A whiff of the future: functions of phenylalanine-derived aroma compounds and advances in their industrial production.

Plants produce myriad aroma compounds-odorous molecules that are key factors in countless aspects of the plant's life cycle, including pollinator attraction and communication within and between plants. For humans, aroma compounds convey accurate information on food type, and are vital for assessing the environment. The phenylpropanoid pathway is the origin of notable aroma compounds, such as raspberry ketone and vanillin. In the last decade, great strides have been made in elucidating this pathway with the identification of numerous aroma-related biosynthetic enzymes and factors regulating metabolic shunts. These scientific achievements, together with public acknowledgment of aroma compounds' medicinal benefits and growing consumer demand for natural products, are driving the development of novel biological sources for wide-scale, eco-friendly, and inexpensive production. Microbes and plants that are readily amenable to metabolic engineering are garnering attention as suitable platforms for achieving this goal. In this review, we discuss the importance of aroma compounds from the perspectives of humans, pollinators and plant-plant interactions. Focusing on vanillin and raspberry ketone, which are of high interest to the industry, we present key knowledge on the biosynthesis and regulation of phenylalanine-derived aroma compounds, describe advances in the adoption of microbes and plants as platforms for their production, and propose routes for improvement.

Unbalanced fatty acid diets impair discrimination ability of honey bee workers to damaged and healthy brood odors.

Nutrition supports social insect colonies by regulating both individual performance and colony growth. In honey bee colonies, task-related behaviors such as nursing and foraging are partially mediated by nutrition. Young workers (nurses) consume almost all of the pollen in the hive, while foragers consume mostly nectar. Pollen provides vital proteins and lipids, consumed by nurse bees for approximately 1 week post-eclosion. The role that lipids play in the physiology and behavior of adult bees is gaining significant attention. Recent research suggests that diets with balanced ratios of fatty acids increase olfactory learning in honey bees. Olfaction is crucial for young worker bees to perform brood care and cell cleaning behaviors, which is important for hive health and disease control. Thus, we targeted the early adult, pollen-feeding stage to examine how fatty acids affect cognition to hive-relevant odors. We fed young workers (days 0-9) diets balanced or unbalanced in their ratio of essential fatty acids (ω-6:3) sourced from pollen or cooking oils. We then measured their ability to learn healthy and damaged brood odors, as well as their ability to discriminate between the two. Workers fed balanced diets could learn and discriminate between brood odors better than workers fed unbalanced diets. Consumption of both diet types decreased with age, but their cognitive effects remained. These results suggest that diet affects young worker cognitive development, which may affect task-related behaviors and colony hygiene.

Spatial patterning of scent in petunia corolla is discriminated by bees and involves the ABCG1 transporter.

Floral guides are patterned cues that direct the pollinator to the plant reproductive organs. The spatial distribution of showy visual and olfactory traits allows efficient plant-pollinator interactions. Data on the mechanisms underlying floral volatile patterns or their interactions with pollinators are lacking. Here we characterize the spatial emission patterns of volatiles from the corolla of the model plant Petunia × hybrida and reveal the ability of honeybees to distinguish these patterns. Along the adaxial epidermis, in correlation with cell density, the petal base adjacent to reproductive organs emitted significantly higher levels of volatiles than the distal petal rim. Volatile emission could also be differentiated between the two epidermal surfaces: emission from the adaxial side was significantly higher than that from the abaxial side. Similar emission patterns were also observed in other petunias, Dianthus caryophyllus (carnation) and Argyranthemum frutescens (Marguerite daisy). Analyses of transcripts involved in volatile production/emission revealed lower levels of the plasma-membrane transporter ABCG1 in the abaxial versus adaxial epidermis. Transient overexpression of ABCG1 enhanced emission from the abaxial epidermis to the level of the adaxial epidermis, suggesting its involvement in spatial emission patterns in the epidermal layers. Proboscis extension response experiments showed that differences in emission levels along the adaxial epidermis, that is, petal base versus rim, detected by GC-MS are also discernible by honeybees.

Effect of diet lipids and omega-6:3 ratio on honey bee brood development, adult survival and body composition.

Lipids have a key role in a variety of physiological functions in insects including energy, reproduction, growth and development. Whereas most of the required fatty acids can be synthesized endogenously, omega-3 and omega-6 polyunsaturated fatty acids (PUFA) are essential fatty acids that must be acquired through nutrition. Honey bees (Apis mellifera) obtain lipids from pollen, but different pollens vary in nutritional composition, including of PUFAs. Low floral diversity and abundance may expose bees to nutritional stress. We tested the effect of total lipids concentration and their omega-6:3 ratio on aspects of honey bee physiology: brood development, adult longevity and body fatty acids composition. All three parameters were affected by dietary lipid concentration and omega-6:3 ratio. Higher lipid concentration in diet increased brood production, and high omega-6:3 ratio increased mortality rate and decreased brood rearing. Fatty acid analysis of the bees showed that the amount of lipids and the omega-6:3 ratio in their body generally reflected the composition of the diet on which they fed. Consistent with previous findings of the importance of a balanced omega-6:3 ratio diet for learning performance, we found that such a balanced PUFA diet, with above threshold total lipid composition, is also necessary for maintaining proper colony development.

A Transmissible RNA Pathway in Honey Bees.

Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive.

Natural Variation in Flower Color and Scent in Populations of Eruca sativa (Brassicaceae) Affects Pollination Behavior of Honey Bees.

Plants of Eruca sativa Mill. (Brassicaceae) from desert and Mediterranean populations in Israel differ in flower color and size. In the desert habitat, the population has higher abundance of flowers with cream color and longer petals, whereas in the Mediterranean habitat, the population has higher abundance of flowers with yellow and shorter petals. Choice experiments with honey bee foragers (Apis mellifera Linn., Apidae, Hymenoptera), the main pollinator in the natural habitat in Israel, confirmed that they are more attracted to the yellow flower morph than to the cream one. A proboscis extension response test indicated that honey bees are able to discriminate between flower scents of the desert and Mediterranean populations. Considering the advantage of plants of the yellow morph in attracting pollinators, we further tested in a common garden experiment whether these possess higher fitness than plants of the desert population. Indeed, a significant association was found between flower color and fruit set, and seed mass. In general, our results provide evidence for ecotypic differentiation between populations imposed by pollinators. The advantage of the yellow color morph in attracting pollinators may explain its dominance among plants of the Mediterranean population. We discuss why the cream color morph may be dominant in the desert habitat, considering the possibility of different pollinators, tradeoffs between traits, or pleiotropy.

Omega-6:3 Ratio More Than Absolute Lipid Level in Diet Affects Associative Learning in Honey Bees.

Floral pollen is a major source of honey bee nutrition that provides them with micro- and macro-nutrients, including proteins, fatty acids, vitamins, and minerals. Different pollens vary in composition, including in the essential fatty acids, alpha-linolenic acid (omega-3) and linoleic acid (omega-6). Monocultures, prevalent in modern agriculture, may expose honey bee colonies to unbalanced omega-6:3 diets. The importance of omega-3 in the diet for adequate learning and cognitive function, with a focus on suitable omega-6:3 ratio, is well documented in mammals. We have recently shown, for the first time in invertebrates, the importance of omega-3 in diets for associative learning ability in honey bees. In the current work, we examine the effect of the absolute amount of omega-3 in diet compared to the omega-6:3 ratio on honey bee associative learning. We fed newly emerged bees for 1 week on different artificial diets, which had lipid concentration of 1, 2, 4, or 8%, with omega-6:3 ratios of 0.3, 1, or 5, respectively. We then tested the bees in a proboscis-extension response olfactory conditioning assay. We found that both omega-6:3 ratio and total lipid concentration affected learning. The most detrimental diet for learning was that with a high omega-6:3 ratio of 5, regardless of the absolute amount of omega-3 in the diet. Bees fed an omega-6:3 ratio of 1, with 4% total lipid concentration achieved the best performance. Our results with honey bees are consistent with those found in mammals. Best cognitive performance is achieved by a diet that is sufficiently rich in essential fatty acids, but as long as the omega-6:3 ratio is not high.

Random peptide mixtures as new crop protection agents.

Many types of crops are severely affected by at least one important bacterial disease. Chemical control of bacterial plant diseases in the field vastly relies on copper-based bactericides, yet with limited efficacy. In this study, we explored the potential of two random peptide mixture (RPM) models as novel crop protection agents. These unique peptide mixtures consist of random combination of l-phenylalanine and l- or d-lysine (FK-20 and FdK-20, respectively) along the 20 mer chain length of the peptides. Both RPMs displayed powerful bacteriostatic and bactericidal activities towards strains belonging to several plant pathogenic bacterial genera, for example, Xanthomonas, Clavibacter and Pseudomonas. In planta studies in the glasshouse revealed that RPMs significantly reduced disease severity of tomato and kohlrabi plants infected with Xanthomonas perforans and Xanthomonas campestris pv. campestris respectively. Moreover, RPM effects on reduction in disease severity were similar to those exerted by the commercial copper-based bactericide Kocide 2000 that was applied at a 12-fold higher concentration of the active compound relative to the RPM treatments. Importantly, the two tested RPM compounds had no toxic effect on survival of bees and Caco-2 mammalian cells. This study demonstrates the potential of these innovative RPMs to serve as crop protection agents against crop diseases caused by phytopathogenic bacteria.

Nutritional Physiology and Ecology of Honey Bees.

Honey bees feed on floral nectar and pollen that they store in their colonies as honey and bee bread. Social division of labor enables the collection of stores of food that are consumed by within-hive bees that convert stored pollen and honey into royal jelly. Royal jelly and other glandular secretions are the primary food of growing larvae and of the queen but are also fed to other colony members. Research clearly shows that bees regulate their intake, like other animals, around specific proportions of macronutrients. This form of regulation is done as individuals and at the colony level by foragers.

Omega-3 deficiency impairs honey bee learning.

Deficiency in essential omega-3 polyunsaturated fatty acids (PUFAs), particularly the long-chain form of docosahexaenoic acid (DHA), has been linked to health problems in mammals, including many mental disorders and reduced cognitive performance. Insects have very low long-chain PUFA concentrations, and the effect of omega-3 deficiency on cognition in insects has not been studied. We show a low omega-6:3 ratio of pollen collected by honey bee colonies in heterogenous landscapes and in many hand-collected pollens that we analyzed. We identified Eucalyptus as an important bee-forage plant particularly poor in omega-3 and high in the omega-6:3 ratio. We tested the effect of dietary omega-3 deficiency on olfactory and tactile associative learning of the economically highly valued honey bee. Bees fed either of two omega-3-poor diets, or Eucalyptus pollen, showed greatly reduced learning abilities in conditioned proboscis-extension assays compared with those fed omega-3-rich diets, or omega-3-rich pollen mixture. The effect on performance was not due to reduced sucrose sensitivity. Omega-3 deficiency also led to smaller hypopharyngeal glands. Bee brains contained high omega-3 concentrations, which were only slightly affected by diet, suggesting additional peripheral effects on learning. The shift from a low to high omega-6:3 ratio in the Western human diet is deemed a primary cause of many diseases and reduced mental health. A similar shift seems to be occurring in bee forage, possibly an important factor in colony declines. Our study shows the detrimental effect on cognitive performance of omega-3 deficiency in a nonmammal.

Nutritional aspects of honey bee-collected pollen and constraints on colony development in the eastern Mediterranean.

Pollen is the main protein and lipid source for honey bees (Apis mellifera), and nutritionally impoverished landscapes pose a threat to colony development. To determine colony nutritional demands, we analyzed a yearly cycle of bee-collected pollen from colonies in the field and compared it to colony worker production and honey bee body composition, for the first time in social insects. We monitored monthly bee production in ten colonies at each of seven sites throughout Israel, and trapped pollen bi-monthly in five additional colonies at each of four of these sites. Pollen mixtures from each sampling date and site were analyzed for weight, total protein, total fatty acids (FAs), and FA composition. Compared to more temperate climates, the eastern Mediterranean allows a relatively high yearly colony growth of ca. 300,000-400,000 bees. Colonies at higher elevation above sea level showed lower growth rates. Queen egg-laying rate did not seem to limit growth, as peaks in capped brood areas showed that queens lay a prolific 2000 eggs a day on average, with up to 3300 eggs in individual cases. Pollen uptake varied significantly among sites and seasons, with an overall annual mean total 16.8kg per colony, containing 7.14kg protein and 677g fat. Overall mean pollen protein content was high (39.8%), and mean total FA content was 3.8%. Production cost, as expressed by the amount of nutrient used per bee, was least variable for linoleic acid and protein, suggesting these as the best descriptive variables for total number of bees produced. Linolenic acid levels in pollen during the autumn were relatively low, and supplementing colonies with this essential FA may mitigate potential nutritional deficiency. The essentiality of linoleic and linolenic acids was consistent with these FAs' tendency to be present at higher levels in collected pollen than in the expected nutrients in bee bodies, demonstrating a well-developed adjustment between pollinator nutritional demands and the nutritional value of food offered by pollinated plants.

Amino acid and carbohydrate tradeoffs by honey bee nectar foragers and their implications for plant-pollinator interactions.

Honey bees are important pollinators, requiring floral pollen and nectar for nutrition. Nectar is rich in sugars, but contains additional nutrients, including amino acids (AAs). We tested the preferences of free-flying foragers between 20 AAs at 0.1% w/w in sucrose solutions in an artificial meadow. We found consistent preferences amongst AAs, with essential AAs preferred over nonessential AAs. The preference of foragers correlated negatively with AA induced deviations in pH values, as compared to the control. Next, we quantified tradeoffs between attractive and deterrent AAs at the expense of carbohydrates in nectar. Bees were attracted by phenylalanine, willing to give up 84units sucrose for 1unit AA. They were deterred by glycine, and adding 100 or more units of sucrose could resolve to offset 1unit AA. In addition, we tested physiological effects of AA nutrition on forager homing performance. In a no-choice context, caged bees showed indifference to 0.1% proline, leucine, glycine or phenylalanine in sucrose solutions. Furthermore, flight tests gave no indication that AA nutrition affected flight capacity directly. In contrast, low carbohydrate nutrition reduced the performance of bees, with important methodological implications for homing studies that evaluate the effect of substances that may affect imbibition of sugar solution. In conclusion, low AA concentrations in nectar relative to pollen suggest a limited role in bee nutrition. Most of the 20 AAs evoked a neutral to a mild deterrent response in bees, thus it seems unlikely that bees respond to AAs in nectar as a cue to assess nutritional quality. Nonetheless, free choice behavior of foraging bees is influenced, for instance by phenylalanine and glycine. Thus, AAs in nectar may affect plant-pollinator interactions and thereby exhibit a selective pressure on the flora in the honey bee habitat.

Nectar minerals as regulators of flower visitation in stingless bees and nectar hoarding wasps.

Various nectar components have a repellent effect on flower visitors, and their adaptive advantages for the plant are not well understood. Persea americana (avocado) is an example of a plant that secretes nectar with repellent components. It was demonstrated that the mineral constituents of this nectar, mainly potassium and phosphate, are concentrated enough to repel honey bees, Apis mellifera, a pollinator often used for commercial avocado pollination. Honey bees, however, are not the natural pollinator of P. americana, a plant native to Central America. In order to understand the role of nectar minerals in plant-pollinator relationships, it is important to focus on the plant's interactions with its natural pollinators. Two species of stingless bees and one species of social wasp, all native to the Yucatan Peninsula, Mexico, part of the natural range of P. americana, were tested for their sensitivity to sugar solutions enriched with potassium and phosphate, and compared with the sensitivity of honey bees. In choice tests between control and mineral-enriched solutions, all three native species were indifferent for mineral concentrations lower than those naturally occurring in P. americana nectar. Repellence was expressed at concentrations near or exceeding natural concentrations. The threshold point at which native pollinators showed repellence to increasing levels of minerals was higher than that detected for honey bees. The results do not support the hypothesis that high mineral content is attractive for native Hymenopteran pollinators; nevertheless, nectar mineral composition may still have a role in regulating flower visitors through different levels of repellency.

Comparative evaluations of reward dimensions in honey bees: evidence from two-alternative forced choice proboscis-extension conditioning.

A major challenge in understanding choice behaviour is determining how subjects evaluate alternatives that differ along multiple dimensions. Of particular interest is whether similar dimensions are compared to each other or whether each alternative is assigned an absolute value (utility). We assumed that choice proportions would follow Weber's effect, according to which discrimination is proportional to relative difference (difference/mean). We tested honey bees in a two-alternative forced choice (2AFC) paradigm of proboscis-extension response (PER) conditioning. Subjects were conditioned over six trials to associate each of two odours with sucrose solution rewards and then tested in a choice trial between the two alternatives. Each group of subjects was tested in one treatment, and there were four treatments in each of six experiments. Rewards differed in delay, duration of feeding, and sucrose concentration. In each treatment, the high-profitability alternative was better than the low-profitability alternative along a single dimension, but between treatments of each experiment values in another dimension monotonically increased. Proboscis-response proportions during the conditioning phase tended to be greater for the high-profitability alternative, and choice proportions for it in the choice phase ranged between 0.72 and 0.89 in the 24 treatments. We show for the first time that harnessed bees are sensitive to reward delay. Preferences did not differ statistically between the different treatments of any of the experiments. The results support comparative evaluation of alternatives and are pertinent to multi-attribute choice, with implications for context-dependent preferences. We also discuss the potential advantages of the 2AFC PER simultaneous choice assay.

PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers.

• Floral scent is a complex trait of biological and applied significance. To evaluate whether scent production originating from diverse metabolic pathways (e.g. phenylpropanoids and isoprenoids) can be affected by transcriptional regulators, Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) transcription factor was introduced into Rosa hybrida. • Color and scent profiles of PAP1-transgenic and control (ß-glucuronidase-expressing) rose flowers and the expression of key genes involved in the production of secondary metabolites were analyzed. To evaluate the significance of the scent modification, olfactory trials were conducted with both humans and honeybees. • In addition to increased levels of phenylpropanoid-derived color and scent compounds when compared with control flowers, PAP1-transgenic rose lines also emitted up to 6.5 times higher levels of terpenoid scent compounds. Olfactory assay revealed that bees and humans could discriminate between the floral scents of PAP1-transgenic and control flowers. • The increase in volatile production in PAP1 transgenes was not caused solely by transcriptional activation of their respective biosynthetic genes, but probably also resulted from enhanced metabolic flux in both the phenylpropanoid and isoprenoid pathways. The mechanism(s) governing the interactions in these metabolic pathways that are responsible for the production of specialized metabolites remains to be elucidated.

Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population.

The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.

Selection and breeding of honey bees for higher or lower collection of avocado nectar.

Intensive activity of honey bees, Apis mellifera L., is essential for high fruit set in avocado, Persea americana Mill., orchards, but even when hives are located inside the orchard, many bees still search for alternative blooms. We tested for a possible genetic component for a preference of avocado bloom relative to competing bloom. The honey from each hive was extracted at the end of the avocado bloom and the concentration of perseitol, a carbohydrate that is unique to avocado, was analyzed as a measure for avocado foraging. During the first year, five bee strains were compared in three different sites in Israel. Significant differences were found between strains in honey perseitol concentrations, suggesting differences in their efficiency as avocado pollinators, although these differences were site dependent. At two sites, colonies with the highest and lowest perseitol concentrations were selected as parental "high" and "low" lines. Queens were raised from the selected colonies and were instrumentally inseminated by drones from other colonies of this line. During the second and third years, colonies with inseminated queens were introduced to the avocado orchards, together with the selected colonies still surviving from the previous year. Colonies of the high line had greater perseitol concentrations than those of the low line. Selected colonies that survived from the previous year performed consistently vis-à-vis perseitol concentration, in the second year of testing. Heritability value of 0.22 was estimated based on regression of offspring on midparent. The results reveal a heritable component for willingness of honey bees to collect avocado nectar.

Optic flow informs distance but not profitability for honeybees.

How do flying insects monitor foraging efficiency? Honeybees (Apis mellifera) use optic flow information as an odometer to estimate distance travelled, but here we tested whether optic flow informs estimation of foraging costs also. Bees were trained to feeders in flight tunnels such that bees experienced the greatest optic flow en route to the feeder closest to the hive. Analyses of dance communication showed that, as expected, bees indicated the close feeder as being further, but they also indicated this feeder as the more profitable, and preferentially visited this feeder when given a choice. We show that honeybee estimates of foraging cost are not reliant on optic flow information. Rather, bees can assess distance and profitability independently and signal these aspects as separate elements of their dances. The optic flow signal is sensitive to the nature of the environment travelled by the bee, and is therefore not a good index of flight energetic costs, but it provides a good indication of distance travelled for purpose of navigation and communication, as long as the dancer and recruit travel similar routes. This study suggests an adaptive dual processing system in honeybees for communicating and navigating distance flown and for evaluating its energetic costs.

Perceptual accuracy and conflicting effects of certainty on risk-taking behaviour.

The 'certainty effect' is a notable violation of expected utility theory by decision makers. It shows that people's tendency to select the safer of two prospects increases when this prospect provides a good outcome with certainty (for example, people prefer a monetary gain of 3 with certainty over 4 with a probability of 0.8, but do not prefer 3 with a probability of 0.25 over 4 with a probability of 0.2). Subsequent work on experience-based decision making in rats extended the certainty effect to other animals, suggesting its generality across different species and different decision-making mechanisms. However, an attempt to replicate this study with human subjects showed a surprising 'reversed certainty effect', namely, the tendency to prefer the safer option decreases when this prospect is associated with certainty (and people now prefer 4 with a probability of 0.8 over 3 with certainty). Here we show that these conflicting results can be explained by perceptual noise and that the certainty effect can be restored experimentally by reducing perceptual accuracy. Using complementary experiments in humans and honeybees (Apis mellifera), we show that by manipulating perceptual accuracy in experience-based tasks, both the certainty and the reversed certainty effects can be exhibited by humans and other animals: the certainty effect emerges when it is difficult to discriminate between the different rewards, whereas the reversed certainty effect emerges when discrimination is easy. Our results fit a simple process-based model of matching behaviour, capable of explaining the certainty effect in humans and other animals that make repeated decisions based on experience. This mechanism should probably be distinguished from those involved in the original certainty effect that was exhibited by human subjects in single description-based problems.

Parameters of variable reward distributions that affect risk sensitivity of honey bees.

We investigated risk sensitivity with harnessed honey bees in a proboscis-extension conditioning paradigm. We conditioned each subject to turn its head and extend its proboscis towards one of two presented odors; one odor was associated with a constant reward and the other with a variable reward that was either low or high, with probabilities P and (1-P), respectively. Reward values and probabilities were set so that the expected value of the variable alternative was equal to that of the constant one. We performed six experimental conditions in which variability was in reward volume and three conditions in which variability was in reward concentration. The experiments were designed to systematically test the effect of various parameters that describe the reward distributions on levels of risk sensitivity. Risk aversion was greatest when variability was in reward volume, and the variable distribution included zero rewards and had a high coefficient of variation (CV=s.d./mean). The variance itself did not affect risk sensitivity. Subjects were risk indifferent when the variable distribution did not include zero rewards, however these distributions were positively skewed. The independent effects of zero rewards and distribution skew remain to be tested. Subjects were risk indifferent in conditions where variability was in reward concentration, but concentration range was limited and these distributions did not include zero rewards and were skewed. We conclude that risk aversion to variability in reward amount is a robust phenomenon for some reward distributions. A systematic evaluation of the effect of various reward distribution parameters on choice behavior should complement functional and mechanistic approaches.