Worker Honeybees Research Articles

Preparation of Ophthalmic Microemulsions Containing Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation

Royal jelly is a natural substance secreted by worker honeybees that possesses antioxidant, anti-inflammatory, and other biological activities. The purpose of this study was to formulate microemulsions with incorporated Lithuanian royal jelly for possible ophthalmic delivery and to evaluate the quality of the microemulsions in vitro. The oil in water type microemulsions were prepared by the oil titration method, incorporating royal jelly, surfactant, co-surfactant, oil, and water. Physicochemical characteristics of the microemulsions and the quantity of 10-hydroxy-2-decenoic acid released in vitro were assessed. The in vitro assessment of prepared microemulsions formulations was performed with the Statens Seruminstitut rabbit cornea (SIRC) cell culture model. The results revealed that the droplet size of all microemulsion formulations was 67.88–124.2 nm and the polydispersity index was lower than 0.180. In the in vitro release study, the release of 10-hydroxy-2-decenoic acid depended on the amount of royal jelly incorporated and on the ratio of surfactant and co-surfactant in formulations. The in vitro tests with the SIRC cell culture line have shown that all formulations were found non-irritating.

The trophocytes and oenocytes of worker and queen honey bees (Apis mellifera) exhibit distinct age-associated transcriptome profiles.

Despite the identical genomic context, trophocytes and oenocytes in worker bees exhibit aging-related phenotypes, in contrast to the longevity phenotypes in queen bees. To explore this phenomenon at the molecular level, we evaluated the age-associated transcriptomes of trophocytes and oenocytes in worker bees and queen bees using high-throughput RNA-sequencing technology (RNA-seq). The results showed that (i) while gene expression profiles were different between worker and queen bees, they remained similar between young and old counterparts; (ii) worker bees express a high proportion of low-abundance genes, whereas queen bee transcriptomes display a high proportion of moderate-expression genes; (iii) genes were upregulated to a greater extent in queen bees vs. worker bees; and (iv) distinct aging-related and longevity-related candidate genes were found in worker and queen bees. These results provide new insights into the cellular aging and longevity of trophocytes and oenocytes in honey bees. Identification of aging-associated biomarker genes also constitutes a basis for translational research of aging in higher organisms.

Identification of Mesophylic Bacterial Flora in Deceased Worker Adults of Apis mellifera caucasia (Pollmann, 1889)

Apis mellifera, widely farmed around the world, is the most economically important species within the genus Apis. While the microbiota of live honey bees have been extensively examined, bacteria found in deceased honey bees (which might indicate infection or opportunistic pathogens) is in contrast poorly studied. Therefore, we decided to investigate the mesophilic bacterial flora of dead honey bees. So, in September 2013, dead adult worker honey bees were collected from 12 different cities, most of which were in the border provinces of Turkey. We identified bacterial isolates at the species level by using different morphological, biochemical, physical and molecular methods, in conjunction with molecular phylogenetic analysis. We constructed phylogenetic trees for isolated bacteria with the MEGA 6.0 program and neighbor-joining trees were reconstructed based on 16S rDNA gene sequences. The phylogenetic trees indicated that isolates DE003, DE007, DE011, DE001, DE019 and DE016, DE029 could be new members of the genera Erwinia, Acidovorax, Hydrogenophaga and Bacillus genus, respectively. In the bioassay study results, we observed that DE019 Hydrogenophaga sp. (64.7%) and DE004 Klebsiella grimontii (73.3%) had lethal effects on the honey bees. The other mortalities ranged from 10% to 25% (p>0.05), and according to a One-Way ANOVA analysis DE004 and DE019 significantly affect the A. mellifera caucasia in adult worker honey bees. This study is the first report of Hydrogenophaga as honey bee pathogen.

The Effect of An Alternative Diet Fermented by Bee Bread Microorganisms on Hypopharyngeal Glands Development and Acini Size of Honey Bee Workers, (Apis mellifera L.)

The hypopharyngeal glands of worker bees located in the head; consist of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct. The glands contribute to the production of the royal jelly fed to queens and larvae. They are highly sensitive to the quantity and quality of the food as pollen and pollen substitutes that the nurse bee consumes. The role of the worker honey bee Apis mellifera L. changes depending on age after eclosion (age polyethism): young workers (nurse bees) take care of their brood by synthesizing and secreting brood food (royal jelly), while older workers (foragers) forage for nectar and process it into honey. In our experiment, we tested how diets impact hypopharyngeal gland development and their acini size, where our diets compared were (bee bread diet; unfermented diet; fermented diet in a simulation method for nature; and sucrose syrup). Also, we mentioned understanding the role of these glands in hive health. For this study, we have examined the morphogenesis of the hypopharyngeal gland during different ages of workers honeybee Apis mellifera L. that fed on the different diets; we measured the size of glandular acini in a robust measure. These results obtained indicated that the hypopharyngeal gland development has flexibility and can depending on the condition of the colony as the pollen substitute diet we prepared in the periods of food shortage in nature. This described the hypothesis that feeding plays an important role in the development of HG according to diet nutritional values, ensuring the importance of the fermentation process for the better health of honey bees.

A computational approach to tracking age-based task frequency distributions of Apis mellifera worker cohorts

We present a model for tracking future task frequency distributions of individual adult worker honey bee (Apis mellifera L.) cohorts in a colony based on the composition of immatures at a specific time period. The user inputs the proportions of brood comb occupied by eggs, larvae, capped brood, or non-brood/empty cells. The model captured an experimental example where a brood comb is inserted into a surrogate colony and the individuals then emerge and express their adult lives. This model has clear application to many other experiments such as sublethal pesticide exposure. The model output is the expected proportion of cohort members engaged in a focal task at any given day post-insertion.

Tachykinin signaling inhibits task-specific behavioral responsiveness in honeybee workers.

Behavioral specialization is key to the success of social insects and leads to division of labor among colony members. Response thresholds to task-specific stimuli are thought to proximally regulate behavioral specialization, but their neurobiological regulation is complex and not well understood. Here, we show that response thresholds to task-relevant stimuli correspond to the specialization of three behavioral phenotypes of honeybee workers in the well-studied and important Apis mellifera and Apis cerana. Quantitative neuropeptidome comparisons suggest two tachykinin-related peptides (TRP2 and TRP3) as candidates for the modification of these response thresholds. Based on our characterization of their receptor binding and downstream signaling, we confirm a functional role of tachykinin signaling in regulating specific responsiveness of honeybee workers: TRP2 injection and RNAi-mediated downregulation cause consistent, opposite effects on responsiveness to task-specific stimuli of each behaviorally specialized phenotype but not to stimuli that are unrelated to their tasks. Thus, our study demonstrates that TRP signaling regulates the degree of task-specific responsiveness of specialized honeybee workers and may control the context specificity of behavior in animals more generally.

Nuclear and mitochondrial markers: molecular characterization of Africanized Apis mellifera queens as royal jelly producers

Apis mellifera scutellata was introduced in Brazil in 1956. This subspecies originated from Africanized polyhybrid honey bees (A. mellifera), and quickly spread due to its high adaptability, reproductive behavior, foraging ability, and defensiveness. Good management practices have been developed that allow for good beekeeping performance of this introduced subspecies in Brazil. The selection of queens that produce royal jelly has made it possible to identify molecular markers to select the best matrices for the development of bee breeding programs to increase royal jelly production. We evaluated the ability of different major royal jelly protein-3 (mrjp3) microsatellite markers to verify the polymorphism of selected royal jelly-producing queens, as well as the mitochondrial DNA haplotypes of these queens to identify their African or European origin. We verified the maternal origins of the honey bee workers in eight colony matrices of Africanized A. mellifera belonging to a breeding program of FEI, UEM aimed at producing royal jelly. Analyses were performed for the identification of microsatellite mrjp3 markers in the nuclear DNA, and restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) was used to search for markers in the mtDNA (mitochondrial DNA). Six alleles were found for the mrjp3 locus, and it was possible to use these to infer the genotypes of queens from seven colonies. Throughout the selection period, the main alleles (C, D, and E) remained present in the genomes of queens producing royal jelly. Queens selected for royal jelly production contained a predominance of African mtDNA. Microsatellite markers and mtDNA can be used in bee breeding programs to ensure the genetic origin of the queens used and verify the efficiency of Africanized A. mellifera bee breeding programs.

Honeybee workers with higher reproductive potential have a greater learning ability

Learning ability, which allows individuals to adjust their behaviour to changing environmental conditions, has a considerable positive impact on individual fitness. However, in addition to benefits, learning also incurs a cost, which means that investment in learning and maintaining learned skills can lead to trade-offs impacting other biological functions. Here, we tested whether a trade-off exists between learning skills and reproductive potential in honeybee workers. For this purpose, we compared learning ability between two groups of workers that differed in reproductive potential—normal and rebel workers. The results showed that workers with high reproductive potential (rebels), measured according to the number of ovarioles in the ovary, learned faster than normal workers with low reproductive potential. Moreover, by performing separate regression analyses within the rebel and non-rebel worker groups, we found that the reproductive potential of workers was positively correlated with their learning ability. The results show that in honeybees, there is no trade-off in resource allocation between two costly biological functions, learning and reproduction.

You are what you eat: relative importance of diet, gut microbiota and nestmates for honey bee, Apis mellifera, worker health

In eusocial honey bees, Apis mellifera, diet, gut microbiota and nestmates can all contribute to the health of freshly emerged individual workers, but their relative importance for longevity and body weight is currently unknown. Here, we show that diet is most relevant, followed by gut microbiota and the presence of nestmates. Freshly emerged workers were randomly assigned to eight treatments (with or without honey/pollen, protein-substitute lactalbumin, antibiotic tetracycline and nestmates for 24 h) and maintained under standardised laboratory conditions. Longevity and food consumption were measured daily and fresh body weight was assessed at day 7. The data show a significantly better survival and a higher body weight in workers supplied with honey/pollen. Survival was higher in the lactalbumin treatments compared to the ones restricted to sucrose only, but lower compared to those with honey/pollen, highlighting the importance of micronutrients. In contrast, antibiotic treatment had a significant negative effect on longevity and body weight, which may be explained by inactivated gut microbiota and/or toxicity of the antibiotics. There was no positive effect of nestmates, probably due to the short exposure period. In contrast, nestmates showed a negative effect on survival in antibiotic-treated workers, possibly by transmitting pathogens and antibiotic-induced gut dysbiosis. In conclusion, a macro- and micronutrient-rich diet appears to be the key to individual honey bee worker health. Providing an optimal diet and possibly gut microbiota appears to be a promising way to promote managed A. mellifera health.

Probiotic capability of novel lactic acid bacteria isolated from worker honey bees gut microbiota.

The study aimed to evaluate the probiotic and safety properties of lactic acid bacterial (LAB) strains isolated from the gut microbiota of honey bee Apis mellifera L., since this source remains a promising reservoir of microbial diversity. A total of five bacterial isolates were molecularly identified using 16S rRNA gene sequencing as Enterococcus faecalis-HBE1, Lactobacillus brevis-HBE2, Enterococcus faecalis-HBE3, Enterococcus faecalis-HBE4 and Lactobacillus casei-HBE5. Gut tolerance conditions (low pH and bile salt) were evaluated. Exopolysaccharides (EPS) production, hemolytic, antioxidant activity, resistance toward antibiotics and technological characteristics (starter activity, pH and proteolysis) were examined. The five isolates showed a high survival rate (>95%), under gastrointestinal tract conditions indicating excellent potential for application as probiotics. The isolates showed no hemolytic activities and good acidification rates in the range of pH 4.6-4.98 after incubation at 37°Cfor 24h.The isolates exhibited promising proteolytic activity as well as DPPH radical scavenging activity in the range of 16.52-59.39%. All the tested isolates had the capability to produce exopolysaccharides except Lactobacillus casei-HBE5. These results put forward that lactic acid bacterial strain isolated from honey bee workers can be considered as promising candidates for future applications as starter cultures and could constitute new potential probiotics for the production of functional dietary products promoting health benefits.

Reproductive activation in honeybee (Apis mellifera) workers protects against abiotic and biotic stress.

Social insect reproductives exhibit exceptional longevity instead of the classic trade-off between somatic maintenance and reproduction. Even normally sterile workers experience a significant increase in life expectancy when they assume a reproductive role. The mechanisms that enable the positive relation between the antagonistic demands of reproduction and somatic maintenance are unclear. To isolate the effect of reproductive activation, honeybee workers were induced to activate their ovaries. These reproductively activated workers were compared to controls for survival and gene expression patterns after exposure to Israeli Acute Paralysis Virus or the oxidative stressor paraquat. Reproductive activation increased survival, indicating better immunity and oxidative stress resistance. After qPCR analysis confirmed our experimental treatments at the physiological level, whole transcriptome analysis revealed that paraquat treatment significantly changed the expression of 1277 genes in the control workers but only two genes in reproductively activated workers, indicating that reproductive activation preemptively protects against oxidative stress. Significant overlap between genes that were upregulated by reproductive activation and in response to paraquat included prominent members of signalling pathways and anti-oxidants known to affect ageing. Thus, while our results confirm a central role of vitellogenin, they also point to other mechanisms to explain the molecular basis of the lack of a cost of reproduction and the exceptional longevity of social insect reproductives. Thus, socially induced reproductive activation preemptively protects honeybee workers against stressors, explaining their longevity. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Assessing Agrochemical Risk to Mated Honey Bee Queens.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method's intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

Evaluating the Impact of Post-Emergence Weed Control in Honeybee Colonies Located in Different Agricultural Surroundings.

Simple SummaryThe honeybee Apis mellifera is one of the main pollinators in agricultural ecosystems and therefore they are exposed to pesticides and the reduction of floral diversity. Cytochrome P450 monooxygenases are enzymes involved in xenobiotic detoxification used by organisms, including insects. In this study, we evaluated honeybee foraging activity and the expression profiles of several cytochromes, before and after the administration of a mixture of three of the most used herbicides in the region during the summer season. Additionally, we investigated whether colonies located in three distinct agricultural environments with different crop/wild flora proportions are affected similarly or differently by herbicide administration. We found that the expression of several cytochrome P450 genes decreased significantly in larvae after post-emergence weed control and they showed significant differences between apiaries in the case of honeybee workers. Besides, we found significant positive correlations between pollen collection and some pesticide detoxification genes. Our results support that diversity and quality of resource availability as well as the presence of herbicides affect colonies’ nutritional state and bee health. Moreover, their detoxification response shows that larvae are more harmed than adults in these agricultural ecosystems.The honeybee Apis mellifera is exposed to agricultural intensification, which leads to an improved reliance upon pesticide use and the reduction of floral diversity. In the present study, we assess the changes in the colony activity and the expression profile of genes involved in xenobiotic detoxification in larvae and adult honeybees from three apiaries located in agricultural environments that differ in their proportion of the crop/wild flora. We evaluated these variables before and after the administration of a mixture of three herbicides during the summer season. The expression of several cytochrome P450 monooxygenases decreased significantly in larvae after post-emergence weed control and showed significant differences between apiaries in the case of honeybee workers. Principal component analysis (PCA) revealed that colonies located in the plot near to a wetland area exhibited a different relative gene expression profile after herbicide application compared with the other plots. Moreover, we found significant positive correlations between pollen collection and the pesticide detoxification genes that discriminated between plots in the PCA. Our results suggest that nutrition may modify herbicide impact on honeybees and that larvae are more harmed than adults in agroecosystems, a factor that will alter the colonies’ population growth at the end of the blooming period.

Diel rhythmicity of alcohol‐induced intoxication in the honeybee workers

AbstractAmong the honeybee workers, extranidal foragers are more rhythmic in their diel activity and more likely to encounter alcohol than intranidal nurses. Foragers are more active and potentially exposed to alcohol during the day, while nurses work around the clock and probably never encounter alcohol. Here, we took advantage of these differences and checked for a connection between the effects of alcohol and period of the day. We hypothesized that foragers would show higher intoxication resistance to alcohol than nurses during the day. Furthermore, we hypothesized that only in foragers the intoxication resistance to alcohol would be different between day and night. We detected higher intoxication resistance to alcohol in older, forager‐age bees, than in younger, nurse‐age bees during the day, which confirmed the first hypothesis. However, the intoxication resistance differed between day and night in both worker types, which provided no support for the second hypothesis. Surprisingly, we found the intoxication resistance to be highest during the night in both types of workers. Reported differences in the intoxication resistance showed no relation to differences in alcohol absorption between day and night, thus the exact mechanistic explanation for the diel oscillation in alcohol influence on bees remained unclear.

Toxicological and morphological analysis of Africanized Apis mellifera selected for tolerance to the neonicotinoid thiamethoxam

Honeybees are the insect most used for pollination purposes due to its efficient characteristics for this function, which reflects in positive aspects for both nature and man. The expansion of agriculture and the development of agrochemicals to combat pests has had negative impacts on honeybee health, causing its disappearance around the world. This research aimed to evaluate the effects of honeybee exposure to the neonicotinoid insecticide thiamethoxam on its survival rate, as well as on morphological and histological changes in the midgut of adult workers from the F4 generation of Apis mellifera queens tolerant to thiamethoxam and non-tolerant worker honeybees. After the bioassays, the midgut was removed for morphological evaluation. The results showed that F4 bees were more tolerance to thiamethoxam and exhibited less significant morphological changes when compared to the non-tolerant control group.

Pollen Source Affects Development and Behavioral Preferences in Honey Bees.

Simple SummaryPollinators adjust their foraging preference based on the pollen cues of foraging plants. Honey bees, for example, prefer to collect one type of pollen from plants that bloom at the same time. In northern China, apricot and pear trees are the two main foraging plants in the early spring. However, honey bees tend to collect pollen from apricot trees. It is interesting to understand what affects the foraging decision of honey bees regarding these two pollen types. In this study, we observed the foraging preference of Apis mellifera workers with respect to apricot and pear pollen under laboratory conditions. The effect of pollen on the development of the hypopharyngeal gland (HG) and ovary was measured. The number of visits made to apricot pollen was significantly higher than that to pear pollen. Furthermore, the response of the HG and ovary to these two pollens was different. The development of the HG was significantly affected by pollen diet treatments. However, there was no significant difference in the ovarian development of caged workers supplied with the two different pollen diets. Overall, honey bees showed a significant preference for apricot pollen over pear pollen. Compared with the ovary, the HG of honey bee workers may be more sensitive to pollen nutrition.With the availability of various plants in bloom simultaneously, honey bees prefer to collect some pollen types over others. To better understand pollen’s role as a reward for workers, we compared the digestibility and nutritional value of two pollen diets, namely, pear (Pyrus bretschneideri Rehd.) and apricot (Armeniaca sibirica L.). We investigated the visits, pollen consumption, and pollen extraction efficiency of caged Apis mellifera workers. Newly emerged workers were reared, and the effects of two pollen diets on their physiological status (the development of hypopharyngeal glands and ovaries) were compared. The choice-test experiments indicated a significant preference of A. mellifera workers for apricot pollen diets over pear pollen diets (number of bees landing, 29.5 ± 8.11 and 9.25 ± 5.10, p < 0.001 and pollen consumption, 0.052 ± 0.026 g/day and 0.033 ± 0.013 g/day, p < 0.05). Both pollen diets had comparable extraction efficiencies (67.63% for pear pollen and 67.73% for apricot pollen). Caged workers fed different pollen diets also exhibited similar ovarian development (p > 0.05). However, workers fed apricot pollen had significantly larger hypopharyngeal glands than those fed pear pollen (p < 0.001). Our results indicated that the benefits conferred to honey bees by different pollen diets may influence their foraging preference.

Transcriptomic analysis suggests candidate genes for hygienic behavior in African-derived Apis mellifera honeybees

Hygienic behavior is a social defense mechanism against parasites and pathogens in honeybees. We studied the genetic basis of hygienic behavior in African-derived Apis mellifera by performing RNA sequencing on brains of individual honeybee workers observed performing hygienic behavior, in order to identify expression changes linked with this behavior. We also used the transcriptome data to search for single nucleotide polymorphism (SNP) variation in genes previously associated with the trait in other A. mellifera populations. The analysis revealed 49 differentially expressed genes (DEG), most of them upregulated during hygienic acts. One DEG, Apidaecin, also showed SNP variation across samples, providing a promising candidate gene for both expression-based and heritable variation in hygienic behavior. We also identified 27 additional SNPs in the coding regions of five candidate genes previously linked with honeybee hygienic behavior. These results provide a useful starting place to analyze the genetic basis of hygienic behavior in African-derived honeybees.

Formulation of Liposomes Containing Royal Jelly and Their Quality Assessment.

Royal jelly, a gelatinuous consistency bee product produced and secreted by the hypopharyngeal and mandibular glands of worker honeybees, is beneficial in the treatment of dermatological conditions, likely through its content of the fatty acid 10-hydroxy-2-decenoic acid (10-HDA). However, 10-HAD poorly penetrates into skin. Thus, in this work, we produced royal jelly incorporated liposomes with the aim of increasing skin penetration of 10-HDA. Lipid nanocarriers were prepared by the thin lipid-film hydration method. Size and polydispersity index of the nanocarrier particles, and their stability over 30 days were measured. The effects of royal jelly and 10-HDA liposomal formulations on the viability of immortalized human keratinocyte cells were tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The skin penetration of 10-HDA from liposomal formulations and royal jelly solution was studied in vitro with Franz type vertical diffusion cells using porcine skin as limiting membrane. As result, small liposomes were achieved, and the efficacy of the obtained nanoformulations was examined by means of in vitro cell assays with a HaCaT immortalized human keratinocyte cell culture line. Finally, the skin penetration experiments showed that liposomal incorporation greatly increased 10-HDA penetration into skin layers.

Vitellogenin expression in the ovaries of adult honeybee workers provides insights into the evolution of reproductive and social traits.

Social insects are notable for having two female castes that exhibit extreme differences in their reproductive capacity. The molecular basis of these differences is largely unknown. Vitellogenin (Vg) is a powerful antioxidant and insulin-signalling regulator used in oocyte development. Here we investigate how Royal Jelly (the major food of honeybee queens) and queen mandibular pheromone (a major regulator of worker fertility), affect the longevity and reproductive status of honey bee workers, the expression of Vg, its receptor VgR and associated regulatory proteins. We find that Vg is expressed in the ovaries of workers and that workers fed a queen diet of Royal Jelly have increased Vg expression in the ovaries. Surprisingly, we find that expression of Vg is not associated with ovary activation in workers, suggesting that this gene has potentially acquired non-reproductive functions. Therefore, Vg expression in the ovaries of honeybee workers provides further support for the Ovarian Ground Plan Hypothesis, which argues that genes implicated in the regulation of reproduction have been co-opted to regulate behavioural differences between queens and workers.

Consumption of field-realistic doses of a widely used mito-toxic fungicide reduces thorax mass but does not negatively impact flight capacities of the honey bee (Apis mellifera)

Commercial beekeepers in many locations are experiencing increased annual colony losses of honey bees (Apis mellifera), but the causes, including the role of agrochemicals in colony losses, remain unclear. In this study, we investigated the effects of chronic consumption of pollen containing a widely-used fungicide (Pristine®), known to inhibit bee mitochondria invitro, which has recently been shown to reduce honey bee worker lifespan when field-colonies are provided with pollen containing field-realistic levels of Pristine®. We fed field colonies pollen with a field-realistic concentration of Pristine® (2.3ppm) and a concentration two orders of magnitude higher (230ppm). To challenge flight behavior and elicit near-maximal metabolic rate, we measured flight quality and metabolic rates of bees in two lower-than-normal air densities. Chronic consumption of 230 but not 2.3ppm Pristine® reduced maximal flight performance and metabolic rates, suggesting that the observed decrease in lifespans of workers reared on field-realistic doses of Pristine®-laced pollen is not due to inhibition of flight muscle mitochondria. However, consumption of either the 230 or 2.3ppm dose reduced thorax mass (but not body mass), providing the first evidence of morphological effects of Pristine®, and supporting the hypothesis that Pristine® reduces forager longevity by negatively impacting digestive or nutritional processes.