Sucrose Sensitivity Research Articles

Oral administration of phytochemicals protects honey bees against cognitive and motor impairments induced by the insecticide fipronil.

Pollution produced by exposure to pesticides is a major concern for food security because the negative impacts on pollinators. Fipronil, an insecticide broadly used around the globe has been associated with the ongoing decline of bees. With a characteristic neuroactive toxicodynamic, fipronil leads to cognitive and motor impairments at sublethal dosages. Despite of regional bans, multilevel strategies are necessary for the protection of pollinators. Recent evidence suggests that specific nutrients in the diets of bees may induce protection against insecticides. Here, we evaluated whether the administration of three phytochemicals, namely rutin, kaempferol and p-coumaric acid provide protection to the Africanized honey bee Apis mellifera against oral administration of realistic dosages of fipronil. We tested the potential impairment produced by fipronil and the protection induced by the phytochemicals in learning, 24h memory, sucrose sensitivity and motor control. We found that the administration of fipronil induced a concentration-dependent impairment in learning and motor control, but not 24h memory or sucrose sensitivity across a 24h window. We also found that the administration of rutin, p-coumaric acid, kaempferol and the mixture was innocuous and generally offered protection against the impairments induced by fipronil. Overall, our results indicate that bees can be prophylactically protected against insecticides via nutrition, providing an alternative to the ongoing conflict between the use of insecticides and the decline of pollinators. As the studied phytochemicals are broadly present in nectar and pollen, our results suggest that the nutritional composition, and not only its production, should be considered when implementing strategies of conservation via gardens and co-cropping.

Determining the sweet detection threshold of COVID-19 patients during infection and recovery periods.

The loss of taste and smell is a common symptom of COVID-19, affecting individuals' quality of life and nutritional status. Detecting sweet thresholds during infection and recovery periods can assist in implementing dietary modifications and nutritional strategies for these patients. To investigate the changes and differences in sweet detection thresholds of confirmed COVID-19 patients on Day 1, Day 10, and Day 14 of the infection and recovery periods. The demographic factors such as gender, smoking status, BMI, and age group were abstracted on Excel sheet from the medical health records for confirmed COVID-19 patients, who were admitted to King Fahad General Hospital in Jeddah, Saudi Arabia, a COVID-19 care facility, from September 2021 to July 2022. Sweet detection thresholds were determined using a pair-wise comparison procedure and sugar solutions with varying concentrations, arranged in ascending order and presented to participants until the lowest detected concentration was noted after three consecutive positive detections, with the median just noticeable difference (JND) value calculated as the population average threshold. Sensory tests were conducted on COVID-19 patients during their infection and recovery periods to evaluate their taste sensation thresholds. The demographic factors of gender, smoking status, BMI, and age group were considered in the analysis. A total of 37 patients who met the inclusion criteria of the study were enrolled as participants. Significant variances in sweet detection thresholds were observed among the COVID-19 patients, with consistent decreases over the three testing days, indicating increasing sucrose sensitivity. Infected men showed significant returns to sweet detection thresholds on Day 14 compared to women, while infected smokers exhibited greater recoveries than non-smokers. Overweight patients had consistently elevated thresholds and recovery rates that were comparable to those of normal-weight patients by Day 14, while younger patients had lower thresholds than their older counterparts. On Day 14, the thresholds had significantly recovered to a level comparable to that of healthy individuals (approximately 0.23%). These findings suggest that sweet detection thresholds can be used as a marker for assessing the progression and recovery of COVID-19 patients. These findings highlight the importance of recognizing and managing alterations in sweet detection thresholds promptly in COVID-19 patients, as this could positively impact dietary management, nutritional recommendations, and interventions during infection and recovery periods.

MicroRNA ame-let-7 targets Amdop2 to increase sucrose sensitivity in honey bees (Apis mellifera)

BackgroundAs an important catecholamine neurotransmitter in invertebrates and vertebrates, dopamine plays multiple roles in the life of the honey bee. Dopamine receptors (DA), which specifically bind to dopamine to activate downstream cascades, have been reported to be involved in honey bee reproduction, division of labour, as well as learning and motor behaviour. However, how dopamine receptors regulate honey bee behavior remains uninvestigated.ResultsThe expression level of Amdop2 in the brain increased with the age of worker bees, which was just the opposite trend of ame-let-7. Inhibition of ame-let-7 through feeding an inhibitor upregulated Amdop2 expression; conversely, overexpression of ame-let-7 through a mimic downregulated Amdop2. Moreover, knockdown of Amdop2 in forager brain led to significantly higher sucrose responsiveness, which is similar to the phenotype of overexpression of ame-let-7. Finally, we confirmed that ame-let-7 directly targets Amdop2 in vitro by a luciferase reporter assay.Conclusionsame-let-7 is involved in the dopamine receptor signaling pathway to modulate the sucrose sensitivity in honey bees. Specifically, it down-regulates Amdop2, which then induces higher responses to sucrose. These results further unraveled the diverse mechanisms of the dopamine pathway in the regulation of insect behavior.

Sugar responsiveness could determine foraging patterns in yellowjackets

Sympatric-related species often exhibit resource partitioning. This can occur through different mechanisms, such as behavioral, morphological, and sensory variations, leading to qualitative, temporal, or spatial differences in resource exploitation, such as consuming different types of food. Sensory-based niche partitioning could be the underlying mechanism through which closely related species effectively reduce niche overlap. Here we ask whether variations in sensory responses to carbohydrates could reflect differences in the foraging patterns of two Vespula species present in Patagonia. For this, we established (i) the response thresholds toward carbohydrate solutions of foraging V. germanica and V. vulgaris in the laboratory, (ii) the sugar concentration of foraged carbohydrates in the field, and (iii) possible effects of incoming sugar concentration and performance at individual and colony levels. Results indicate a higher sucrose response threshold in V. germanica than V. vulgaris. Field results indicate that higher carbohydrate concentrations foraged by V. germanica, with 57% of V. germanica foragers returning with concentrations above 50% w/w, while only 23% of V. vulgaris foragers did so. These differences in sucrose sensitivity and foraging patterns positively correlate with colony size, irrespective of the species. Our results suggest that competition could be reduced in these closely related invasive social wasp species through sensory differences in their sugar perception levels, which would lead to them foraging different carbohydrate sources. This study suggests that sensory niche partitioning could promote species coexistence in these social wasps.

Triangular Silver Nanoparticles Synthesis: Investigating Potential Application in Materials and Biosensing

Triangular silver nanoplates (TSNPs) exhibit unique optical and antimicrobial properties due to their shape, sharp edges, and vertices. In this study, TSNPs were incorporated into biopolymer blends (bacterial cellulose (BC) with polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate (PHB)). Antimicrobial activity of materials was tested against Escherichia coli ATCC 95922 and Staphylococcus aureus ATCC 25923 (106 CFU/mL). After incubation (24 h at 37 °C, 100 rpm), optical density was measured at 630 nm. In order to assess biosensing applications, specifically fibronectin (Fn) behavior, TSNPs were protected with gold (AuTSNP) and analyzed via sucrose sensitivity test and monitored by localized surface plasmon resonance (LSPR). Additionally, AuTSNPs were coated with polyethylene glycol (PEGAuTSNP). Fibronectin functionalization of PEGAuTSNPs and pH-conformation was monitored (FnPEGAuTSNP). Eventually, adequate Fn and anti-Fn antibody concentrations were determined. BC/PHB/TSNPs showed antimicrobial activity against E. coli and S. aureus with 80 and 95% of growth inhibition, respectively. The sucrose sensitivity test indicated that the LSPRλmax of the spectra is directly proportional to the sucrose concentration. LSPRλmax of Fn-PEGAuTSNPs at pH 7 and pH 4 were measured at 633 and 643 nm, respectively. A total of 5 µg of Fn was determined to be adequate concentration, while 0.212 mg/mL of anti-Fn antibody indicatied system saturation.

Changes in resource perception throughout the foraging visit contribute to task specialization in the honey bee Apis mellifera

Division of labor is central to the ecological success of social insects. Among foragers of the honey bee, specialization for collecting nectar or pollen correlates with their sensitivity to sucrose. So far, differences in gustatory perception have been mostly studied in bees returning to the hive, but not during foraging. Here, we showed that the phase of the foraging visit (i.e. beginning or end) interacts with foraging specialization (i.e. predisposition to collect pollen or nectar) to modulate sucrose and pollen sensitivity in foragers. In concordance with previous studies, pollen foragers presented higher sucrose responsiveness than nectar foragers at the end of the foraging visit. On the contrary, pollen foragers were less responsive than nectar foragers at the beginning of the visit. Consistently, free-flying foragers accepted less concentrated sucrose solution during pollen gathering than immediately after entering the hive. Pollen perception also changes throughout foraging, as pollen foragers captured at the beginning of the visit learned and retained memories better when they were conditioned with pollen + sucrose as reward than when we used sucrose alone. Altogether, our results support the idea that changes in foragers' perception throughout the foraging visit contributes to task specialization.

Thiacloprid impairs honeybee worker learning and memory with inducing neuronal apoptosis and downregulating memory-related genes

Neonicotinoids are among the most widely used insecticides in the world and are recognized as a potential cause of pollinator decline. Previous studies have demonstrated that the neonicotinoid thiacloprid has adverse effects on foraging and memory behaviors. However, there is no direct evidence linking thiacloprid-induced neuronal cell damage in the brains of honeybees to learning and memory dysfunction. Adult honeybee (Apis mellifera L.) workers were chronically exposed to sub-lethal concentrations of thiacloprid. We discovered that thiacloprid negatively affected their survival, food consumption, and body weight. In addition, sucrose sensitivity and memory performance were impaired. We evaluated the apoptosis of honeybee brain cells using TUNEL (Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling) and Caspase-3 assays, which revealed that thiacloprid increases the dose-dependent apoptosis of neurons in the mushroom bodies (MB) and antennal lobes (AL). We also determined the abnormal transcripts of multiple genes, including vitellogenin (Vg), immune system genes (apidaecin and catalase), and memory-associated genes (pka, creb, Nmdar1, Dop2, Oa1, Oa-2R, and Oa-3R). These results indicate that exposure to sublethal concentrations of thiacloprid cause abnormal expression of memory-related genes and apoptosis of brain cells in the AL and MB, which may contribute to the memory disorder induced by thiacloprid exposure.

Study of relationships between taste receptor gene (<i>TAS1R2</i>) polymorphism rs4920566 and sugar sensitivity and food preference of sweet products

BACKGROUND : Increased consumption of sweet and high-calorie foods leads to weight gain in humans and the development of metabolic syndrome. Great attention is given to a personalized approach to nutrition correction based on genetic testing. The genetic basis for human taste sensitivity to sweet stimuli remains is understudied. The role of the g.18853330 A>G (rs4920566) polymorphism of the TAS1R2 gene in preference for sweets has not been fully studied. AIM : To investigate the possible relationship between rs4920566 polymorphism in TAS1R2 gene and sensitivity to natural sugars and food preference of high-calorie sweet foods in humans. MATERIALS AND METHODS : A single-sample experimental study was carried out. The study participants were conditionally healthy students who voluntarily agreed to conduct it. Sugar sensitivity was assessed in two tasting tests. Sucrose sensitivity thresholds (STS) were determined by staircase procedure (solutions: 8.0 to 500 mM/l). To assess food preferences for sweet foods, specially designed questionnaires with a checklist of products were used. Genomic DNA samples from all study participants were obtained from buccal epithelial cells. Genomic DNA was extracted from buccal epithelial cells using the adsorption of DNA with an inorganic sorbent in the presence of a chaotropic agent. Typing of the rs4920566 polymorphism in TAS1R2 gene was performed using asymmetric real-time PCR. RESULTS : The study included 26 young men and 110 young women which the average age was 20.8±4.8 (SD) years. Gender was a significant factor affecting the taste perception of the sweetness sugars (maltose, glucose, sucrose and fructose) in students. Young men more often than young women could not distinguish the taste of four sugars (β=-2.93(0.99), p=0.003). The rs4920566 polymorphism in TAS1R2 gene did not affect the ability of students to distinguish the taste of four natural sugars. It was found that the variation series of STS values for sucrose in young women with the A allele (16.0[16.0–31.0] versus 16.0[8.0–16.0] mmol/l, p cor =0.002) and A/G genotype (16.0[16.0–31.0] vs. 16.0[8.0–16.0] mmol/l, p cor =0.010) were significantly lower compared to young men. Separately, in young women with the G/G genotype the range of STS is shifted towards higher concentrations of sucrose compared with women with the A/G genotype (16.0[16.0–31.0] vs. 16.0[8.0 -16.0] mmol/l, p cor =0.039). Testing the hypothesis about the randomness of the relationship between the rs4920566 genotypes of the TAS1R2 gene and the liking rating of foods with a sweet taste using Kruskal-Wallis test did not allow us to conclude that the A/A, A/G and G/G genotypes of the TAS1R2 gene influence the choice of sugary high-calorie foods in students. CONCLUSION : Our results are consistent with the literature data and confirm that rs4920566 polymorphism of TAS1R2 gene cannot be an informative marker for the diagnosis of metabolic conditions associated with the consumption of high-calorie sweet foods. Likely, its functions are related to the mechanisms of neurotransduction of the sweet taste signal.

How foraging preference and activity level of bumble bees contribute to colony flexibility under resource demand

Groups of cooperative organisms, including humans, benefit from the contributions of both specialist and generalist individuals. Group responses to resource or task demand emerge from the specific actions of individuals within the group. Bumble bee colonies need to respond to resource demands for both pollen and nectar for colony growth and reproduction. Bumble bee workers have been shown to display differences in foraging preferences for these essential resources and in activity level. This study investigated the responses of Bombus terrestris individuals to resource demand by subjecting colonies to different pollen and sucrose storage scenarios, considering their individual foraging preferences and activity levels. The sucrose sensitivity and olfactory learning ability of individuals were then compared along a spectrum of foraging preference (between pollen and sucrose foraging) using the proboscis extension reflex paradigm. When sucrose was in demand, individuals with a preference for sucrose showed the largest upregulation in foraging bouts. Individual preference for pollen and activity level correlated with the upregulation of foraging bouts when pollen was in demand. This study detected no relationship between body size, sucrose preference and worker sucrose sensitivity or olfactory learning ability, tested with proboscis extension reflex conditioning. This study highlights the potential value of foraging preferences in bumble bee colonies but also the flexibility these colonies have with a small foraging workforce. • Bumble bees with high sucrose preference upregulate to fill sucrose demands. • Pollen preference and individual activity level predict upregulation in pollen demand. • Body size and sucrose preference do not predict worker sucrose sensitivity. • Body size and sucrose preference do not predict odour learning performance. • Workload is not evenly split between individuals.

Macronutrient balance has opposing effects on cognition and survival in honey bees

Abstract Malnutrition in vertebrates, including humans, is known to affect brain development. Protein deprivation in the early stages of life dramatically impairs cognitive abilities. In insects, dietary protein deficiency influences several physiological and behavioural traits, but it is unknown how protein to carbohydrate (P:C) ratio simultaneously affects survival and cognitive performance. We used the geometric framework approach to examine the effects of P:C ratio in the diet of newly emerged honey bees on their survival, sucrose sensitivity, and learning and memory by conditioning of the proboscis extension response. The P:C ratio in the diet did not affect sucrose sensitivity, whereas it affected lifespan and cognition differently. While lifespan peaked at P:C ratio of 1:19, learning and short‐term memory peaked at P:C ratios of 1:9 and 1:4. Protein deprivation slightly reduced bee lifespan but highly impaired learning and short‐term memory. On the other hand, an excess of protein dramatically reduced bee lifespan with lesser effects on learning and short‐term memory. Bees were only able to form an early long‐term memory when they had been eating a protein diet (P:C ratio of 1:9) during 24 hr before the test, regardless of the diet eaten previously. Our study shows that nutrition affects honey bee cognitive abilities, but that these effects, unlike in vertebrates, can be quickly reversed with an appropriate diet. Read the free Plain Language Summary for this article on the Journal blog.

Acute and chronic ingestion of polyethylene (PE) microplastics has mild effects on honey bee health and cognition

The massive use of plastic has contributed to huge quantities of hazardous refuse at a global scale and represents one of the most prominent issues of the Anthropocene. Microplastics (MPs) have been detected in almost all environments and pose a potential threat to a variety of plant and animal species. Many studies have reported a variety of effects, from negligible to detrimental, of MPs to aquatic organisms. Conversely, much less is known about their effect on terrestrial biota, and particularly on animal behavior and cognition. We assessed the oral toxicity of polyethylene (PE) MPs at three different concentrations (0.5, 5, and 50 mg L−1), and at different timescales (1 day and 7 days of exposure) and tested for their effects on survival, food intake, sucrose responsiveness, habituation to sucrose and appetitive olfactory learning and memory in the honey bee Apis mellifera. We found that workers were not completely unaffected by acute and prolonged ingestion of this polymer. A significant effect of PE on bee mortality was found for the highest concentration but not for lower ones. PE affected feeding behavior in a concentration-dependent manner, with bees consuming more food than controls when exposed to low concentration PE. Regarding our behavioral and cognitive experiments, the high concentration PE was found to affect only bees’ ability to respond consistently to sucrose but not sucrose sensitivity, habituation to sucrose or learning and memory abilities, even for prolonged exposure to PE. While these last results may look somewhat encouraging, we discussed why caution is warranted before ruling out the possibility that PE particles at environmental concentrations are harmful to honey bees.

Distinct Roles of Honeybee Gut Bacteria on Host Metabolism and Neurological Processes.

ABSTRACTThe honeybee possesses a limited number of bacterial phylotypes that play essential roles in host metabolism, hormonal signaling, and feeding behavior. However, the contribution of individual gut members in shaping honeybee brain profiles remains unclear. By generating gnotobiotic bees which were mono-colonized by a single gut bacterium, we revealed that different species regulated specific modules of metabolites in the hemolymph. Circulating metabolites involved in carbohydrate and glycerophospholipid metabolism pathways were mostly regulated by Gilliamella, while Lactobacillus Firm4 and Firm5 mainly altered amino acid metabolism pathways. We then analyzed the brain transcriptomes of bees mono-colonized with these three bacteria. These showed distinctive gene expression profiles, and genes related to olfactory functions and labor division were upregulated by Lactobacillus. Interestingly, differentially spliced genes in the brains of gnotobiotic bees largely overlapped with those of bees unresponsive to social stimuli. The differentially spliced genes were enriched in pathways involved in neural development and synaptic transmission. We showed that gut bacteria altered neurotransmitter levels in the brain. In particular, dopamine and serotonin, which show inhibitory effects on the sensory sensitivity of bees, were downregulated in bacteria-colonized bees. The proboscis extension response showed that a normal gut microbiota is essential for the taste-related behavior of honeybees, suggesting the contribution of potential interactions among different gut species to the host’s physiology. Our findings provide fundamental insights into the diverse functions of gut bacteria which likely contribute to honeybee neurological processes.IMPORTANCE The honeybee possesses a simple and host-restricted gut community that contributes to the metabolic health of its host, while the effects of bacterial symbionts on host neural functions remain elusive. We found that the colonization of specific bee gut bacteria regulates distinct circulating metabolites enriched in carbohydrate, amino acid, and glycerophospholipid metabolic pathways. The brains of bees colonized with different gut members display distinct transcriptomic profiles of genes crucial for bee behaviors and division of labor. Alternative splicing of genes related to disordered bee behaviors is also mediated. The presence of gut bacteria promotes sucrose sensitivity with major neurotransmitters being regulated in the brain. Our findings demonstrate how individual bee gut species affect host behaviors, highlighting the gut-brain connections important for honeybee neurobiological and physiological states.

Evaluation of masticatory behavior and taste sensitivity after pacifier removal in preschool children: a 1-year follow-up.

The aim was to investigate the influence of pacifier removal on the development of masticatory function and taste sensitivity in preschool children. Sixty children (mean age 48.2months) were divided into two groups: pacifier group (n = 28) and a control group (n = 32), which were evaluated and followed up for a period of 12months (at baseline, 6months, and 1year). Masticatory and swallowing functions were assessed using the Mastication Observation and Evaluation (MOE) protocol and Orofacial Myofunctional Rating (MBGR), respectively. Detection thresholds for sucrose and urea were measured by the staircase method. The two-way ANOVA mixed model was used for time*group interaction analysis. MOE scores improved significantly over time in both groups, although a significant difference between groups persisted after 1year. On the other hand, swallowing scores were significantly different at baseline, but within 1year, the scores were no longer different between groups. Chewing time and the number of cycles were not different between groups and both decreased after 1year. Sucrose sensitivity was significantly greater in the control group at baseline and changed over time (p < 0.05), being no longer different between groups after 6months. Bitter sensitivity did not differ over time nor between groups. Detection threshold for sucrose differed significantly between children with and without pacifier habit at a mean age of 42months. Total masticatory function did not self-correct after sucking habit removal within a 1-year period. Children with pacifier habit showed important changes in masticatory function that did not self-correct 1year after cessation of the habit, highlighting the need for prevention and habit interruption as early as possible.

Effects of cypermethrin as a model chemical on life cycle and biochemical responses of the tropical stingless bee Meliponula bocandei Spinola, 1853

The tropical stingless bee, Meliponula bocandei Spinola, 1853 is an important pollinator in West Africa but there is no study on the effect of pesticides on this species. This study assessed the effects of cypermethrin, a common pyrethroid on survival, taste (sucrose sensitivity), and biochemical responses of M. bocandei. The biochemical markers were superoxide dismutase [SOD], catalase [CAT], glutathione S-transferase (GST), reduced glutathione [GSH]), and acetylcholinesterase [AChE]) as well as glucose, trehalose and total protein concentrations. Test temperature was optimized by acclimatizing adult worker bees collected from a pristine natural colony to different temperature regimes in the laboratory and fed with sucrose solution. The optimized temperature (22°C) for survival and sucrose consumption was adopted for the toxicity test. The 24–48 h oral lethal dose (LD50) and 24–96 h indirect contact lethal concentration (LC50) of cypermethrin on the bees was in the range of 0.66–0.76 µg/ml and 92.24–223.69 µg/ml respectively. Also, the overall PER response reduced below 50% in bees that were orally exposed to high doses of cypermethrin. There was significant decrease in glucose, trehalose, total protein and GSH concentrations in bees when compared with the control. Also, significant alterations were observed in SOD, CAT, GST and AChE activity for both oral and contact exposure pathways. In conclusion, cypermethrin modulated the activities of biomarkers of oxidative stress and altered the level of energy metabolites in the bees.

Enantioselective Olfactory Effects of the Neonicotinoid Dinotefuran on Honey Bees (Apis mellifera L.).

Sublethal exposure to neonicotinoids affects honey bee olfaction, but few studies have investigated the sublethal effects of the enantioselective neonicotinoid dinotefuran on honey bee olfaction. This study assessed the sublethal olfactory toxicity of dinotefuran enantiomers to honey bees. Compared to R-dinotefuran, S-dinotefuran had higher acute oral toxicity, sucrose sensitivity effects, octopamine concentrations, lower learning ability, and memory effects on honey bees. High-throughput circular RNA sequencing of the honey bee brain revealed that R-dinotefuran caused more gene regulatory changes than S-dinotefuran. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that the SERCA, Kca, and Maxik genes may be related to the enantioselective effects of dinotefuran isomers on honey bee olfaction. These results indicated that the current ecotoxicological safety knowledge about chiral dinotefuran effects on honey bees should be amended.

Honeybee rebel workers preferentially respond to high concentrations of sucrose

The honeybee is one of several eusocial species with a complex set of anatomical, physiological, and behavioural traits that are correlated with foraging preferences. Sensory sensitivity to sucrose is associated with the foraging preferences of workers, with individuals that are less sensitive to sugar preferring to forage for nectar and those that are highly sensitive to sucrose preferring to forage for pollen. In this study, we test this hypothesis by comparing the sucrose responsiveness of genetically similar normal and rebel honeybee workers, which present different foraging preferences. We found that rebels, which prefer to forage for nectar and have a higher reproductive potential than normal workers, are characterised by sucrose sensitivity that is lower than that of normal workers. Moreover, we show that in both rebel and normal workers, sensitivity to sugar in nectar is lower in workers when their ovaries contain more ovarioles. The results confirm the prediction that bees present a foraging preference depending on their response thresholds to sucrose in solution. However, compared with previous studies, our investigation does not support the assumption that reproductive workers are more sensitive to sucrose concentrations in nectar.

A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

Acute sublethal exposure to toxic heavy metals alters honey bee (Apis mellifera) feeding behavior

Heavy metal toxicity is an ecological concern in regions affected by processes like mining, industry, and agriculture. At sufficiently high concentrations, heavy metals are lethal to honey bees, but little is known about how sublethal doses affect honey bees or whether they will consume contaminated food. We investigated whether honey bees reject sucrose solutions contaminated with three heavy metals – cadmium, copper, and lead – as a measure of their ability to detect the metals, and whether ingesting these metals altered the bees’ sucrose sensitivity. The metals elicited three different response profiles in honey bees. Cadmium was not rejected in any of the assays, and ingesting cadmium did not alter sucrose sensitivity. Copper was rejected following antennal stimulation, but was readily consumed following proboscis stimulation. Ingestion of copper did not alter sucrose sensitivity. Lead appeared to be palatable at some concentrations and altered the bees’ sensitivity to and/or valuation of sucrose following antennal stimulation or ingestion of the metal. These differences likely represent unique mechanisms for detecting each metal and the pathology of toxicity. The bees’ ability to detect and consume these toxic metals highlights the risk of exposure to these elements for bees living in or near contaminated environments.

Lateralization of Sucrose Responsiveness and Non-associative Learning in Honeybees.

Lateralization is a fundamental property of the human brain that affects perceptual, motor, and cognitive processes. It is now acknowledged that left–right laterality is widespread across vertebrates and even some invertebrates such as fruit flies and bees. Honeybees, which learn to associate an odorant (the conditioned stimulus, CS) with sucrose solution (the unconditioned stimulus, US), recall this association better when trained using their right antenna than they do when using their left antenna. Correspondingly, olfactory sensilla are more abundant on the right antenna and odor encoding by projection neurons of the right antennal lobe results in better odor differentiation than those of the left one. Thus, lateralization arises from asymmetries both in the peripheral and central olfactory system, responsible for detecting the CS. Here, we focused on the US component and studied if lateralization exists in the gustatory system of Apis mellifera. We investigated whether sucrose sensitivity is lateralized both at the level of the antennae and the fore-tarsi in two independent groups of bees. Sucrose sensitivity was assessed by presenting bees with a series of increasing concentrations of sucrose solution delivered either to the left or the right antenna/tarsus and measuring the proboscis extension response to these stimuli. Bees experienced two series of stimulations, one on the left and the other on the right antenna/tarsus. We found that tarsal responsiveness was similar on both sides and that the order of testing affects sucrose responsiveness. On the contrary, antennal responsiveness to sucrose was higher on the right than on the left side, and this effect was independent of the order of antennal stimulation. Given this asymmetry, we also investigated antennal lateralization of habituation to sucrose. We found that the right antenna was more resistant to habituation, which is consistent with its higher sucrose sensitivity. Our results reveal that the gustatory system presents a peripheral lateralization that affects stimulus detection and non-associative learning. Contrary to the olfactory system, which is organized in two distinct brain hemispheres, gustatory receptor neurons converge into a single central region termed the subesophagic zone (SEZ). Whether the SEZ presents lateralized gustatory processing remains to be determined.

Effect of Radiation on Sucrose Detection Thresholds of Mice.

Radiotherapy is one of the most common treatments for head and neck cancers, with an almost obligate side effect of altered taste (Conger AD. 1973. Loss and recovery of taste acuity in patients irradiated to the oral cavity. Radiat Res. 53:338-347.). In mice, targeted irradiation of the head and neck causes transient repression of proliferation of basal epithelial cells responsible for taste cell replacement, leading to a temporary depletion of taste sensory cells within taste buds, including Type II taste cells involved in detection of sweet stimuli (Nguyen HM, Reyland ME, Barlow LA. 2012. Mechanisms of taste bud cell loss after head and neck irradiation. J Neurosci. 32:3474-3484.). These findings suggest that irradiation may elevate sucrose detection thresholds, peaking at 7 days postirradiation when loss of Type II cells is greatest. To test this hypothesis, sucrose detection thresholds (concentration detected in 50% of presentations) were measured in mice for 15 days after treatment of: 1) irradiation while anesthetized, 2) anesthetic alone, or 3) saline. Mice were trained to distinguish water from several concentrations of sucrose. Mice were irradiated with one 8 Gy dose (RADSOURCE-2000 X-ray Irradiator) to the nose and mouth while under 2,2,2-tribromethanol anesthesia (Avertin). Unexpectedly, mice given anesthesia showed a small elevation in sucrose thresholds compared to saline-injected mice, but irradiated mice show significantly elevated sucrose thresholds compared to either control group, an effect that peaked at 6-8 days postirradiation. The timing of loss and recovery of sucrose sensitivity generally coincides with the reported maximal reduction and recovery of Type II taste cells (Nguyen HM, Reyland ME, Barlow LA. 2012. Mechanisms of taste bud cell loss after head and neck irradiation. J Neurosci. 32:3474-3484.). Thus, even a single dose of irradiation can significantly alter detection of carbohydrates, an important consideration for patients undergoing radiotherapy.