Masami Sasaki

Associative visual learning, color discrimination, and chromatic adaptation in the harnessed honeybee Apis mellifera L.

We studied associative visual learning in harnessed honeybees trained with monochromatic lights associated with a reward of sucrose solution delivered to the antennae and proboscis, to elicit the proboscis extension reflex (PER). We demonstrated five properties of visual learning under these conditions. First, antennae deprivation significantly increased visual acquisition, suggesting that sensory input from the antennae interferes with visual learning. Second, covering the compound eyes with silver paste significantly decreased visual acquisition, while covering the ocelli did not. Third, there was no significant difference in the visual acquisition between nurse bees, guard bees, and foragers. Fourth, bees conditioned with a 540-nm light stimulus exhibited light-induced PER with a 618-nm, but not with a 439-nm light stimulus. Finally, bees conditioned with a 540-nm light stimulus exhibited PER immediately after the 439-nm light was turned off, suggesting that the bees reacted to an afterimage induced by prior adaptation to the 439-nm light that might be similar to the 540-nm light.

CpG Methylation in the Hexamerin 110 Gene in the European Honeybee, Apis mellifera

Abstract The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), has a full set of machinery for functional CpG methylation of its genome. A recent study demonstrated that DNA methylation in the honeybee is involved in caste differentiation. In this study, the expression and methylation of the hexamerin 110 gene (Hex110), which encodes a storage protein, was analyzed. High levels of the Hex110 transcript were expressed in both worker and queen larvae. Low levels of this transcript were also detected in adult fat bodies, and the expression level was higher in the queen than in the worker. Bisulfite sequencing revealed that the Hex110 gene is overall methylated at a low level, with a limited number of CpG sites methylated at relatively high levels. These highly methylated sites were exclusively located in the exon regions. The average methylation rate of the Hex110 gene was higher in the adult stage than in the larval stage. Furthermore, several CpG sites were differentially methylated between the worker and queen larvae. These observations suggest that the methylation of the Hex110 gene is regulated at the developmental stage and in a caste-dependent manner.

Heat production by balling in the Japanese honeybee,Apis cerana japonica as a defensive behavior against the hornet,Vespa simillima xanthoptera (Hymenoptera: Vespidae)

As an effective counterattack strategy against predacious hornets, especiallyVespa simillima xanthoptera, workers ofApis cerana japonica showed a distinct balling reaction, usually involving 180–300 bees. This produced heat for as long as 20 min, giving rise to temperatures inside the ball higher than 46°C, which is lethal to the hornet but not to the bees.

Insect signalling: Components of giant hornet alarm pheromone

Up to 74 people die each year in Japan after being stung by Hymenopteran insects, with hornets (Vespa spp.) being among the worst offenders. Here we identify a volatile, multi-component alarm pheromone in the venom of the worlds largest hornet, V. mandarinia, and use field bioassays to show that 2-pentanol is its principal active component, and that 3-methyl-1-butanol and 1-methylbutyl 3-methylbutanoate act synergistically with it. The compound 1-methylbutyl 3-methylbutanoate, which may also be a foraging-site-marking pheromone, elicits a strong defensive reaction in the sympatric prey hornet V. simillima xanthoptera. As these chemicals are sometimes used in food flavourings and as fragrances in cosmetics, it is possible that they might provoke a seemingly unwarranted hornet attack on humans.

Functional Flexibility of the Honey Bee Hypopharyngeal Gland in a Dequeened Colony

Abstract The role of the worker honey bee Apis mellifera L. changes depending on age after eclosion (age polyethism): young workers (nurse bees) take care of their brood by synthesizing and secreting brood food (royal jelly), while older workers (foragers) forage for nectar and process it into honey. Previously, we showed that the major proteins synthesized in the hypopharyngeal gland of the worker change from brood food proteins to α-glucosidase at the single secretory cell level in parallel with this age polyethism [Kubo et al., J. Biochem. 119, 291–295 (1996); Ohashi et al., Eur. J. Biochem. 249, 797–802 (1997)]. Here, we examined whether the function of the hypopharyngeal gland has flexibility depending on the colony conditions, by creating a dequeened colony in which the older workers were compelled to feed the drone larvae. It was found that most of the older workers in the dequeened colony synthesized brood food proteins as did nurse bees. Furthermore, the percentage of workers that synthesized brood food proteins was maintained at 80–90% of the total workers for at least two months, as in a normal colony. These results indicate that the function of the hypopharyngeal gland cells of the worker has flexibility and can, if necessary, be maintained as that of the nurse bee, depending on the condition of the colony.

Reduced expression of major royal jelly protein 1 gene in the mushroom bodies of worker honeybees with reduced learning ability

The learning ability of European honeybees, Apis mellifera, develops with age. However, when worker bees are isolated from their colony and are fed only sucrose solution, their learning development is hindered. This rearing method has allowed us to compare worker bees of the same age but with different learning abilities. In this study, we examined the influence of this rearing condition on gene expression in the mushroom body, which is the insect brain center involved in learning and memory. A differential display experiment comparing worker bees maintained in a hive with those reared in isolation showed that the expression of the major royal jelly protein (mrjp) 1 gene was reduced in the isolated worker bees. MRJP1 is synthesized in the hypopharyngeal gland and serves a nutritional function in larval and queen food. Our results suggest that mrjp1 is also important in brain function, possibly involved in the development of learning ability.ZusammenfassungDie Lernfähigkeiten der Westlichen Honigbiene, Apis mellifera, entwickeln sich mit zunehmendem Alter der Arbeiterin weiter. Das Duftlernen ist ein Beispiel hierfür. Wenn junge Arbeiterinnen allerdings in Isolation ohne ihr Volk gehalten werden und ausschließlich mit Zuckerlösung gefüttert werden, wird ihre Lernentwicklung behindert. Diese Aufzuchtmethode erlaubte uns, Arbeiterinnen desselben Alters mit unterschiedlichen Lernfähigkeiten zu untersuchen. Während unserer Studie untersuchten wir den Einfluss der Aufzuchtbedingung auf die Genexpression im Pilzkörper, eines Bereiches im Insektengehirn, dem eine zentrale Rolle beim Lernen und Gedächtnis zukommt. Ein differential display Experiment, bei dem Bienen aus dem Volk mit isoliert aufgezogenen Arbeiterinnen verglichen wurden, zeigt, dass die Expression des major royal jelly protein (mrjp1) in isolierten Bienen deutlich reduziert ist. Frisch geschlüpfte Bienen wurden hierzu einzeln 8 Tage lang bei 32 °C im Dunkeln in Glasfläschchen gehalten und mit Zuckerwasser gefüttert. Mithilfe der Fluorescence differential display (FDD) Technik mit 16 primer sets wurde die Genexpression im Pilzkörper gemessen. Von insgesamt 604 Banden haben wir 13 Banden gefunden, die bei sozial aufgewachsenen Bienen eine höhere Expressionsintensität aufwiesen als in den isolierten Bienen. Eine Bande von etwa 1,1 kbp identifizierten wir als mrjp1. Diesen Befund konnten wir mit einer quantitativen real-time PCR bestätigen. Unsere Ergebnisse deuten an, dass mrjp1 wichtig für die Gehirnfunktion ist und möglicherweise an der Entwicklung von Lernfähigkeiten beteiligt ist.

Oriental orchid (Cymbidium pumilum) attracts drones of the Japanese honeybee (Apis cerana japonica) as pollinators

The discovery that drones of the Japanese honeybee (Apis cerana japonica) pollinate the oriental orchid (Cymbidium pumilum) is reported. Drones are attracted to the orchid flower aroma mainly during their mating flights in April through May. Some drones cluster on the flower racemes and others insert their heads deep into the flowers. Drones with pollinia on their scutellum visit other orchids, which facilitates pollination. Individual workers and swarming colonies are also strongly attracted by the flower aroma, but the allopatric western honeybee (Apis mellifera) is not attracted.

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.

Notes on paternal care and sibling cannibalism in the giant water bug, Lethocerus deyrolli (Heteroptera: Belostomatidae)

Males of the giant water bug, Lethocerus deyrolli, care for egg masses on vegetation above the water surface. They supply the developing eggs with water and guard them against predators. In the present study, mechanisms by which paternal care is extended were found. Males were found situated just below the water on the natal substrate (usually a stick), and the first instar nymphs were aggregated around the substrate. When disturbed, the males showed aggressive behavior, threatening the intruder with their forelegs. Nymphs up to 12 h old did not attack the offered sibling nymphs or anuran larvae, which are common prey in the field. The 24 h‐old nymphs attacked both prey animals; however, they preferred anuran larvae. Cannibalistic behavior in the nymphs was well developed 72 h after hatching, when the nymphs had already dispersed from the natal substrate. The suppression of sibling cannibalism in younger nymphs would promote the maintenance of tight nymphal aggregations and consequently extend male care in this predatory species.

Honeybee foragers adjust crop contents before leaving the hive

Upon leaving the hive, foragers carry a small amount of honey, which they subsequently consume to generate energy for flight. We investigated the relationship between waggle-phase duration and crop volume in foragers (both dancers and dance followers) leaving the hive. Our findings indicate that these variables were positively correlated in the two types of bee, suggesting that they were able to adjust the amount of food that they carry depending on the distance to a food source. We also found that dance followers left the hive with a larger amount of honey than dancers. We suggest two possible explanations: (1) dance followers have less information about the location of the food source than dancers, who have a better knowledge of the surrounding area; or (2) honeybees lack a precise calibration method for estimating energy needs from waggle-run duration. The effect of foraging experience was confirmed: bees decreased their honey load at departure with repeated trips to a sugar-syrup feeder. Honeybees showed a different pattern of change when the feeder provided soybean flour as a pollen substitute, possibly because honeybees use honey not only as an energy source but also as “glue” to form “balls” of pollen on their hind legs. Based on our observations that followers of sugar-syrup foragers carry a different amount of honey in their crop than followers of soybean-followers, we suggest that waggle dancers also convey information concerning food type.