Learning of a mimic odor combined with nectar nonsugar compounds enhances honeybee pollination of a commercial crop.

The increasing demand on pollination services leads food industry to consider new strategies for management of pollinators to improve their efficiency in agroecosystems. Recently, it was demonstrated that feeding beehives food scented with an odorant mixture mimicking the floral scent of a crop (sunflower mimic, SM) enhanced foraging activity and improved recruitment to the target inflorescences, which led to higher density of bees on the crop and significantly increased yields. Besides, the oral administration of nonsugar compounds (NSC) naturally found in nectars (caffeine and arginine) improved short and long-term olfactory memory retention in conditioned bees under laboratory conditions. To test the effect of offering of SM-scented food supplemented with NSC on honeybees pollinating sunflower for hybrid seed production, in a commercial plantation we fed colonies SM-scented food (control), and SM-scented food supplemented with either caffeine, arginine, or a mixture of both, in field realistic concentrations. Their foraging activity was assessed at the hive and on the crop up to 90 h after treatment, and sunflower yield was estimated prior to harvest. Our field results show that SM + Mix-treated colonies exhibited the highest incoming rates and densities on the crop. Additionally, overall seed mass was significantly higher by 20% on inflorescences close to these colonies than control colonies. Such results suggest that combined NSC potentiate olfactory learning of a mimic floral odor inside the hive, promoting faster colony-level foraging responses and increasing crop production.

Managed honeybees and South American bumblebees exhibit complementary foraging patterns in highbush blueberry.

Despite Apis mellifera being the most widely managed pollinator to enhance crop production, they are not the most suitable species for highbush blueberries, which possess restrictive floral morphology and require buzz-pollination. Thus, the South American bumblebee Bombus pauloensis is increasingly managed as an alternative species in this crop alongside honeybees. Herein, we evaluated the foraging patterns of the two species, concerning the potential pollen transfer between two blueberry co-blooming cultivars grown under open high tunnels during two seasons considering different colony densities. Both managed pollinators showed different foraging patterns, influenced by the cultivar identity which varied in their floral morphology and nectar production. Our results demonstrate that both species are efficient foragers on highbush blueberry and further suggest that they contribute positively to its pollination in complementary ways: while bumblebees were more effective at the individual level (visited more flowers and carried more pollen), the greater densities of honeybee foragers overcame the difficulties imposed by the flower morphology, irrespective of the stocking rate. This study supports the addition of managed native bumblebees alongside honeybees to enhance pollination services and emphasizes the importance of examining behavioural aspects to optimize management practices in pollinator-dependent crops.

Combined secondary compounds naturally found in nectars enhance honeybee cognition and survival.

The alkaloid caffeine and the amino acid arginine are present as secondary compounds in nectars of some flower species visited by pollinators. Each of these compounds affects honeybee appetitive behaviours by improving foraging activity and learning. While caffeine potentiates responses of mushroom body neurons involved in honeybee learning processes, arginine acts as precursor of nitric oxide, enhancing the protein synthesis involved in memory formation. Despite existing evidence on how these compounds affect honeybee cognitive ability individually, their combined effect on this is still unknown. We evaluated acquisition and memory retention in a classical olfactory conditioning procedure, in which the reward (sucrose solution) contained traces of caffeine, arginine or a mixture of the two. The results indicate that the presence of the single compounds and their most concentrated mixture increases bees' learning performance. However, memory retention, measured in the short and long term, increases significantly only in those treatments offering combinations of the two compounds in the reward. Additionally, the most concentrated mixture triggers a significant survival rate in the conditioned bees. Thus, some nectar compounds, when combined, show synergistic effects on cognitive ability and survival in an insect.

Honey bee and native solitary bee foraging behavior in a crop with dimorphic parental lines.

Insect pollination is issential for hybrid seed production systems, among which, introduced and native bees are the primary pollinating agents transferring pollen from male fertile (MF) to male sterile (MS) lines. On a highly dimorphic sunflower (Helianthus annuus) crop, we assessed the foraging behavior of solitary Melissodes bees and honey bees Apis mellifera. We found that Melissodes spp. were dominant in and showed fidelity to MF plants, gathering sunflower pollen efficiently throughout the day. In contrast, honey bees dominated on MS lines, mostly gathered nectar and exhibited high floral constancy, even after interacting with a second visitor. Also, honey bees carried sunflower pollen on their bodies while visiting MS inflorescences. This study highlights the need for a thorough understanding of the factors involved in a pollinator-dependent agroecosystem crop to assess the contribution of native bees on pollination of crops which offer resources spatially separated in two highly dimorphic parental lines.

Appetitive behavior of the honey bee Apis mellifera in response to phenolic compounds naturally found in nectars.

The honey bee is the most frequently used species in pollination services for diverse crops. In onion crops (Allium cepa), however, bees avoid visiting certain varieties, being attracted differently to male sterile (MS) and fertile (OP) lines. These differences might be based on the phenolic profiles of the cultivars' nectars. To understand the relationship between nectar composition and pollinator attraction to different onion lines, we tested sensory and cognitive abilities and palatability in honey bees exposed to MS and OP onion nectars and sugar solutions mimicking them. We evaluated the proboscis extension response (PER) after antennal contact (unconditioned response) to MS or OP onion nectars, finding no statistical differences, which indicates similar gustatory perception for the two nectars. We also performed food uptake assays to test palatability of different artificial nectars, considering their flavonoids and potassium content. The presence of potassium decreased the palatability of the artificial nectars. Finally, we evaluated the bees' cognitive abilities when the reward (unconditioned stimulus) offered during conditioning PER assays presents differences in composition. We found that potassium by itself impaired learning; however, such impairment was even higher when naringenin and quercetin were added in the unconditioned stimulus (MS nectar mimic). Interestingly, potassium together with luteolin (OP nectar mimic) improved learning. Our study demonstrates that the differences in the nectars' flavonoid profiles combined with their high potassium content could explain the previously reported differences in attractiveness between onion lines, suggesting an important role of nectar compounds other than sugars for the attractiveness of flowers to pollinators.

Odor Learning and Its Experience-Dependent Modulation in the South American Native Bumblebee Bombus atratus (Hymenoptera: Apidae).

Learning about olfactory stimuli is essential in bumblebees' life since it is involved in orientation, recognition of nest sites, foraging efficiency and food yield for the colony as a whole. To evaluate associative learning abilities in bees under controlled environmental conditions, the proboscis extension response (PER) assay is a well-established method used in honey bees, stingless bees and successfully adapted to bumblebees of the genus Bombus. However, studies on the learning capacity of Bombus atratus (Hymenoptera: Apidae), one of the most abundant native species in South America, are non-existent. In this study, we examined the cognitive abilities of worker bees of this species, carrying out an olfactory PER conditioning experiment. Bumblebees were able to learn a pure odor when it was presented in paired association with sugared reward, but not when odor and reward were presented in an unpaired manner. Furthermore, if the bees were preexposed to the conditioned odor, the results differed depending on the presence of the scent either as a volatile in the rearing environment or diluted in the food. A decrement in learning performance results from the non-reinforced pre-exposure to the to-be-conditioned odor, showing a latent inhibition phenomenon. However, if the conditioned odor has been previously offered diluted in sugared reward, the food odor acts as a stimulus that improves the learning performance during PER conditioning. The native bumblebee B. atratus is thus a new hymenopteran species capable of being trained under controlled experimental conditions. Since it is an insect increasingly reared for pollination service, this knowledge could be useful in its management in crops.

Filling dynamics of the Brindley's glands in the blood-sucking bug Triatoma infestans (Hemiptera: Reduviidae).

The filling dynamics of exocrine defensive glands is an important component of the defensive capacity of an insect in its natural environment. We studied the filling state and reloading rate of the Brindley's glands in the haematophagous Chagas disease vector Triatoma infestans (Hemiptera: Reduviidae). Quantitative analyses of isobutyric acid, the main secretion component, were carried out with glands dissected from adults under different scenarios of development, number of discharging events and feeding conditions. The alarm-pheromone function of the gland secretion was also assessed in bioassays with conspecific nymphs. Although pharate adults have their glands completely developed, these were not full until imaginal ecdysis. If kept undisturbed, the adults maintained a constant gland load, and discharged about 75% of the gland contents upon one disturbance event. While the glands can be discharged several times, full replenishing was not complete after one week, unless the insect had access to food. The escape behavior of nymphs in bioassays correlated with the chemical analyses, with nymphs showing significant avoidance only toward gland discharges from undisturbed or disturbed/fed adults. The results are discussed in reference to the feeding frequency and gregarious behavior of T. infestans under natural conditions, which suggest a relevant role of the filling dynamics of the Brindley's glands in the intraspecific communication of the insect.

The main component of an alarm pheromone of kissing bugs plays multiple roles in the cognitive modulation of the escape response.

Innate responses in animals can be modulated by experience. Disturbed adults of the triatomine bug Triatoma infestans release an alarm pheromone (AP) that elicits an escape response in conspecific larvae. The main component of this AP, the isobutyric acid (IsoAc), alone has already shown to generate an escape response in this species. However, not much is known about the modulation of this behavior by non-associative and associative cognitive processes. We present here evidences of the cognitive capacities of T. infestans larvae in an escape context under different conditioning paradigms, including IsoAc in different roles. We show that: (1) the duration of a pre-exposure to IsoAc plays a main role in determining the type of non-associative learning expressed: short time pre-exposures elicit a sensitization while a longer pre-exposure time triggers a switch from repellence to attractiveness; (2) a simple pre-exposure event is enough to modulate the escape response of larvae to the AP and to its main component: IsoAc; (3) IsoAc and the AP are perceived as different chemical entities; (4) an association between IsoAc and an aversive stimulus can be created under a classical conditioning paradigm; (5) an association between IsoAc and a self-action can be generated under an operant conditioning. These results evince that IsoAc can attain multiple and different cognitive roles in the modulation of the escape response of triatomines and show how cognitive processes can modulate a key behavior for surviving, as it is the escaping response in presence of a potential danger in insects.

Dislodgement effect of natural semiochemicals released by disturbed triatomines: a possible alternative monitoring tool.

The quick detection of domestic and peridomestic triatomines in their environments becomes difficult without the use of dislodgement substances that flush them out from their shelters. At present, tetramethrin 0.2% is being widely used in control programs. Although it is an efficient dislodging agent, its toxicity might affect the health of captured triatomines, of other insects and, to a lesser extent, of other animals, including humans. Here, we tested if semiochemicals released by disturbed adults of Triatoma infestans and/or Rhodnius prolixus can make larvae of the same species exit from their refuges. In a walking olfactometer we found that: 1) larvae of T. infestans were repelled by the odors released by disturbed adults of their own species and of R. prolixus, 2) larvae of R. prolixus did not change their behavior in the presence of odors released by adults of both species, and 3) activity levels were not modulated by these odors in any of both species. Besides, in pseudo-natural conditions we found an increased flushing-out activity of larvae of T. infestans when their shelters were sprayed with isobutyric acid or 3-pentanol, and of larvae of R. prolixus when sprayed with 3-methyl-1-butanol. We succeeded in this work to dislodge larvae of triatomines from artificial shelters using natural volatile compounds, allowing the capture of live bugs for further investigations (e.g., xenodiagnosis or genetic studies) and favoring ecological aspects (e.g., minimizing environmental insecticide-contamination and non-targeted mortality).