queenless. However, queens from different colonies of M. rubra adopted by queenless populations of workers in spring, control their brood‐rearing behaviour perfectly. M. rubra workers from different colonies bring gynes to maturity from female sexual larvae at different average sizes. When workers from two such sources are mixed in equal proportions, the size of gyne larva produced after a week's culture corresponds with that of one of the worker populations; it is not intermediate in size. Also, large workers can rear larger gyne‐larvae than small workers of the same age. This is only true if the workers have been living with queens all the time from emergence as an imago to the moment the experiment was set. Size mixtures only achieve the same size larvae as a pure culture of small workers would. A possible reason for this is that small workers exclude the larger ones from the nursery areas of the nest.

The importance of daylength and queens for larval care by young workers of the ant Myrmica rubra L.

ABSTRACT. Workers whilst still less than a month from emergence rear early stage larvae better in a 16h day than in one of 14h. The callows must perceive the diel rhythm directly because there are no foragers to give them a clue, and the larvae are kept in the dark and cannot respond directly. This, and the study of egg‐laying by young workers that preceded it, indicates that the extra hours daylight enable the workers to avoid the reproductive diapause that normally prepares them for hibernation. They retain active ovaries, ingest and share food with larvae either as trophic eggs or as a regurgitate from the crop.In late summer, callows can rear sexual larvae in spring condition. Queens have a small negative effect on this, expressed as a smaller, though still gyne, pupa produced a little earlier. This weak response to queens develops into a strong negative one at a constant temperature of either 10 or 20°C. The workers then behave as they normally do in spring, and feed a watery regurgitate (dietary control) or, if this fails to arrest sexual development, physically harass the larvae by biting them in the ventral thorax (manipulative control). Large workers then result instead of gynes.

Queen number and the production of sexuals in the fire ant, Solenopsis invicta (Hymenoptera: Formicidae)

To investigate the possibility of queen control over the production of sexuals in polygyne colonies of the fire ant, Solenopsis invicta, large colonies were divided into polygyne (P) and monogyne (M) or queenless (Q-) halves. Sexual larvae were evident in the M and Q- halves 3 to 4 days after colony division, whereas sexual forms failed to develop in all but one of the 32 P halves examined. Both male and female sexuals were produced in abundance in all M (n=25) and Q- (n=7) halves. Evidently, individuals capable of sexualization are present in colonies with many functional queens but are normally prevented from developing. Electrophoretic and morphometric analyses indicated that both haploid and diploid males were produced in the Q- halves, although diploids far outnumbered haploids. It thus appears that queens exert control over all potential and genetically determined sexuals regardless of sex or ploidy. The timing of the appearance of sexual forms following colony division suggests that queen control may be pheromonally mediated and inhibits the growth of sexuals late in larval development. An experiment in which the queens from M and P halves of colonies were exchanged demonstrated the reversible nature of this inhibition within colonies, but also suggested that once individual larvae develop beyond a critical point they are no longer subject to queen control. Despite seasonal variation in the production of sexuals in the field, no substantial differences between colonies collected in the summer and fall were found in their response to colony manipulations. The interaction of colony weight and number of queens present prior to colony division was associated with the number of males produced in the Q- halves, but no factors examined were associated with the number of females produced in these halves, or with the number of males or females produced in the M halves.

Evidence of pheromonal queen control over the production of male and female sexuals in the fire ant,Solenopsis invicta

Two hypotheses that could explain social regulation of the production of sexuals inSolenopsis invicta were investigated: (1) differences in worker/larva ratios; and (2) pheromonal regulation by queens. Small laboratory units (fragments of multiple-queen, i.e. polygyne, colonies) were found to be capable of producing sexuals and to be sensitive in this regard to differences in queen number; the presence of even a single queen inhibited the numbers of sexuals (male and female) produced in comparison with queenless controls. To determine whether differences in worker/larva ratios were involved in this inhibition, a greater number of eggs than could be laid by a single queen was added daily to queenless units. These units produced more sexuals than similar units containing one queen, but did not differ from queenless controls which did not receive eggs. These results rule out the possibility that differences in worker/ larva ratios significantly affected the production of sexuals in such experimental units. They also exclude the possibility that chemical cues were transmitted by queens via their eggs. To test the hypothesis that queen pheromones were involved, freshly killed corpses of functional (egg-laying) queens were added daily to queenless units. These effectively inhibited the production of sexuals, although not as effectively as living queens. On the other hand, corpses of alate virgin (non-egg-laying) queens did not inhibit the production of sexuals. The addition of live queens to previously queenless units in which large sexual larvae had developed resulted in the execution of most of these larvae by workers, indicating that queen control over the production of sexuals can act retroactively after larvae are sexualized. A similar but less pronounced result was obtained by the addition of functional queen corpses. These results provide evidence that functional queens exert control over the production of sexuals inS. invicta through pheromones that influence the behavior of workers toward both male and female larvae.