Honeybees fail to discriminate floral scents in a complex learning task after consuming a neonicotinoid pesticide.

Neonicotinoids are pesticides used to protect crops but with known secondary influences at sublethal doses on bees. Honeybees use their sense of smell to identify the queen and nestmates, to signal danger and to distinguish flowers during foraging. Few behavioural studies to date have examined how neonicotinoid pesticides affect the ability of bees to distinguish odours. Here, we used a differential learning task to test how neonicotinoid exposure affects learning, memory and olfactory perception in foraging-age honeybees. Bees fed with thiamethoxam could not perform differential learning and could not distinguish odours during short- and long-term memory tests. Our data indicate that thiamethoxam directly impacts the cognitive processes involved in working memory required during differential olfactory learning. Using a combination of behavioural assays, we also identified that thiamethoxam has a direct impact on the olfactory perception of similar odours. Honeybees fed with other neonicotinoids (clothianidin, imidacloprid, dinotefuran) performed the differential learning task, but at a slower rate than the control. These bees could also distinguish the odours. Our data are the first to show that neonicotinoids have compound specific effects on the ability of bees to perform a complex olfactory learning task. Deficits in decision making caused by thiamethoxam exposure could mean that this is more harmful than other neonicotinoids, leading to inefficient foraging and a reduced ability to identify nestmates.

Honey Bees (Apis mellifera) Show a Preference for the Consumption of Ethanol.

BACKGROUND: Alcohol abuse and alcoholism are significant global issues. Honey bees are excellent models for learning and other complex behaviors; furthermore, they share many behavioral responses to ethanol (EtOH) with humans and animal models. We develop a 2-feeder choice assay to determine whether honey bees will self-administer and preferentially consume solutions containing EtOH. METHODS: Gustatory responsiveness to EtOH is determined using the proboscis extension reflex and consumption assays. A 2-feeder choice assay is used to examine preference for the consumption of EtOH. Survival assays assess the metabolic and toxic effects of EtOH consumption. RESULTS: Honey bees find the taste of EtOH to be aversive when in water, but addition of sucrose masks the aversive taste. Even though the taste of EtOH is not appetitive, honey bees preferentially consume sucrose solutions containing 1.25 to 2.5% EtOH in a dose-dependent manner. Based on survival assays, honey bees may not be able to derive caloric value from EtOH, and EtOH concentrations of 2.5% or higher lead to significant increases in mortality. CONCLUSIONS: Honey bees will self-administer EtOH and show a preference for consuming solutions containing EtOH. Bees may not be able to efficiently utilize EtOH as an energy source, but EtOH-dependent increases in mortality complicate separating the effects of caloric value and toxicity.

The buzz on caffeine in invertebrates: effects on behavior and molecular mechanisms.

A number of recent studies from as diverse fields as plant-pollinator interactions, analyses of caffeine as an environmental pollutant, and the ability of caffeine to provide protection against neurodegenerative diseases have generated interest in understanding the actions of caffeine in invertebrates. This review summarizes what is currently known about the effects of caffeine on behavior and its molecular mechanisms in invertebrates. Caffeine appears to have similar effects on locomotion and sleep in both invertebrates and mammals. Furthermore, as in mammals, caffeine appears to have complex effects on learning and memory. However, the underlying mechanisms for these effects may differ between invertebrates and vertebrates. While caffeine's ability to cause release of intracellular calcium stores via ryanodine receptors and its actions as a phosphodiesterase inhibitor have been clearly established in invertebrates, its ability to interact with invertebrate adenosine receptors remains an important open question. Initial studies in insects and mollusks suggest an interaction between caffeine and the dopamine signaling pathway; more work needs to be done to understand the mechanisms by which caffeine influences signaling via biogenic amines. As of yet, little is known about whether other actions of caffeine in vertebrates, such as its effects on GABAA and glycine receptors, are conserved. Furthermore, the pharmacokinetics of caffeine remains to be elucidated. Overall behavioral responses to caffeine appear to be conserved amongst organisms; however, we are just beginning to understand the mechanisms underlying its effects across animal phyla.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Octopamine modulates activity of neural networks in the honey bee antennal lobe.

Neuronal plasticity allows an animal to respond to environmental changes by modulating its response to stimuli. In the honey bee (Apis mellifera), the biogenic amine octopamine plays a crucial role in appetitive odor learning, but little is known about how octopamine affects the brain. We investigated its effect in the antennal lobe, the first olfactory center in the brain, using calcium imaging to record background activity and odor responses before and after octopamine application. We show that octopamine increases background activity in olfactory output neurons, while reducing average calcium levels. Odor responses were modulated both upwards and downwards, with more odor response increases in glomeruli with negative or weak odor responses. Importantly, the octopamine effect was variable across glomeruli, odorants, odorant concentrations and animals, suggesting that the octopaminergic network is shaped by plasticity depending on an individual animal's history and possibly other factors. Using RNA interference, we show that the octopamine receptor AmOA1 (homolog of the Drosophila OAMB receptor) is involved in the octopamine effect. We propose a network model in which octopamine receptors are plastic in their density and located on a subpopulation of inhibitory neurons in a disinhibitory pathway. This would improve odor-coding of behaviorally relevant, previously experienced odors.

Chronic nicotine exposure influences learning and memory in the honey bee.

In insects, nicotine activates nicotinic acetylcholine receptors, which are expressed throughout the central nervous system. However, little work has been done to investigate the effects of chronic nicotine treatment on learning or other behaviors in non-herbivorous insects. To examine the effects of long term nicotine consumption on learning and memory, honey bees were fed nicotine containing solutions over four days. Bees were able to detect nicotine at 0.1 mM in sucrose solutions, and in a no choice assay, bees reduced food intake when nicotine was 1 mM or higher. Treatment with a low dose of nicotine decreased the proportion of bees able to form an associative memory when bees were conditioned with either a massed or spaced appetitive olfactory training paradigm. On the other hand, higher doses of nicotine increased memory retention and the proportion of bees responding to the odor during 10 min and 24 h recall tests. The reduction in nicotine containing food consumed may also impact response levels during learning and recall tests. These data suggest that long term exposure to nicotine has complex effects on learning and memory.

Tyramine and its Amtyr1 receptor modulate attention in honey bees (Apis mellifera).

Animals must learn to ignore stimuli that are irrelevant to survival and attend to ones that enhance survival. When a stimulus regularly fails to be associated with an important consequence, subsequent excitatory learning about that stimulus can be delayed, which is a form of nonassociative conditioning called 'latent inhibition'. Honey bees show latent inhibition toward an odor they have experienced without association with food reinforcement. Moreover, individual honey bees from the same colony differ in the degree to which they show latent inhibition, and these individual differences have a genetic basis. To investigate the mechanisms that underly individual differences in latent inhibition, we selected two honey bee lines for high and low latent inhibition, respectively. We crossed those lines and mapped a Quantitative Trait Locus for latent inhibition to a region of the genome that contains the tyramine receptor gene Amtyr1 [We use Amtyr1 to denote the gene and AmTYR1 the receptor throughout the text.]. We then show that disruption of Amtyr1 signaling either pharmacologically or through RNAi qualitatively changes the expression of latent inhibition but has little or slight effects on appetitive conditioning, and these results suggest that AmTYR1 modulates inhibitory processing in the CNS. Electrophysiological recordings from the brain during pharmacological blockade are consistent with a model that AmTYR1 indirectly regulates at inhibitory synapses in the CNS. Our results therefore identify a distinct Amtyr1-based modulatory pathway for this type of nonassociative learning, and we propose a model for how Amtyr1 acts as a gain control to modulate hebbian plasticity at defined synapses in the CNS. We have shown elsewhere how this modulation also underlies potentially adaptive intracolonial learning differences among individuals that benefit colony survival. Finally, our neural model suggests a mechanism for the broad pleiotropy this gene has on several different behaviors.

To efficiently navigate their environment, animals must pay attention to cues associated with events important for survival while also dismissing meaningless signals. The difference between relevant and irrelevant stimuli is learned through a range of complex mechanisms that includes latent inhibition. This process allows animals to ignore irrelevant stimuli, which makes it more difficult for them to associate a cue and a reward if that cue has been unrewarded before. For example, bees will take longer to 'learn' that a certain floral odor signals a feeding opportunity if they first repeatedly encountered the smell when food was absent. Such a mechanism allows organisms to devote more attention to other stimuli which have the potential to be important for survival. The strength of latent inhibition ­ as revealed by how quickly and easily an individual can learn to associate a reward with a previously unrewarded stimulus ­ can differ between individuals. For instance, this is the case in honey bee colonies, where workers have the same mother but may come from different fathers. Such genetic variation can be beneficial for the hive, with high latent inhibition workers being better suited for paying attention to and harvesting known resources, and their low latent inhibition peers for discovering new ones. However, the underlying genetic and neural mechanisms underpinning latent inhibition variability between individuals remained unclear. To investigate this question, Latshaw et al. cross-bred bees from high and low latent inhibition genetic lines. The resulting progeny underwent behavioral tests, and the genome of low and high latent inhibition individuals was screened. These analyses revealed a candidate gene, Amtyr1, which was associated with individual variations in the learning mechanism. Further experiments showed that blocking or disrupting the production the AMTYR1 protein led to altered latent inhibition behavior as well as dampened attention-related processing in recordings from the central nervous system. Based on these findings, a model was proposed detailing how varying degrees of Amtyr1 activation can tune Hebbian plasticity, the brain mechanism that allows organisms to regulate associations between cues and events. Importantly, because of the way AMTYR1 acts in the nervous system, this modulatory role could go beyond latent inhibition, with the associated gene controlling the activity of a range of foraging-related behaviors. Genetic work in model organisms such as fruit flies would allow a more in-depth understanding of such network modulation.

GABA signaling affects motor function in the honey bee.

GABA is the most common inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. In insects, inhibition plays important roles at the neuromuscular junction, in the regulation of central pattern generators, and in the modulation of information in higher brain processing centers. Additionally, increasing our understanding of the functions of GABA is important since GABAA receptors are the targets of several classes of pesticides. To investigate the role of GABA in motor function, honey bee foragers were injected with GABA or with agonists or antagonists specific for either GABAA or GABAB receptors. Compounds that activated either type of GABA receptor decreased activity levels. Bees injected with the GABAA receptor antagonist picrotoxin lost the ability to right themselves, whereas blockade of GABAB receptors led to increases in grooming. Injection with antagonists of either GABAA or GABAB receptors resulted in an increase in extended wing behavior, during which bees kept their wings out at right angles to their body rather than folded along their back. These data suggest that the GABA receptor types play distinct roles in behavior and that GABA may affect behavior at several different levels.

CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly.

CRISPR Cas9 genome editing allows researchers to modify genes in a multitude of ways including to obtain deletions, epitope-tagged loci, and knock-in mutations. Within 6 years of its initial application, CRISPR-Cas9 genome editing has been widely employed, but disadvantages to this method, such as low modification efficiencies and off-target effects, need careful consideration. Obtaining custom donor vectors can also be expensive and time-consuming. This chapter details strategies to overcome barriers to CRISPR-Cas9 genome editing as well as recent developments in employing this technique.

Influence of sugar experience during development on gustatory sensitivity of the honey bee.

The level of response to sugar plays a role in many aspects of honey bee behavior including age dependent polyethism and division of labor. Bees may tune their sensitivity to sugars so that they maximize collection of high quality nectar, but they must also be able to collect from less profitable sources when high quality food is scarce. However, our understanding of the mechanisms by which bees can change their responsiveness to different sugars remains incomplete. To investigate the plasticity of sensitivity to sugar, bees were raised on different sugars either in vitro or in colonies. Bees raised in the incubator on diets containing mostly either fructose or glucose showed significantly more responsiveness to the majority sugar. In contrast, bees raised in colonies that only foraged on fructose or glucose responded equally well to both sugars. These data suggest that developmental plasticity for responses to sugar is masked by the feeding of worker jelly to larvae and young bees. The production of worker jelly from secretions of the hypopharyngeal and mandibular glands by nurse bees ensures that both glucose and fructose are experienced by young bees so that they respond to both sugars and will be able to exploit all future food sources.

Acute ethanol ingestion impairs appetitive olfactory learning and odor discrimination in the honey bee.

Invertebrates are valuable models for increasing our understanding of the effects of ethanol on the nervous system, but most studies on invertebrates and ethanol have focused on the effects of ethanol on locomotor behavior. In this work we investigate the influence of an acute dose of ethanol on appetitive olfactory learning in the honey bee (Apis mellifera), a model system for learning and memory. Adult worker honey bees were fed a range of doses (2.5%, 5%, 10%, or 25%) of ethanol and then conditioned to associate an odor with a sucrose reward using either a simple or differential conditioning paradigm. Consumption of ethanol before conditioning significantly reduced both the rate of acquisition and the asymptotic strength of the association. Honey bees also exhibited a dose dependent reduction in arousal/attention during conditioning. Consumption of ethanol after conditioning did not affect recall 24h later. The observed deficits in acquisition were not due to the affect of ethanol on gustatory sensitivity or motor function. However, honey bees given higher doses of ethanol had difficulty discriminating amongst different odors suggesting that ethanol consumption influences olfactory processing. Taken together, these results demonstrate that an acute dose of ethanol affects appetitive learning and olfactory perception in the honey bee.

Nutritional value and taste play different roles in learning and memory in the honey bee (Apis mellifera).

Honey bees will learn to respond to an odor when their antennae are stimulated with sucrose, even if they are not fed during the conditioning phase. However, if they are not fed, the memory of this association is significantly reduced 24 h after conditioning. These results suggest that stimulation of proboscis with sucrose and/or the nutritional quality of the reward plays an important role in establishing a long lasting memory. Three sugars, xylose, sorbitol and mannitol, are used to investigate the relationship among learning, sensory perception and nutritional value. The proboscis extension reflex is used to show that honey bees cannot taste these sugars, whereas mortality data suggest that bees can metabolize all three sugars. Feeding with sorbitol or xylose during olfactory associative conditioning restores robust 24 h memories. However, when given a free choice between consuming sucrose alone or sucrose supplemented with these nutritional sugars, bees did not show a preference for food containing the higher nutritional content. Furthermore, bees did not ingest solutions containing only the tasteless sugar even when it was the only food source. Together, these results suggest that nutritional content and not just sensory information is important for establishing long term memories, but that bees may not be able to assess nutritional content when it is disassociated from taste.

Acute ethanol ingestion produces dose-dependent effects on motor behavior in the honey bee (Apis mellifera).

Ethanol consumption produces characteristic behavioral states in animals that include sedation, disorientation, and disruption of motor function. Using individual honey bees, we assessed the effects of ethanol ingestion on motor function via continuous observations of their behavior. Consumption of 1 M sucrose solutions containing a range of ethanol doses led to hemolymph ethanol levels of approximately 40-100 mM. Using ethanol doses in this range, we observed time and dose-dependent effects of ethanol on the percent of time our subjects spent walking, stopped, or upside down, and on the duration and frequency of bouts of behavior. The effects on grooming and flying behavior were more complex. Behavioral recovery from ethanol treatment was both time and ethanol dose dependent, occurring between 12 and 24 h post-ingestion for low doses and at 24-48 h for higher doses. Furthermore, the amount of ethanol measured in honey bee hemolymph appeared to correlate with recovery. We predict that the honey bee will prove to be an excellent model system for studying the influence of ethanol on the neural mechanisms underlying behavior.

Octopamine and tyramine influence the behavioral profile of locomotor activity in the honey bee (Apis mellifera).

The biogenic amines octopamine and tyramine are believed to play a number of important roles in the behavior of invertebrates including the regulation of motor function. To investigate the role of octopamine and tyramine in locomotor behavior in honey bees, subjects were injected with a range of concentrations of octopamine, tyramine, mianserin or yohimbine. Continuous observation of freely moving worker bees was used to examine the effects of these treatments on the amount of time honey bees spent engaged in different locomotor behaviors such as walking, grooming, fanning and flying. All treatments produced significant shifts in behavior. Decreases in time spent walking and increases in grooming or stopped behavior were observed for every drug. However, the pattern of the shift depended on drug, time after injection and concentration. Flying behavior was differentially affected with increases in flying seen in octopamine treated bees, whereas those receiving tyramine showed a decrease in flying. Taken together, these data provide evidence that octopamine and tyramine modulate motor function in the honey bee perhaps via interaction with central pattern generators or through effects on sensory perception.

Developmental expression of a tyramine receptor gene in the brain of the honey bee, Apis mellifera.

This study reveals that the tyramine receptor gene, Amtyr1, is expressed in the developing brain, as well as in the brain of the adult worker honey bee. Changes in levels of Amtyr1 expression were examined using Northern analysis. Age-related increases in Amtyr1 transcript levels were observed not only during metamorphic adult development, but also in the brain of the adult worker bee. RNA in situ hybridization revealed the pattern of Amtyr1 expression. Cell bodies staining intensely for tyramine receptor-gene transcript were observed throughout the somata rind, with well-defined clusters of cells associated with developing mushroom bodies, optic lobes, and antennal lobes of the brain. Staining for Amtyr1 transcript was particularly intense within the three major divisions of mushroom body intrinsic neurons (outer compact, noncompact, and inner compact cells), suggesting that Amtyr1 is highly expressed in these structures. Activation of AmTYR1 receptors heterologously expressed in insect (Spodoptera frugiperda) cells led to a reduction in intracellular levels of cAMP similar to that reported for AmTYR1 receptors expressed in mammalian (HEK 293) cells (Blenau et al. [2000] J Neurochem 74:900-908). Taken together, these results suggest that AmTYR1 receptors may play a role in the developing brain as well as in the brain of the adult worker bee. The actions of tyramine are likely to be mediated, at least in part, via the cAMP-signaling pathway.

Characterization of a D2-like dopamine receptor (AmDOP3) in honey bee, Apis mellifera.

Dopamine is an important neurotransmitter in vertebrate and invertebrate nervous systems and is widely distributed in the brain of the honey bee, Apis mellifera. We report here the functional characterization and cellular localization of the putative dopamine receptor gene, Amdop3, a cDNA clone isolated and identified in previous studies as AmBAR3 (Apis mellifera Biogenic Amine Receptor 3). The Amdop3 cDNA encodes a 694 amino acid protein, AmDOP3. Comparison of AmDOP3 to Drosophila melanogaster sequences indicates that it is orthologous to the D2-like dopamine receptor, DD2R. Using AmDOP3 receptors expressed in HEK293 cells we show that of the endogenous biogenic amines, dopamine is the most potent AmDOP3 agonist, and that activation of AmDOP3 receptors results in down regulation of intracellular levels of cAMP, a property characteristic of D2-like dopamine receptors. In situ hybridization reveals that Amdop3 is widely expressed in the brain but shows a pattern of expression that differs from that of either Amdop1 or Amdop2, both of which encode D1-like dopamine receptors. Nonetheless, overlaps in the distribution of cells expressing Amdop1, Amdop2 and Amdop3 mRNAs suggest the likelihood of D1:D2 receptor interactions in some cells, including subpopulations of mushroom body neurons.

Developmental changes in expression patterns of two dopamine receptor genes in mushroom bodies of the honeybee, Apis mellifera.

The expression patterns of two dopamine receptor genes, Amdop1 and Amdop2, in the developing mushroom bodies of the honeybee brain were determined by using in situ hybridisation. Both genes were expressed throughout pupal development, but their patterns of expression in the three major divisions of mushroom body intrinsic neurons (outer compact cells, noncompact cells, and inner compact cells) were quite distinct. Amdop1 expression could be detected in all three mushroom body cell groups throughout development. Staining for Amdop1 mRNA was particularly intense in newly born Kenyon cells, suggesting that levels of Amdop1 expression are higher in newborn cells than in more mature mushroom body neurons. This was not the case for Amdop2. Amdop2 expression in the mushroom bodies was restricted to inner and outer compact cells during most of pupal development, appearing in noncompact cells only late in metamorphosis or at adult eclosion. In contrast to the case with Amdop1, staining for Amdop2 mRNA was observed in glial cells. Expression of Amdop2 in glial cells was detected only at early stages of glial cell development, when the cells are reported to be actively dividing. This study not only implicates dopamine in the development of honeybee mushroom bodies but also suggests different roles for the two dopamine receptors investigated.

Analysis of two D1-like dopamine receptors from the honey bee Apis mellifera reveals agonist-independent activity.

Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.

Molecular traces of alternative social organization in a termite genome.

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

Invertebrate models of alcoholism.

For invertebrates to become useful models for understanding the genetic and physiological mechanisms of alcoholism related behaviors and the predisposition towards alcoholism, several general requirements must be fulfilled. The animal should encounter ethanol in its natural habitat, so that the central nervous system of the organism will have evolved mechanisms for responding to ethanol exposure. How the brain adapts to ethanol exposure depends on its access to ethanol, which can be regulated metabolically and/or by physical barriers. Therefore, a model organism should have metabolic enzymes for ethanol degradation similar to those found in humans. The neurons and supporting glial cells of the model organism that regulate behaviors affected by ethanol should share the molecular and physiological pathways found in humans, so that results can be compared. Finally, the use of invertebrate models should offer advantages over traditional model systems and should offer new insights into alcoholism-related behaviors. In this review we will summarize behavioral similarities and identified genes and mechanisms underlying ethanol-induced behaviors in invertebrates. This review mainly focuses on the use of the nematode Caenorhabditis elegans, the honey bee Apis mellifera and the fruit fly Drosophila melanogaster as model systems. We will discuss insights gained from those studies in conjunction with their vertebrate model counterparts and the implications for future research into alcoholism and alcohol-induced behaviors.