Changes in the gut microbiota of honey bees associated with jujube flower disease.

Honeybees are prone to poisoning after collecting jujube nectar during the jujube flowering period ('honeybee's jujube flower disease'). To explore the mechanism of honeybee poisoning, the gut microbiota of honeybees undergoing the disease were characterised based on amplicon sequencing of the 16 S rRNA gene. Our results showed that the composition and diversity of the gut microbiota were significantly altered in diseased honeybees. We observed a decrease in the relative abundance of Proteobacteria and increased abundances of Firmicutes and Actinobacteria in the midgut and hindgut of diseased honeybees. Moreover, linear discriminant analysis (LDA) effect size revealed significantly selected enrichment of Fructobacillus and Snodgrassella in the midguts from diseased honeybees and Lactobacillus, Bifidobacterium, and Snodgrassella in the hindguts from diseased honeybees. Tax4Fun anylasis indicated that the functional potential of the diseased honeybee gut bacterial community was significantly changed relative to the healthy honeybee. Carbohydrate metabolism, nucleotides metabolism, amino acid synthesis metabolism, coenzyme and vitamins metabolism were increased, while energy metabolism and xenobiotic biodegradation and metabolism were decreased in the diseased honeybees. These results provide a new perspective for evaluating the response of honeybees to jujube flower disease based on changes in the intestinal microflora.

Linden (Tilia cordata) associated bumble bee mortality: Metabolomic analysis of nectar and bee muscle.

Linden (Tilia spp.), a profusely flowering temperate tree that provides bees with vital pollen and nectar, has been associated with bumble bee (Bombus spp.) mortality in Europe and North America. Bee deaths have been attributed, with inadequate evidence, to toxicity from mannose in nectar or starvation due to low nectar in late blooming linden. Here, we investigated both factors via untargeted metabolomic analyses of nectar from five T. cordata trees beneath which crawling/dead bumble bees (B. vosnesenskii) were observed, and of thoracic muscle of 28 healthy foraging and 29 crawling bees collected from linden trees on cool mornings (< 30°C). Nectar contained the pyridine alkaloid trigonelline, a weak acetylcholinesterase inhibitor, but no mannose. Principal component analysis of muscle metabolites produced distinct clustering of healthy and crawling bees, with significant differences (P<0.05) in 34 of 123 identified metabolites. Of these, TCA (Krebs) cycle intermediates were strongly represented (pathway analysis; P<0.01), suggesting that the central metabolism is affected in crawling bees. Hence, we propose the following explanation: when ambient temperature is low, bees with energy deficit are unable to maintain the thoracic temperature required for flight, and consequently fall, crawl, and ultimately, die. Energy deficit could occur when bees continue to forage on linden despite limited nectar availability either due to loyalty to a previously energy-rich source or trigonelline-triggered memory/learning impairment, documented earlier with other alkaloids. Thus, the combination of low temperature and nectar volume, resource fidelity, and alkaloids in nectar could explain the unique phenomenon of bumble bee mortality associated with linden.

Bumblebee Rejection of Toxic Pollen Facilitates Pollen Transfer.

Many bees are effective pollen collectors; however, pollen grains collected by bees for larval food are lost for plant sexual reproduction. Recognition of these conflicting interests between bees and flowers is essential for understanding of reproduction for both bees and flowers [1-3]. Plant defense compounds in pollen may function to reduce pollen waste by deterring ineffective pollinators [4-6], but this hypothesis remains unexamined. Here, we provide evidence that secondary metabolites in pollen function as chemical defense by deterring some bees from gathering pollen. In two Dipsacus species, a defense compound, dipsacus saponin [7], occurs in pollen but not in nectar. We observed that bumblebees disliked grooming bitter-tasting pollen with a high saponin content. Manipulation of saponin concentrations in nectar and measurements of corbicular pollen showed that the bumblebee species differed in their tolerance to saponin. Those species susceptible to saponin groomed little Dipsacus pollen into their pollen loads, and their ungroomed pollen was observed to be effectively delivered to stigmas. By rewarding bees with edible nectar, but not pollen, plants solve the conflict of pollen partitioning between sexual and reward functions. Ungroomed toxic pollen on the bee body promotes pollen transfer efficiency, facilitating pollination.

Consequences of toxic secondary compounds in nectar for mutualist bees and antagonist butterflies.

Attraction of mutualists and defense against antagonists are critical challenges for most organisms and can be especially acute for plants with pollinating and non-pollinating flower visitors. Secondary compounds in flowers have been hypothesized to adaptively mediate attraction of mutualists and defense against antagonists, but this hypothesis has rarely been tested. The tissues of milkweeds (Asclepias spp.) contain toxic cardenolides that have long been studied as chemical defenses against herbivores. Milkweed nectar also contains cardenolides, and we have examined the impact of manipulating cardenolides in nectar on the foraging choices of two flower visitors: generalist bumble bees, Bombus impatiens, which are mutualistic pollinators, and specialist monarch butterflies, Danaus plexippus, which are herbivores as larvae and ineffective pollinators as adults. Although individual bumble bees in single foraging bouts showed no avoidance of cardenolides at the highest natural concentrations reported for milkweeds, a pattern of deterrence did arise when entire colonies were allowed to forage for several days. Monarch butterflies were not deterred by the presence of cardenolides in nectar when foraging from flowers, but laid fewer eggs on plants paired with cardenolide-laced flowers compared to controls. Thus, although deterrence of bumble bees by cardenolides may only occur after extensive foraging, a primary effect of nectar cardenolides appears to be reduction of monarch butterfly oviposition.

Pyrethroids and Nectar Toxins Have Subtle Effects on the Motor Function, Grooming and Wing Fanning Behaviour of Honeybees (Apis mellifera).

Sodium channels, found ubiquitously in animal muscle cells and neurons, are one of the main target sites of many naturally-occurring, insecticidal plant compounds and agricultural pesticides. Pyrethroids, derived from compounds found only in the Asteraceae, are particularly toxic to insects and have been successfully used as pesticides including on flowering crops that are visited by pollinators. Pyrethrins, from which they were derived, occur naturally in the nectar of some flowering plant species. We know relatively little about how such compounds--i.e., compounds that target sodium channels--influence pollinators at low or sub-lethal doses. Here, we exposed individual adult forager honeybees to several compounds that bind to sodium channels to identify whether these compounds affect motor function. Using an assay previously developed to identify the effect of drugs and toxins on individual bees, we investigated how acute exposure to 10 ng doses (1 ppm) of the pyrethroid insecticides (cyfluthrin, tau-fluvalinate, allethrin and permethrin) and the nectar toxins (aconitine and grayanotoxin I) affected honeybee locomotion, grooming and wing fanning behaviour. Bees exposed to these compounds spent more time upside down and fanning their wings. They also had longer bouts of standing still. Bees exposed to the nectar toxin, aconitine, and the pyrethroid, allethrin, also spent less time grooming their antennae. We also found that the concentration of the nectar toxin, grayanotoxin I (GTX), fed to bees affected the time spent upside down (i.e., failure to perform the righting reflex). Our data show that low doses of pyrethroids and other nectar toxins that target sodium channels mainly influence motor function through their effect on the righting reflex of adult worker honeybees.