Timing of male territorial flight and foraging of the large carpenter bee Xylocopa appendiculata related to serotonin in the brain.

To determine how males of the large carpenter bee, Xylocopa appendiculata, maximize access to females while minimizing energy cost and acquiring energy for territorial flights, we investigated the times of territorial flights by males and foraging by males and females. Males were present continuously in territories from 8:00 to 12:00. They approached, chased, and excluded conspecific males from their territories. In the laboratory, males showed higher locomotor and flight activities in the morning and lower activities in the afternoon. Both males and females visited flowers from 8:00 to 16:00, but the most frequent visits were earlier in females (10:00-12:00) than in males (12:00-13:00). Relative body weights in territorial males often increased. These results indicate that the males time their territorial flights to maximize contact with females and obtain nectar as fuel between and after the territorial flights. The time-related territorial flight in males might be based on a time-keeping system in the brain. Brain levels of serotonin and its precursor tryptophan were significantly higher in males collected at 16:00 than at 11:00, suggesting a relation between time-related territorial flight and serotonin synthesis in the brain.

Honeybee colonies provide foragers with costly fuel to promote pollen collection.

Honeybee pollen foragers departing the hive carry concentrated nectar to use as fuel for flight and glue for forming pollen loads. Since nectar is concentrated by in-hive bees at the cost of time and energy, using concentrated nectar increases the cost of foraging at the colony level. This experimental study explored the potential benefit to honeybees of using concentrated nectar for pollen collection by diluting nectar carried by pollen foragers from the hive. Mass feeding with 30% sugar solution successfully reduced the crop-load-sugar concentration in putative pollen foragers departing the hive, but while those bees tended to increase the crop-load volume, such increase did not fully compensate for the decreased amount of dissolved sugars in the crop load. Feeding 30% sugar solution reduced the pollen load dry weight by approx. 10-20% compared to the unfed control and to another test group fed 60% sugar solution. In addition, the pollen load size and sugar concentration of crop load remaining in returning pollen foragers was positively correlated. These results clearly show the advantage to honeybees of using concentrated nectar for pollen foraging.

Adjustment of fuel loads in stingless bees (Melipona subnitida).

Eusocial bee foragers leave their nest with nectar as flight fuel, therewith reducing the risk of starvation during a foraging trip. Yet, the extra mass results in an increase of energetic expenditure for flight. Thus, bees should tune their fuel loads to the respective foraging situation. In the present study, we investigated the fuel adjustment in the Brazilian stingless bee Melipona subnitida (Apidae, Meliponini). Specifically, we examined whether foragers of this species increase their fuel loads when they have low expectation for nectar collection during a foraging trip. Crop load measurements revealed that nectar foragers carried significantly less fuel on departing the nest than foragers collecting either pollen, clay, or resin. Surprisingly, 75% of nectar foragers left the nest without any detectable amount of nectar, which suggests that the majority of bees collected at nearby nectar sources and avoided an increase in foraging costs. Moreover, foragers increased their fuel loading when repeatedly experiencing empty food sources that had previously been rewarding. These results support our hypothesis and demonstrate that the capability of fuel adjustment is not restricted to honey bees.

Caste-specific development of the dopaminergic system during metamorphosis in female honey bees.

Caste-specific differences in the dopaminergic systems of social insects assist in maintaining caste-specific behavior. To determine how caste differences in the honey bee occur during metamorphosis, a number of comparative analyses between castes were performed including comprehensive quantification of: levels of dopamine and its metabolite in the brain, the gene expression levels of enzymes involved in dopamine biosynthesis and conversion as well as expression levels of dopamine receptors and a dopamine transporter. Dopamine levels standardized to the protein contents of a whole brain at the day of eclosion in queens were 3.6-fold higher than those in workers. Dopamine levels increased until eclosion (7 days) in queens, whereas those in workers increased until 5-6 days before eclosion and then maintained until eclosion (10 days). These caste-specific dopamine dynamics in the brain were supported by the higher expression of genes (Amddc and Amth) encoding enzymes involved in dopamine synthesis in queens. The distribution of cells expressing Amddc in the brain revealed that soma clusters of dopaminergic cells were similar between castes at 7-8 days after pupation, suggesting the upregulation of Amddc expression in some cells in queens rather than addition of cell clusters. In contrast, genes encoding dopamine receptors were downregulated in queens or showed similar expression levels between castes. The expression of genes encoding dopamine transporters did not differ between castes. These results reveal the developmental process of caste-specific dopaminergic systems during metamorphosis in the honey bee, suggesting caste-specific behavior and division of reproduction in this highly eusocial species.

Male scent-marking pheromone of Bombus ardens ardens (Hymenoptera; Apidae) attracts both conspecific queens and males.

To explore the role of the volatiles emitted from male labial gland (LG) of the bumblebee Bombus ardens ardens, we investigated the responses of virgin queens and males to volatiles using a gas chromatography-electroantennographic detector (GC-EAD) system and Y-tube olfactometer. GC-EAD analysis revealed that citronellol, the main compound detected in the male LG, caused clear electrophysiological responses in the antennae of B. a. ardens virgin queens and males although two minor compounds elicited antennal responses when applied in a high concentration. Behavioral tests using a Y-tube olfactometer showed that queens and males were significantly attracted to both LG extracts and citronellol more than to the solvent alone. This is the first study to demonstrate that citronellol as a major compound of male scent-marking pheromone in B. a. ardens functions as a sex attractant for queens. The results also suggest that this compound has another function as a trail marker used by males.

The mechanism controlling phenotypic plasticity of body color in the desert locust: some recent progress.

Schistocerca gregaria exhibits density-dependent body color polyphenism. Nymphs occurring at low population densities show green-brown polyphenism. They show phase polyphenism and develop black patterns at high population densities. Recent studies suggest a third type of polyphensim, that is, homochromy, a response to background color. Laboratory experiments that considered homochromy suggest that humidity is not directly involved in green-brown polyphenism and that odor from other individuals does not induce black patterns. Black patterns can be induced in isolated nymphs by video images of locusts and tadpoles. Juvenile hormone and [His7]-corazonin control body color in locusts. The gene encoding the latter has been identified for S. gregaria and Locusta migratoria, and its key role in controlling black patterning has been demonstrated.

Honeybee males use highly concentrated nectar as fuel for mating flights.

Honeybees use nectar held in the crop as their main source of energy for flight but the mass of the crop nectar load may be a cost burden. This study investigated whether males of the honeybee Apis mellifera adjust their nectar fuel load and concentration to enhance the success of mating flights. When the crop content was compared between males staying in the hive and those departing, the latter group had the larger volume (median, 5.0µl; range, 0.0-17.8µl) and higher concentration (median, 71.6%; range, 49.0%-77.6%), indicating that departing males load concentrated nectar as fuel before mating flights. Moreover, the crop nectar concentration was significantly higher in departing males than in departing workers. These results suggest that concentrated nectar is advantageous to males because it provides more sugar for energy at lower mass and secures longer or more effective mating flights for higher chance of reproductive success. No significant effect of age was detected in crop volume, and concentration and amount of dissolved sugars in the crop content. In addition, laboratory experiments showed that males had only about 5µl of nectar in the crop soon after feeding, irrespective of fed volume (5-15µl), suggesting they do not hold much nectar in the crop but send it rapidly to the midgut, unlike workers.

Nectar loads as fuel for collecting nectar and pollen in honeybees: adjustment by sugar concentration.

When honeybee foragers leave the nest, they receive nectar from nest mates for use as fuel for flight or as binding material to build pollen loads. We examined whether the concentration of nectar carried from the nest changes with the need for sugar. We found that pollen foragers had more-concentrated nectar (61.8 %) than nectar foragers (43.8 %). Further analysis revealed that the sugar concentration of the crop load increased significantly with waggle duration, an indicator of food-source distance, in both groups of foragers. Crop volume also increased with waggle duration. The results support our argument that foragers use concentrated nectar when the need for sugar is high and suggest that they precisely adjust the amount of sugar in the crop by altering both volume and nectar concentrations. We also investigated the impact of the area where foragers receive nectar on the crop load concentration at departure. Although nectar and pollen foragers tend to load nectar at different areas in the nest, area did not have a significant effect on crop load concentration. Departing foragers showed an average of 2.2 momentary (<1 s) begging trophallactic contacts before leaving the nest. They might be rejecting nectar with inappropriate concentrations during these contacts.

Honey loading for pollen collection: regulation of crop content in honeybee pollen foragers on leaving hive.

Before foraging honeybees leave the hive, each bee loads its crop with some amount of honey "fuel" depending on the distance to the food source and foraging experience. For pollen collection, there is evidence that foragers carry additional honey as "glue" to build pollen loads. This study examines whether pollen foragers of the European honeybee Apis mellifera regulate the size of the crop load according to food-source distances upon leaving the hive and how foraging experience affects load regulation. The crop contents of bees foraging on crape myrtle Lagerstroemia indica, which has no nectary, were larger than those foraging on nectar from other sources, confirming a previous finding that pollen foragers carry glue in addition to fuel honey from the hive. Crop contents of both waggle dancers and dance followers showed a significant positive correlation with waggle-run durations. These results suggest that bees carry a distance-dependent amount of fuel honey in addition to a fixed amount of glue honey. Crop contents on leaving the hive were statistically larger in dancers than followers. Based on these results, we suggest that pollen foragers use information obtained through foraging experience to adjust crop contents on leaving the hive.

Effects of barley chromosome addition to wheat on behavior and development of Locusta migratoria nymphs.

Locusta migratoria feeds on various Poaceae plants but barley. Barley genes related to feeding deterrence may be useful for developing novel resistant crops. We investigated the effects of barley cultivar Betzes, wheat cultivar Chinese Spring (CS), and six barley chromosome disomic addition lines of wheat (2H-7H) on locomotor activity, feeding behavior, survival and development of L. migratoria nymphs. Locomotor activity was similar in nymphs kept with wheat and 2H-7H in an actograph, whereas it was generally high in those kept with barely. No-choice and choice feeding tests suggested that barley genes related to inhibition of feeding by L. migratoria are located on barley chromosomes 5H and 6H and those related to the palatability of plants on chromosomes 2H, 5H and 6H. Rearing experiments suggested the presence of barley genes negatively affecting the survival and growth of locust nymphs on chromosomes 5H and 2H, respectively, and the effects are phase-dependent.

Caste differences in dopamine-related substances and dopamine supply in the brains of honeybees (Apis mellifera L.).

We determined the mechanisms underlying caste differences in the brain levels of dopamine-related substances in adult honeybees (Apis mellifera L.). Brain levels of dopamine, DOPA (a dopamine precursor), and N-acetyldopamine (a dopamine metabolite, NADA) were significantly higher in three-day-old virgin queens than same-aged workers. Caste differences in dopamine and NADA levels were also found in the hemolymph. The in vitro enzymatic activities of DOPA decarboxylase (DDC) during dopamine synthesis in brains were not significantly different between castes. The DDC activity in adult queens was mainly found in the brain, but with lower levels of activity detected in the mandibular glands, salivary glands and ovaries. Oral application of DOPA to workers led to DOPA uptake in the brain and significantly higher dopamine and NADA levels in the brain, suggesting that dopamine synthesis could be controlled by the amount of DDC substrate. Royal jelly samples taken from queen cells had a >25-fold higher concentration of dopamine compared with honey samples collected from honey cells. However, oral application of the same concentration of dopamine did not significantly enhance the brain levels of dopamine and NADA. These results suggest that the higher levels of brain dopamine in queens compared with workers can be explained by the higher level of DDC substrate, rather than DDC activity in the brain and other tissues of queens as well as exogenous dopamine in the royal jelly.

Phase-dependent locomotor activity in first-stadium nymphs of the desert locust, Schistocerca gregaria: effects of parental and progeny rearing density.

This study examined the effects of parental and progeny rearing density on locomotor activity of 1st-stadium nymphs of the desert locust, Schistocerca gregaria, using an actograph. Progeny obtained from solitarious (isolated-reared) or gregarious (crowd-reared) locusts were reared in isolation or in a group of 30 nymphs. Crowding after hatching had a slight influence on mean activity shortly after the start of measurements, but no clear effect was detected until day 2, when maximum activity during the 6-24 h of observation was significantly higher than that of the nymphs kept in isolation. On the other hand, the effects of parental rearing density on locomotor activity manifested at all ages examined (0-2 days old). Progeny of gregarious locusts showed consistently higher activity than those of solitarious locusts. In newly hatched nymphs, the effect of parental rearing density was explained by variation in body size at hatching, one of the phase-dependent characteristics. Hatchling body color was also correlated with locomotor activity and body weight. Similar levels of locomotor activity were exhibited when green, solitarious and black, gregarious nymphs were similar in body weight. These results suggested that parental rearing density indirectly influences locomotor activity in the progeny shortly after hatching by affecting their body size as eggs or hatchlings.

Re-examination of the roles of environmental factors in the control of body-color polyphenism in solitarious nymphs of the desert locust Schistocerca gregaria with special reference to substrate color and humidity.

This study re-examines the effects of environmental factors including substrate color, humidity, food quality, light intensity and temperature on the green-brown polyphenism, black patterning and background body color of solitarious (isolated-reared) nymphs of Schistocerca gregaria. All individuals reared in yellow-green or yellow containers became green morphs, whereas those reared in white, ivory-colored, blue, grey, brown, zinc-colored and black containers produced brown morphs in similar proportions. The intensity of black patterns was negatively correlated with the brightness of the substrate color of the containers. Humidity, which previous studies claimed controls green-brown polyphenism in this species, exerted no significant influence on either the green-brown polyphenism or the black patterning. Food quality also had little effect on body color. High temperature tended to inhibit darkening. The background body color on the thorax was greatly influenced by the substrate color of rearing containers and a close correlation was found between these two variables, indicating that, in contrast to what has been suggested by others, this species exhibits homochromy to match the body color to the substrate color of its habitat. Similar responses were observed in another strain, although some quantitative differences occurred between the two strains examined. Based on these results, a new model explaining the control of body-color polyphenism in this locust is proposed and the ecological significance of black patterns in solitarious nymphs is discussed.

Tactile stimuli perceived by the antennae cause the isolated females to produce gregarious offspring in the desert locust, Schistocerca gregaria.

Maternal determination of progeny body size and coloration in the desert locust, Schistocerca gregaria, depends on the crowding conditions experienced during the short sensitive period that occurs two to six days before the deposition of the egg pod. Solitarious (isolated-reared) females produce relatively small eggs that yield solitarious green hatchlings but, females that are exposed to crowded conditions during the sensitive period, produce larger eggs that yield the dark-colored hatchlings characteristic of gregarious forms. The present study aimed to determine the stimuli influencing the maternal determination of progeny characteristics as well as the site at which such stimuli are perceived. By exposing isolated female adults to various combinations of visual, olfactory and tactile stimuli from a crowd of other adults, we found that no crowding effects could be elicited without tactile stimulation. Coating of various body surfaces with nail polish followed by exposure to crowding stimulation suggested that female adults perceive crowding stimuli with their antennae. This finding was supported by another experiment in which the antennae were either removed or covered with wax before the isolated females were exposed to crowded conditions. Neither serotonin nor an antagonist of its receptor affected the density-dependent maternal determination of progeny characteristics when injected into isolated or crowded female adults.

Effects of parental and progeny rearing densities on locomotor activity of 1st-stadium nymphs in the migratory locust, Locusta migratoria: an analysis by long-term monitoring using an actograph.

The effects of parental and progeny rearing densities on locomotor activity in 1st-stadium nymphs of the migratory locust, Locusta migratoria, were observed over a 24- or 36-h period using an actograph. Newly hatched nymphs showed a small activity peak shortly after hatching and the peak level was significantly higher in offspring (gregarious nymphs) of crowd-reared adults than in those (solitarious nymphs) of isolated-reared adults. However, no significant difference was found between the two groups in maximum activity levels exhibited after the initial peak. Post-hatching crowding enhanced locomotor activity during 2-5h of measurements in 2-day-old nymphs. In this case, the parental density resulted in no significant influence on locomotor activity. However, the maximum activity level shown later in the observation period was higher in gregarious nymphs than in solitarious nymphs. Interestingly, this parental effect was more pronounced in nymphs reared in group than in those reared in isolation. The parental density appeared to affect the degree of response to crowding in the progeny. No evidence was found for the phase accumulation in terms of locomotor activity. The variation observed in locomotor activity among geographical populations did not correspond to their phylogenetic relationships.

Dopamine enhances locomotor activity for mating in male honeybees (Apis mellifera L.).

Dopamine plays multiple roles in the regulation of reproduction in female honeybees where it appears to act independently of juvenile hormone (JH). In males the role of dopamine and its relationship to JH control have not been elucidated. In the present study we determined hemolymph levels of dopamine and its metabolite (N-acetyldopamine) in males at post-emergence days 0-16. The development of locomotor and flight activities were recorded over the same period. Hemolymph levels of dopamine and N-acetyldopamine were found to increase at the time of onset of mating flight activity and those of dopamine decreased thereafter. Both locomotor and flight activities increased in parallel with hemolymph dopamine levels but the increased activity levels were maintained following decline of dopamine levels. Brain and meso-metathoracic ganglia levels of dopamine showed a similar developmental profile to hemolymph dopamine levels. Locomotor activities were temporarily inhibited by injection of a dopamine-receptor antagonist (cis(Z)-flupenthixol) into the thorax, and were enhanced by injection of a dopamine-receptor agonist (6,7-ADTN). These results suggest that dopamine regulates locomotor activities for mating and plays a role downstream of JH in premature males in honeybees.

Influence of age and juvenile hormone on brain dopamine level in male honeybee (Apis mellifera): association with reproductive maturation.

Dopamine (DA) is a major functional biogenic amine in insects and has been suggested to regulate reproduction in female honeybees. However, its function has not been investigated in male drones. To clarify developmental changes of DA in drones, brain DA levels were investigated at various ages and showed a similar pattern to the previously reported juvenile hormone (JH) hemolymph titer. The DA level was lowest at emergence and peaked at day 7 or 8, followed by decline. Application of JH analog increased brain DA levels in young drones (2-4-days-old), suggesting regulation of DA by JH in drones. In young drones, maturation of male reproductive organs closely matched the increase in brain DA. The dry weight of testes decreased and that of seminal vesicles increased from emergence to day 8. The dry weight of mucus glands increased up to day 4. Consequently, DA regulated by JH might have reproductive behavior and/or physiological functions in drones.

Depression of brain dopamine and its metabolite after mating in European honeybee (Apis mellifera) queens.

To explore neuro-endocrinal changes in the brain of European honeybee (Apis mellifera) queens before and after mating, we measured the amount of several biogenic amines, including dopamine and its metabolite in the brain of 6- and 12-day-old virgins and 12-day-old mated queens. Twelve-day-old mated queens showed significantly lower amounts of dopamine and its metabolite (N-acetyldopamine) than both 6- and 12-day-old virgin queens, whereas significant differences in the amounts of these amines were not detected between 6- and 12-day-old virgin queens. These results are explained by down-regulation of both synthesis and secretion of brain dopamine after mating. It is speculated that higher amounts of brain dopamine in virgin queens might be involved in activation of ovarian follicles arrested in previtellogenic stages, as well as regulation of their characteristic behaviors.