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The transport of lipids constitutes a vital function in insects and requires the plasma lipoprotein lipophorin. In all insects examined to date, cuticular hydrocarbons are also transported through the hemolymph by lipophorin, and in social insects they play important roles not only in water proofing the cuticle but also in nestmate recognition. High-density lipophorin (HDLp), isolated from Reticulitermes flavipes plasma by KBr gradient ultracentrifugation, contains 66.2% protein and 33.8% lipids; hydrocarbons constitute its major neutral lipid (20.4% of total lipids). Anti-lipophorin serum was generated in rabbit and its specific association with lipophorin, and not with any other plasma proteins, was verified with Western blotting. Immunoprecipitation also confirmed that this antibody specifically recognizes lipophorin, because all hemolymph hydrocarbons of the termites R. flavipes and R. lucifugus and the cockroach Supella longipalpa, which associate only with lipophorin, were recovered in the immunoprecipitated protein. Cross-reactivity of the antiserum with lipophorin from related species was investigated by double immunodiffusion with 10 termite species in the genera Reticulitermes, Coptotermes, Zootermopsis, and Kalotermes, and with five cockroach species. Involvement of lipophorin in hydrocarbon transport was shown by injecting HDLp antiserum into Zootermopsis nevadensis and then monitoring the de novo biosynthesis of hydrocarbons and their transport to the cuticular surface; the antiserum significantly disrupted hydrocarbon transport. ELISA revealed a gradual increase in the lipophorin titer in successively larger R. flavipes workers, and differences among castes in lipophorin titers were highest between nymphs and first instar larvae.

Studies were conducted on the physiological and hormonal changes following the release of alates from developmentally suppressive pheromones produced by mature queens of the fire ant Solenopsis invicta Buren. Winged virgin queens were removed from the pheromonal signal and placed in colony fragments. The time for dealation, degree of ovarian development, and biosynthesis rate and whole body content of juvenile hormone (JH) were measured. The production rate and content of JH were highly correlated. Dealation and the initiation of oviposition corresponded to peak production of JH. JH production rose sharply following separation from the natal nest, peaking after 3 days. After 8 days of isolation, JH production gradually subsided to levels similar to that found in pre-release queens, but began to increase again after 12 days. Mature queens had highly elevated levels of JH relative to recently dealate females, probably reflecting the increased reproductive capability of these older females. The results support the hypothesis that the pheromone released by functional queens inhibits reproduction in virgin alates by suppressing corpora allata activity and the production of JH.

Studies were conducted on the physiological mode of action and mode of perception of a queen pheromone that inhibits dealation (wing-shedding) and ovary development in virgin queens of the fire ant Solenopsis invicta. Winged virgin queens were removed from the pheromonal signal (queen) to compare the response time for dealation, a behavioral response, and ovary development, a physiological response. Dealation was always accompanied by some degree of ovary development, whereas some individuals exhibited slightly developed ovaries before dealation occurred, suggesting that ovary development precedes dealation by several hours to a day. The response time of virgin queens following extinction of the pheromonal signal was highly variable and was related to colony source and body weight. Individuals from monogyne (single functional queen) colonies were more responsive than those from polygyne (multiple functional queens) colonies, a result consistent with the high cumulative levels of queen pheromone in the latter colonies. Virgin queens treated topically with the juvenile hormone (JH) analogue, methoprene, dealated and developed their ovaries in the presence of the pheromone (queen), suggesting that the pheromonal mode of action involves the suppression of JH titers. To obtain more precise information on the physiological processes underlying the inhibition of ovary development, vitellogenin titers were determined for virgin queens and functional (egg-laying) queens. Despite having undeveloped ovaries, virgin queens had vitellogenin titers that were as elevated as those of functional queens. This suggests that the effect of the low JH titers resulting from the primer pheromone is on the uptake of vitellogenin by the oocytes rather than on vitellogenin synthesis. The possible mode of perception of the inhibitory pheromone was also investigated. Virgin queens whose antennae had been removed dealated in the presence of the queen, suggesting that the pheromone acts by stimulating sensory cells in the antennae. The results are incorporated into a general model for the mode of action of the queen primer pheromone.

Termite queens are highly specialized for reproduction, but little is known about the endocrine mechanisms regulating this ability. We studied changes in the endocrinology and ovarian maturation in primary reproductive females of the dampwood termite Zootermopsis angusticollis following their release from inhibitory stimuli produced by mature queens. Winged alates were removed from their natal nest, manually dewinged, then paired in an isolated nest with a reproductive male. Development was tracked by monitoring ovarian development, in vitro rates of juvenile hormone (JH) production by corpora allata, and hemolymph titers of JH and ecdysteroids. The production rate and titer of JH were positively correlated with each other but negatively correlated with ecdysteroid titer. Four days after disinhibition, JH release and titer decreased while ecdysteroid titer increased. The new levels persisted until day 30, after which JH increased and ecdysteroids decreased. Fully mature queens had the highest rates of JH production, the lowest ecdysteroid titers, and the greatest number of functional ovarioles. The results support the hypothesis that JH plays a dual role in termite queens depending on their stage of development; an elevated JH titer in immature alates may maintain reproductive inhibition, but an elevated JH titer in mature queens may stimulate ovarian activity. The decline in JH production and the elevation in ecdysteroid titer correspond to a period of physiological reorganization and activation. The specific function of ecdysteroids is unknown but they may help to modulate the activity of the corpora allata.

The poison sac of the fire ant Solenopsis invicta is the only identified glandular source of pheromones produced by a functional ant queen. This structure, which contains the poison gland, has previously been shown to be the source of a releaser pheromone that mediates queen recognition and tending by workers. The poison sac has also been demonstrated to be the source of a primer pheromone that inhibits winged, virgin queens from shedding their wings (dealating) and developing their ovaries. To determine if the poison sac was the only source of these pheromones, we excised the poison sac from queens and observed whether operated queens retained their pheromonal effects. In a first experiment, the poison sac was removed from functional (egg-laying) queens which were then paired with unoperated nestmate queens in small colonies. Counts of the workers surrounding each queen two weeks after the operation showed that queens without poison sac were as effective as their unoperated nestmates in attracting worker retinues. In a second experiment, we removed the poison sacs of virgin queens which had not yet begun laying eggs and thus had not begun producing queen pheromone. After allowing them to develop their ovaries, these individuals produced amounts of queen recognition pheromone comparable to those secreted by unoperated or sham operated virgin queens as determined by bioassay. Testing the head, thorax and abdomens of functional queens separately revealed that the head was the most attractive region in relation to its relative surface area. Bioassays of extracts of two cephalic glands—the mandibular gland and postpharyngeal gland—showed that the postpharyngeal gland is a second source of the queen recognition pheromone. Finally, we found that virgin queens whose poison sacs were removed before they began producing queen pheromone initiated production of a primer pheromone that inhibits winged virgin queens from dealating, indicating that this pheromonal effect also has an additional but as yet undetermined source. These results parallel those on the honey bee in which several of the pheromonal effects of functional queens appear to have multiple glandular sources.

The viability of eggs laid by individual functional queens from polygyne colonies of the fire ant, Solenopsi:; intricra Buren, was examined by staining for chromatin. In isolation units held in the laboratory inseminated queens laid nearly 100% viable eggs, compared to < 10% for uninseminated queens, Experiments in which the quantity of brood was manipulated demonstrated that the number of brood and workers present has little, if any, effect on the ability of a queen's eggs to form embryos, in contrast to th': stimulatory effect of larvae on oviposition rate. In field colonies, although there was considerable variability among queens, inseminated queens laid a significantly greater proportion of viable eggs than did uninseminated queens. Inseminated queens taken directly from polygyne colonies in the field laid a smaller proportion of viable eggs than those maintained individually for several weeks in the laboratory, suggesting that there is mutual inhibition among cohabiting inseminated queens which affects viable egg pro'duction. Among queens taken from the field, there was no significant correlation between the proportion of viable eggs laid and queen weight, a reliable indicator of oviposition rate. Thus caution should be exercised in equating relative rates of egg production with reproductive success in studies of reproductive roles in social insect colonies.