RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee''s behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.     

Honey bees and wild pollinators differ in their preference for and use of introduced floral resources

Introduced plants may be important foraging resources for honey bees and wild pollinators, but how often and why pollinators visit introduced plants across an entire plant community is not well understood. Understanding the importance of introduced plants for pollinators could help guide management of these plants and conservation of pollinator habitat. We assessed how floral abundance and pollinator preference influence pollinator visitation rate and diversity on 30 introduced versus 24 native plants in central New York. Honey bees visited introduced and native plants at similar rates regardless of floral abundance. In contrast, as floral abundance increased, wild pollinator visitation rate decreased more strongly for introduced plants than native plants. Introduced plants as a group and native plants as a group did not differ in bee diversity or preference, but honey bees and wild pollinators preferred different plant species. As a case study, we then focused on knapweed (Centaurea spp.), an introduced plant that was the most preferred plant by honey bees, and that beekeepers value as a late‐summer foraging resource. We compared the extent to which honey bees versus wild pollinators visited knapweed relative to coflowering plants, and we quantified knapweed pollen and nectar collection by honey bees across 22 New York apiaries. Honey bees visited knapweed more frequently than coflowering plants and at a similar rate as all wild pollinators combined. All apiaries contained knapweed pollen in nectar, 86% of apiaries contained knapweed pollen in bee bread, and knapweed was sometimes a main pollen or nectar source for honey bees in late summer. Our results suggest that because of diverging responses to floral abundance and preferences for different plants, honey bees and wild pollinators differ in their use of introduced plants. Depending on the plant and its abundance, removing an introduced plant may impact honey bees more than wild pollinators.     

Plant evolution can mediate negative effects from honey bees on wild pollinators

1. Pollinators are introduced to agroecosystems to provide pollination services. Introductions of managed pollinators often promote ecosystem services, but it remains largely unknown whether they also affect evolutionary mutualisms between wild pollinators and plants.
2. Here, we developed a model to assess effects of managed honey bees on mutualisms between plants and wild pollinators. Our model tracked how interactions among wild pollinators and honey bees affected pollinator and plant populations.
3. We show that when managed honey bees have a competitive advantage over wild pollinators, or a greater carrying capacity, the honey bees displace the wild pollinator. This leads to reduced plant density because plants benefit less by visits from honey bees than wild pollinators that coevolved with the plants.
4. As wild pollinators are displaced, plants evolve by increasing investment in traits that are attractive for honey bees but not wild pollinators. This evolutionary switch promotes wild pollinator displacement. However, higher mutualism investment costs by the plant to the honey bee can promote pollinator coexistence.
5. Our results show plant evolution can promote displacement of wild pollinators by managed honey bees, while limited plant evolution may lead to pollinator coexistence. More broadly, effects of honey bees on wild pollinators in agroecosystems, and effects on ecosystem services, may depend on the capacity of plant populations to evolve.

     

Olfactory interference during inhibitory backward pairing in honey bees

Background

Restrained worker honey bees are a valuable model for studying the behavioral and neural bases of olfactory plasticity. The proboscis extension response (PER; the proboscis is the mouthpart of honey bees) is released in response to sucrose stimulation. If sucrose stimulation is preceded one or a few times by an odor (forward pairing), the bee will form a memory for this association, and subsequent presentations of the odor alone are sufficient to elicit the PER. However, backward pairing between the two stimuli (sucrose, then odor) has not been studied to any great extent in bees, although the vertebrate literature indicates that it elicits a form of inhibitory plasticity.

Methodology/Principal Findings

If hungry bees are fed with sucrose, they will release a long lasting PER; however, this PER can be interrupted if an odor is presented 15 seconds (but not 7 or 30 seconds) after the sucrose (backward pairing). We refer to this previously unreported process as olfactory interference. Bees receiving this 15 second backward pairing show reduced performance after a subsequent single forward pairing (excitatory conditioning) trial. Analysis of the results supported a relationship between olfactory interference and a form of backward pairing-induced inhibitory learning/memory. Injecting the drug cimetidine into the deutocerebrum impaired olfactory interference.

Conclusions/Significance

Olfactory interference depends on the associative link between odor and PER, rather than between odor and sucrose. Furthermore, pairing an odor with sucrose can lead either to association of this odor to PER or to the inhibition of PER by this odor. Olfactory interference may provide insight into processes that gate how excitatory and inhibitory memories for odor-PER associations are formed.     

Honey bee (Hymenoptera: Apidae) foraging in response to preconditioning with onion flower scent compounds

Onion (Allium cepa L.) seed production has long been plagued with yield problems because of lack of pollination by the honey bee, Apis mellifera L. To attempt to attract more pollinators to the onion seed production field, honey bees were conditioned to associate onion floral odor components with a reward. Isolated nucleus hives of honey bees were fed 30% sucrose solutions scented with a 0.2% solution of onion floral odor compounds. After feeding on these solutions for 6 wk, bees were not found to prefer onion flowers to two competing food sources, carrot and alfalfa flowers, at the 5% significance level. However, there was an overall trend indicating a change in honey bee behavior, with fewer "trained" bees visiting alfalfa and carrot and more visiting onion. Thus, it may be possible to alter honey bee behavior with preconditioning but probably not to a degree that would be economically significant.     

Sucrose acceptance,discrimination and proboscis responses of honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) in the field and the laboratory

Laboratory studies in honey bees have shown positive correlations between sucrose responsiveness, division of labour and learning. We tested the relationships between sucrose acceptance and discrimination in the field and responsiveness in the laboratory. Based on acceptance in the field three groups of bees were differentiated: (1) bees that accept sucrose concentrations >10%, (2) bees that accept some but not all of the sucrose concentrations <10% and water, and (3) bees that accept water and all offered sucrose concentrations. Sucrose acceptance can be described in a model in which sucrose- and water-dependent responses interact additively. Responsiveness to sucrose was tested in the same bees in the laboratory by measuring the proboscis extension response (PER). The experiments demonstrated that PER responsiveness is lower than acceptance in the field and that it is not possible to infer from the PER measurements in the laboratory those concentrations the respective bees accepted in the field. Discrimination between sucrose concentrations was tested in three groups of free-flying bees collecting low, intermediate or high concentrations of sucrose. The experiments demonstrated that bees can discriminate between concentrations differences down to 0.2 relative log units. There exist only partial correlations between discrimination, acceptance and PER responsiveness.     

Farming practices influence wild pollinator populations on squash and pumpkin

Recent declines in managed honey bee, Apis mellifera L., colonies have increased interest in the current and potential contribution of wild bee populations to the pollination of agricultural crops. Because wild bees often live in agricultural fields, their population density and contribution to crop pollination may be influenced by farming practices, especially those used to reduce the populations of other insects. We took a census of pollinators of squash and pumpkin at 25 farms in Virginia, West Virginia, and Maryland to see whether pollinator abundance was related to farming practices. The main pollinators were Peponapis pruinosa Say; honey bees, and bumble bees (Bombus spp.). The squash bee was the most abundant pollinator on squash and pumpkin, occurring at 23 of 25 farms in population densities that were commonly several times higher than that of other pollinators. Squash bee density was related to tillage practices: no-tillage farms hosted three times as great a density of squash bees as tilled farms. Pollinator density was not related to pesticide use. Honey bee density on squash and pumpkin was not related to the presence of managed honey bee colonies on farms. Farms with colonies did not have more honey bees per flower than farms that did not keep honey bees, probably reflecting the lack of affinity of honey bees for these crops. Future research should examine the economic impacts of managing farms in ways that promote pollinators, particularly pollinators of crops that are not well served by managed honey bee colonies.     

Honeybees and nectar nicotine: deterrence and reduced survival versus potential health benefits

Secondary metabolites produced by plants for herbivore defence are often found in floral nectar, but their effect on the foraging behaviour and physiological performance of pollinators is largely unknown. Nicotine is highly toxic to most herbivores, and nicotine-based insecticides may contribute to current pollinator declines. We examined the effects of nectar nicotine on honeybee foraging choices and worker longevity. Free-flying honeybee (Apis mellifera scutellata) workers from six colonies were given a choice between multiple nicotine concentrations (0-1000 μM) in artificial nectar (0.15-0.63 M sucrose). The dose-dependent deterrent effect of nicotine was stronger in lower sugar concentrations, but even the highest nicotine concentrations did not completely repel honeybees, i.e., bees did not stop feeding on these diets. Nicotine in nectar acts as a partial repellent, which may keep pollinators moving between plants and enhance cross-pollination. In the second part of the study, newly emerged workers from 12 colonies were caged and fed one of four nicotine concentrations (0-300 μM) in 0.63 M sucrose for 21 days. Moderate (≤30 μM) nicotine concentrations had no significant detrimental effect, but high nicotine concentrations reduced the survival of caged workers and their nectar storage in the honey comb. In contrast, worker groups that survived poorly on sugar-only diets demonstrated increased survival on all nicotine diets. In the absence of alternative nectar sources, honeybees tolerate naturally occurring nectar nicotine concentrations; and low concentrations can even be beneficial to honeybees. However, high nicotine concentrations may have a detrimental effect on colony fitness.     

Effect of inorganic and organic forms of selenium supplementation on development of larval Heliothis virescens

Selenium (Se) is an essential micronutrient for vertebrates though little is known about the effects on insects. Herbivorous insect larvae acquire Se from plant tissues in the inorganic form of sodium selenate and sodium selenite, and in the organic form of selenoamino acids, selenomethionine, and selenocystine. In this study, we document the effects of dietary supplementation with sodium selenite, sodium selenate, selenocystine, selenomethionine, and selenized yeast on the developmental rate of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Larvae tolerated high levels of Se (500 µg g^?1 Se) as sodium selenate and to a lesser extent as selenocystine. Lower levels of sodium selenite (>1 µg g^?1 Se) caused increased mortality, reduced rates of pupation, more pupal/adult intermediates, and reduced adult emergence. Selenomethionine proved toxic to larvae at levels above 25 µg g^?1 Se, significantly delaying pupation and raising mortality. Provision of Se as selenized yeast, which contains primarily selenomethionine, was also extremely detrimental to larval development and survival. The results indicate that the impact of dietary Se supplement for insects may differ from vertebrates.     

Visual associative learning in restrained honey bees with intact antennae

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee.     

Pollination effectiveness and pollen dispersal in a Rhododendron ferrugineum (Ericaceae) population

Many alpine plants are predominantly outcrossing, thus plant reproductive success is highly dependent on effectiveness of pollinators. How pollinators transfer pollen from one flower to another is of great interest in understanding the genetic structure in plant populations. We studied (1) the role and effectiveness of insect visitors for pollination, and (2) their contribution as pollen vectors for gene dispersal in a Rhododendron ferrugineum population. Various insect visitors were recorded, including Hymenoptera, Diptera, Coleoptera, and Lepidoptera. The most frequent and effective insects were honey bees and bumblebees. Muscid flies were considered as important pollinators, particularly due to their relatively high visitation rate. Syrphid flies, Formicidae, and Coleoptera were ineffective in transporting pollen, while the effectiveness of Lepidoptera and Empididae was negligible. A fluorescence labelling experiment revealed that pollen dispersal was restricted (0 - 2 m) in a dense R. ferrugineum stand and decreased in a leptokurtic fashion. This might lead to geitonogamous self-pollination that could explain the close relationship between individuals found in genetic studies of R. ferrugineum. However, some pollen grains may travel 40 - 45 m, which implies the occurrence of cross-pollination through the foraging activities of bumblebees and honey bees.     

Selenium (Se) from high-selenium broccoli is utilized differently than selenite, selenate and selenomethionine, but is more effective in inhibiting colon carcinogenesis

The reduction in incidence of chemically-induced colon cancer by foods high in selenium (Se) was investigated in Fisher-344 rats. The foods used were high-Se broccoli (produced in a greenhouse by addition of selenate to the media surrounding the plant roots) and a processed high-Se wheat product (made by milling high-Se wheat purchased from a seleniferous area). Weanling rats were fed diets containing different amounts of Se from these foods or from selenium salts (selenite and selenate). Early in the experiment the animals were injected with a chemical carcinogen. After 11 weeks on diets animals were killed and the colons examined for preneoplastic lesions (aberrant crypts foci, ACF). ACF were significantly reduced in animals fed supra-nutritional amounts of Se from broccoli, despite the finding that Se from broccoli was poorly bioavailable. Supra-nutritional amounts of Se from high-Se processed wheat also significantly reduced aberrant crypts (AC), although pure selenomethionine, (the predominant chemical form of Se in wheat), did not significantly reduce AC. These results emphasize the need to study Se in food forms, and not extrapolate from previous studies using pure chemical forms in cancer inhibition studies. They also demonstrate that foods with high Se bioavailability are not necessarily the most efficacious for cancer incidence reduction.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

草甘膦与微塑料对西方蜜蜂生存和糖反应行为的影响

为分析草甘膦与微塑料对西方蜜蜂Apis mellifera存活和行为的影响。本研究利用50%蔗糖溶液配置的400 mg/L草甘膦、50 mg/L微塑料以及两者混合物,分别饲喂1日龄的工蜂;每日记录西方蜜蜂的存活率,20 d后通过伸吻反应测定它们对蜜蜂嗅觉学习行为的影响。结果发现400 mg/L草甘膦处理蜜蜂20 d后,与对照组相比,蜜蜂存活率下降了22%,伸吻比率下降了30%;50 mg/L微塑料处理,与对照组相比,蜜蜂存活率下降了9%,伸吻比率下降了10%;用400 mg/L草甘膦与50 mg/L微塑料协同处理20 d后,与对照组相比,蜜蜂死亡率为50%、伸吻比率下降了45%左右。结果表明草甘膦和微塑料均会导致西方蜜蜂存活率降低,影响其糖反应行为,而两者协同处理对西方蜜蜂的影响更显著。     

Viral Infection Affects Sucrose Responsiveness and Homing Ability of Forager Honey Bees,Apis mellifera L.

Honey bee health is mainly affected by Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition. Interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years. In the present study, the effects of a honey bee virus, Israeli acute paralysis virus (IAPV), on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on proboscis extension response (PER) assays and radio frequency identification (RFID) systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around 10⁷ copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.     

Selenium assimilation and volatilization from dimethylselenoniopropionate by Indian mustard

Earlier work from our laboratory on Indian mustard (Brassica juncea L.) identified the following rate-limiting steps for the assimilation and volatilization of selenate to dimethyl selenide (DMSe): (a) uptake of selenate, (b) activation of selenate by ATP sulfurylase, and (b) conversion of selenomethionine (SeMet) to DMSe. The present study showed that shoots of selenate-treated plants accumulated very low concentrations of dimethylselenoniopropionate (DMSeP). Selenonium compounds such as DMSeP are the most likely precursors of DMSe. DMSeP-supplied plants volatilized Se at a rate 113 times higher than that measured from plants supplied with selenate, 38 times higher than from selenite, and six times higher than from SeMet. The conversion of SeMet to selenonium compounds such as DMSeP is likely to be rate-limiting for DMSe production, but not the formation of DMSe from DMSeP because DMSeP was the rate of Se volatilization from faster than from SeMet and SeMet (but no DMSeP) accumulated in selenite- or SeMet-supplied wild-type plants and in selenate-supplied ATP-sulfurylase transgenic plants. DMSeP-supplied plants absorbed the most Se from the external medium compared with plants supplied with SeMet, selenate, or selenite; they also accumulated more Se in shoots than in roots as an unknown organic compound resembling a mixture of DMSeP and selenocysteine.     

Route-dependent pharmacokinetics, distribution, and placental permeability of organic and inorganic selenium in hamsters

Inorganic selenium (Se) salts (selenite and selenate oxyanions) and the organic selenoamino acids (selenomethionine and seleniferous grains) are teratogenic and embryolethal in domestic and wild birds. Selenium bioaccumulation has been held responsible for reproductive failure among waterfowl at the Kesterson Reservoir (California), the Ouray and Stewart Lake Wildlife Refuges (Utah), and the Carson Sink (Nevada). Anecdotal field and controlled laboratory reports have implicated Se exposure in mammalian embryotoxicity (including human), but developmental toxicity studies in hamsters failed to demonstrate an adverse response, except at maternally toxic doses (Ferm et al., Reprod. Toxicol., in press). Uptake, distribution, and elimination of Se after a single bolus equimolar dose (60 mumol/kg) of selenate or selenomethionine by oral or intravenous administration were compared using day 8 pregnant hamsters. Intravenous selenate was eliminated ten times more rapidly from maternal plasma than oral selenate, but concentrated in liver, kidney, and placenta to the same degree. Intravenous (iv) L-selenomethionine achieved lower maximum circulating total [Se], but it was eliminated more slowly than iv selenate. Larger areas under the plasma and peripheral tissue [Se]:time curve (AUC) after oral or parenteral selenomethionine than after equimolar selenate were consistent with previous studies in rodents and in humans. Embryonic [Se] plateaued at 3 nmol/g after selenate, but embryonic [Se] after selenomethionine continued to accumulate (80 nmol/g) as gestation progressed. The lack of a teratogenic response in hamsters at doses of either selenate or selenomethionine less than those associated with maternal intoxication cannot be attributed to lack of Se accumulation in early embryonic and placental tissue.