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

BACKGROUND: As 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. RESULTS: The 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. CONCLUSIONS: ame-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.

Honey Bee (Apis mellifera) Exposure to Pesticide Residues in Nectar and Pollen in Urban and Suburban Environments from Four Regions of the United States.

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991-1003. © 2022 SETAC.

Mblk-1 regulates sugar responsiveness in honey bee (Apis mellifera) foragers.

Brain transcriptional regulatory network for behavior demonstrates that brain gene expression in the honey bee can be accurately predicted from the expression transcription factors (TFs), but roles for specific TFs are less understood. Mushroom bodies (MBs) are important for learning, memory and sensory integration in the honey bee brain. A TFs, Mblk-1, expressed preferentially in the large-type Kenyon cells of the honeybee MBs is predicted to be involved in brain function by regulating transcription of its target genes in honey bee. However, its function and the mechanism of regulation in behavior of honey bee is still obscure. Here we show that Mblk-1 had significantly higher expression in the brains of forager bees relative to nurse bees. Mblk-1 was significantly inhibited in bees fed small interfering RNA. In addition, inhibition of Mblk-1 decreased sucrose responsiveness in foragers. Finally, we determined that Mblk-1 regulated the messenger RNA of AmGR1. These findings suggest that Mblk-1 may target AmGR1 to regulate the sucrose responsiveness of foragers.

The cell invasion preference of Varroa destructor between the original and new honey bee hosts.

Honey bees (Apis) are important pollinators for food crops and wild plants, but are facing great threats from pathogens and parasites, especially an obligate ectoparasitic mite, Varroa destructor. Cell invasion is a key step for V. destructor to reproduce, and the parasite displays remarkable host preference in this process. Varroa destructor made its host-shift from its original host, the Asian honey bee Apis cerana, to the new host, the European honey bee Apis mellifera several decades ago. However, it remains largely unstudied whether V. destructor shows a cell invasion preference between the two host species. Using cell invasion bioassays on a modified four-well arena, we showed that V. destructor significantly preferred to invade the worker and drone larvae of A. mellifera rather than A. cerana, suggesting that the new host is much more attractive to the parasite than the original one. Using gas chromatography-mass spectrometry (GC-MS), we revealed significant differences between the cuticular hydrocarbon (CHC) profiles of worker and drone larvae of the two bee hosts. The amounts of methyl-branched alkanes and alkenes (unsaturated CHCs), but not n-alkanes, were significantly different, and A. mellifera worker and drone larvae were found to express significantly higher amounts of methyl-alkanes, while A. cerana larvae produced higher amounts of alkenes. Cell invasion bioassays with glass dummies showed that the mites preferred the glass dummies coated with the CHCs of A. mellifera worker or drone larvae, which indicates a role of larval CHCs in mediating the preferential cell invasion of Varroa. The findings from this study extend our understanding of the host preference of V. destructor, and can potentially contribute to the development of effective strategies for mite control.

The Role of Honey Bee Derived Aliphatic Esters in the Host-Finding Behavior of Varroa destructor.

Varroa destructor is an obligate ectoparasite of honey bees and shifted from its original host Apis cerana to the new host Apis mellifera in the first half of the twentieth century. The host shift has resulted in a great threat to the health and survival of A. mellifera colonies worldwide. Chemical signals play a crucial role in all aspects of the Varroa life cycle, including host finding. However, the chemical cues that affect the host finding behavior of Varroa mites are still not fully understood. In this study, we systematically profiled the headspace volatiles of both worker and drone larvae of the two honey bee species by using solid phase micro-extraction coupled to gas chromatography-mass spectrometry (SPME-GC-MS), and then used electrophysiological recording and Y-tube olfactometer bioassay to study the potential roles of the selected compounds. The chemical profiling showed that there were four aliphatic esters, ethyl myristate (EM), methyl palmitate (MP), ethyl palmitate (EP), and ethyl oleate (EO) commonly detected from all four types of larval hosts. Among them, EM was a new substance identified from honey bee headspace volatiles. Results from electrophysiological recordings indicated that all the aliphatic esters could elicit significant responses of Varroa pit organs on its forelegs. Moreover, behavioral analyses revealed that EM could significantly attract V. destructor at a medium dosage (10 µg), while MP had no observable effect on the mites and both EP and EO were able to repel the parasites. Our findings suggest an important role of host-derived aliphatic esters in Varroa host finding, and provide new chemicals for Varroa monitoring and control.

A Meta-Analysis Shows That Screen Bottom Boards Can Significantly Reduce Varroa destructor Population.

Varroa destructor is by far the most serious threat to the western honey bee, Apis mellifera. A screen bottom board, a cultural method for mite control, is a modified bottom board with a screen that allows mites to fall onto a sticky board, or the grass or soil below the screen. Whether or not a screen bottom board can reduce varroa significantly has been controversial. Most studies show a trend of lower varroa populations in colonies with these boards, but the results are usually not statistically significant. To understand whether the negative results have been due to small sample sizes, or because the board is actually ineffective, we conducted a meta-analysis with seven published studies with a total of 145 colonies. Meta-analysis showed that the confidence intervals of the combined effect sizes were negative with a Hedges' g of -1.09 (SE 0.39, 95% CI -2.0 to -0.19, p < 0.01), which suggests that the varroa population in colonies with screen bottom boards is significantly lower compared to those with traditional wooden floors. We thus conclude that the screen bottom board does have a significantly negative impact on the varroa population and can be part of tool kits for mite control.

Reproduction of Distinct Varroa destructor Genotypes on Honey Bee Worker Brood.

Honey bees play important roles in pollination for many crops and wild plants, but have been facing great threats posed by various pathogens and parasites. Among them, Varroa destructor, an obligate ectoparasite of honey bees, is considered the most damaging. Within the last century, V. destructor shifted from the original host, the Asian honey bee Apis cerana to the new host, the European honey bee A. mellifera. However, the reproduction of Varroa mites, especially of different haplotypes in the two hosts, is still largely unknown. In this study, we first investigated the existing Varroa haplotypes in local colonies in southern China, and then compared the reproduction of different haplotypes on the worker brood of both the original and new hosts by artificial inoculation. We confirmed that there are two haplotypes of V. destructor in southern China, one is the Korea haplotype and the other is the China haplotype, and the two types parasitized different honey bee species. Although Varroa females from A. mellifera (Korea haplotype) are able to reproduce on the worker brood of both honey bee species, they showed better reproductive performance in the new host A. mellifera with significantly higher fecundity (number of offspring per mother mite) and reproductive rate (number of adult daughters per mother mite), suggesting that this parasite gains higher fitness after host shift. The data further showed that a short stay of Varroa females inside the A. cerana worker cells decreased their fecundity and especially the reproductive rate in a time-dependent manner, suggesting that the A. cerana worker cells may inhibit Varroa reproduction. In contrast, Varroa mites derived from A. cerana colonies (China haplotype) were entirely sterile in A. mellifera worker cells during two sequential inoculations, while the control mites from A. mellifera colonies (Korea haplotype) reproduced normally. In addition, all the infertile mites were found to defecate on the abdomen of bee pupae. We have revealed that two haplotypes of V. destructor exhibit differential reproduction on the worker brood of the original and new host honey bees, providing novel insights into the diversity and complexity of the reproduction of V. destructor.

Seasonal variation of pollen collected by honey bees (Apis mellifera) in developed areas across four regions in the United States.

For honey bees (Apis mellifera), colony maintenance and growth are highly dependent on worker foragers obtaining sufficient resources from flowering plants year round. Despite the importance of floral diversity for proper bee nutrition, urban development has drastically altered resource availability and diversity for these important pollinators. Therefore, understanding the floral resources foraged by bees in urbanized areas is key to identifying and promoting plants that enhance colony health in those environments. In this study, we identified the pollen foraged by bees in four developed areas of the U.S., and explored whether there were spatial or temporal differences in the types of floral sources of pollen used by honey bees in these landscapes. To do this, pollen was collected every month for up to one year from colonies located in developed (urban and suburban) sites in California, Texas, Florida, and Michigan, except during months of pollen dearth or winter. Homogenized pollen samples were acetolyzed and identified microscopically to the lowest taxonomic level possible. Once identified, each pollen type was classified into a frequency category based on its overall relative abundance. Species richness and diversity indices were also calculated and compared across states and seasons. We identified up to 64 pollen types belonging to 39 plant families in one season (California). Species richness was highest in CA and lowest in TX, and was highest during spring in every state. In particular, "predominant" and "secondary" pollen types belonged to the families Arecaceae, Sapindaceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Fabaceae, Fagaceae, Lythraceae, Myrtaceae, Rhamnaceae, Rosaceae, Rutaceae, Saliaceae, and Ulmaceae. This study will help broaden our understanding of honey bee foraging ecology and nutrition in urban environments, and will help promote the use of plants that serve the dual purpose of providing aesthetic value and nutritious forage for honey bee colonies placed in developed landscapes.

lncRNA profile of Apis mellifera and its possible role in behavioural transition from nurses to foragers.

BACKGROUND: The behavioural transition from nurses to foragers in honey bees is known to be affected by intrinsic and extrinsic factors, including colony demography, hormone levels, brain chemistry and structure, and gene expression in the brain. However, the molecular mechanism underlying this behavioural transition of honey bees is still obscure. RESULTS: Through RNA sequencing, we performed a comprehensive analysis of lncRNAs and mRNAs in honey bee nurses and foragers. Nurses and foragers from both typical colonies and single-cohort colonies were used to prepare six libraries to generate 49 to 100 million clear reads per sample. We obtained 6863 novel lncRNAs, 1480 differentially expressed lncRNAs between nurses and foragers, and 9308 mRNAs. Consistent with previous studies, lncRNAs showed features distinct from mRNAs, such as shorter lengths, lower exon numbers, and lower expression levels compared to mRNAs. Bioinformatic analysis showed that differentially expressed genes were mostly involved in the regulation of sensory-related events, such as olfactory receptor activity and odorant binding, and enriched Wnt and FoxO signaling pathways. Moreover, we found that lncRNAs TCONS_00356023, TCONS_00357367, TCONS_00159909 and mRNAs dop1, Kr-h1 and HR38 may play important roles in behavioural transition in honey bees. CONCLUSION: This study characterized the expression profile of lncRNAs in nurses and foragers and provided a framework for further study of the role of lncRNAs in honey bee behavioural transition.

Genes important for survival or reproduction in Varroa destructor identified by RNAi.

The Varroa mite, (Varroa destructor), is the worst threat to honey bee health worldwide. To explore the possibility of using RNA interference to control this pest, we determined the effects of knocking down various genes on Varroa mite survival and reproduction. Double-stranded RNA (dsRNA) of six candidate genes (Da, Pros26S, RpL8, RpL11, RpP0 and RpS13) were synthesized and each injected into Varroa mites, then mite survival and reproduction were assessed. Injection of dsRNA for Da (Daughterless) and Pros26S (Gene for proteasome 26S subunit adenosine triphosphatase) caused a significant reduction in mite survival, with 3.57% ± 1.94% and 30.03% ± 11.43% mites surviving at 72 h post-injection (hpi), respectively. Control mites injected with green fluorescent protein (GFP)-dsRNA showed survival rates of 81.95% ± 5.03% and 82.36 ± 2.81%, respectively. Injections of dsRNA for four other genes (RpL8, RpL11, RpP0 and RpS13) did not affect survival significantly, enabling us to assess their effect on Varroa mite reproduction. The number of female offspring per mite was significantly reduced for mites injected with dsRNA of each of these four genes compared to their GFP-dsRNA controls. Knockdown of the target genes was verified by real-time polymerase chain reaction for two genes important for reproduction (RpL8, RpL11) and one gene important for survival (Pros26S). In conclusion, through RNA interference, we have discovered two genes important for mite survival and four genes important for mite reproduction. These genes could be explored as possible targets for the control of Varroa destructor in the future.

Quantifying the effects of pollen nutrition on honey bee queen egg laying with a new laboratory system.

Honey bee populations have been declining precipitously over the past decade, and multiple causative factors have been identified. Recent research indicates that these frequently co-occurring stressors interact, often in unpredictable ways, therefore it has become important to develop robust methods to assess their effects both in isolation and in combination. Most such efforts focus on honey bee workers, but the state of a colony also depends on the health and productivity of its queen. However, it is much more difficult to quantify the performance of queens relative to workers in the field, and there are no laboratory assays for queen performance. Here, we present a new system to monitor honey bee queen egg laying under laboratory conditions and report the results of experiments showing the effects of pollen nutrition on egg laying. These findings suggest that queen egg laying and worker physiology can be manipulated in this system through pollen nutrition, which is consistent with findings from field colonies. The results generated using this controlled, laboratory-based system suggest that worker physiology controls queen egg laying behavior. Additionally, the quantitative data generated in these experiments highlight the utility of the system for further use as a risk assessment tool.

Rapid juvenile hormone downregulation in subordinate wasp queens facilitates stable cooperation.

In many cooperatively breeding animals, subordinate group members have lower reproductive capacity than dominant group members. Theory suggests subordinates may downregulate their reproductive capacity because dominants punish subordinates who maintain high fertility. However, there is little direct experimental evidence that dominants cause physiological suppression in subordinates. Here, we experimentally test how social interactions influence subordinate reproductive hormones in Polistes dominula paper wasps. Polistes dominula queens commonly found nests in cooperative groups where the dominant queen is more fertile than the subordinate queen. In this study, we randomly assigned wasps to cooperative groups, assessed dominance behaviour during group formation, then measured levels of juvenile hormone (JH), a hormone that mediates Polistes fertility. Within three hours, lowest ranking subordinates had less JH than dominants or solitary controls, indicating that group formation caused rapid JH reduction in low-ranking subordinates. In a second experiment, we measured the behavioural consequences of experimentally increasing subordinate JH. Subordinates with high JH-titres received significantly more aggression than control subordinates or subordinates from groups where the dominant's JH was increased. These results suggest that dominants aggressively punished subordinates who attempted to maintain high fertility. Low-ranked subordinates may rapidly downregulate reproductive capacity to reduce costly social interactions with dominants. Rapid modulation of subordinate reproductive physiology may be an important adaptation to facilitate the formation of stable, cooperative groups.

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers.

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene-treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene-treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal-aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers' JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.

Genome-wide DNA methylation changes associated with olfactory learning and memory in Apis mellifera.

The honeybee is a model organism for studying learning and memory formation and its underlying molecular mechanisms. While DNA methylation is well studied in caste differentiation, its role in learning and memory is not clear in honeybees. Here, we analyzed genome-wide DNA methylation changes during olfactory learning and memory process in A. mellifera using whole genome bisulfite sequencing (WGBS) method. A total of 853 significantly differentially methylated regions (DMRs) and 963 differentially methylated genes (DMGs) were identified. We discovered that 440 DMRs of 648 genes were hypermethylated and 274 DMRs of 336 genes were hypomethylated in trained group compared to untrained group. Of these DMGs, many are critical genes involved in learning and memory, such as Creb, GABA B R and Ip3k, indicating extensive involvement of DNA methylation in honeybee olfactory learning and memory process. Furthermore, key enzymes for histone methylation, RNA editing and miRNA processing also showed methylation changes during this process, implying that DNA methylation can affect learning and memory of honeybees by regulating other epigenetic modification processes.

Preservation of orange juice using propolis.

Orange juice is one of the most popular and the most consumed fruit juices all over the world, especially in Europe and the chemical food preservatives, such as sodium benzoate, potassium sorbate and their mixtures, have long been used in orange juice sold on the market. Excessive consumption of these preservatives may be hazardous to human health. Propolis, composed of resins collected from plant buds and exudates and mixed with salivary gland secretions and beeswax by honey bee workers, has been used as a human medicine and natural food preservative. We hypothesis that propolis, without alcohol, can serve as an alternative and non-synthetic preservative of orange juice. In this study, the preservative effect of propolis emulsion on orange juice was determined up to 35 days. Propolis emulsion (0.02 g/mL propolis, 12 mL), emulsion control (12 mL containing Tween-80, hydrophilic phospholipid and polyethylene glycol 400), sodium benzoate (0.4 g) and potassium sorbate (0.4 g) was each added to 388, 388, 400 and 400 mL orange juice respectively. Propolis emulsion showed significant inhibition of bacteria growth and l-ascorbic acid degradation. Orange juice pH value, titratable acidity, total phenolic content, color and antioxidant capacity were effectively maintained by propolis emulsion. A control solution with all the same emulsifying agents without propolis did not show these properties. It was concluded that propolis can be used as a natural additive agent in orange juice or other fruit juices as an alternative to chemical preservatives.

The microRNA ame-miR-279a regulates sucrose responsiveness of forager honey bees (Apis mellifera).

Increasing evidence demonstrates that microRNAs (miRNA) play an important role in the regulation of animal behaviours. Honey bees (Apis mellifera) are eusocial insects, with honey bee workers displaying age-dependent behavioural maturation. Many different miRNAs have been implicated in the change of behaviours in honey bees and ame-miR-279a was previously shown to be more highly expressed in nurse bee heads than in those of foragers. However, it was not clear whether this difference in expression was associated with age or task performance. Here we show that ame-miR-279a shows significantly higher expression in the brains of nurse bees relative to forager bees regardless of their ages, and that ame-miR-279a is primarily localized in the Kenyon cells of the mushroom body in both foragers and nurses. Overexpression of ame-miR-279a attenuates the sucrose responsiveness of foragers, while its absence enhances their sucrose responsiveness. Lastly, we determined that ame-miR-279a directly target the mRNA of Mblk-1. These findings suggest that ame-miR-279a plays important roles in regulating honey bee division of labour.

No effect of juvenile hormone on task performance in a bumblebee (Bombus terrestris) supports an evolutionary link between endocrine signaling and social complexity.

A hallmark of insect societies is a division of labor among workers specializing in different tasks. In bumblebees the division of labor is related to body size; relatively small workers are more likely to stay inside the nest and tend ("nurse") brood, whereas their larger sisters are more likely to forage. Despite their ecological and economic importance, very little is known about the endocrine regulation of division of labor in bumblebees. We studied the influence of juvenile hormone (JH) on task performance in the bumblebee Bombus terrestris. We first used a radioimmunoassay to measure circulating JH titers in workers specializing in nursing and foraging activities. Next, we developed new protocols for manipulating JH titers by combining a size-adjusted topical treatment with the allatotoxin Precocene-I and replacement therapy with JH-III. Finally, we used this protocol to test the influence of JH on task performance. JH levels were either similar for nurses and foragers (three colonies), or higher in nurses (two colonies). Nurses had better developed ovaries and JH levels were typically positively correlated with ovarian state. Manipulation of JH titers influenced ovarian development and wax secretion, consistent with earlier allatectomy studies. These manipulations however, did not affect nursing or foraging activity, or the likelihood to specialize in nursing or foraging activity. These findings contrast with honeybees in which JH influences age-related division of labor but not adult female fertility. Thus, the evolution of complex societies in bees was associated with modifications in the way JH influences social behavior.

Socially selected ornaments influence hormone titers of signalers and receivers.

Decades of behavioral endocrinology research have shown that hormones and behavior have a bidirectional relationship; hormones both influence and respond to social behavior. In contrast, hormones are often thought to have a unidirectional relationship with ornaments. Hormones influence ornament development, but little empirical work has tested how ornaments influence hormones throughout life. Here, we experimentally alter a visual signal of fighting ability in Polistes dominulus paper wasps and measure the behavioral and hormonal consequences of signal alteration in signalers and receivers. We find wasps that signal inaccurately high fighting ability receive more aggression than controls and receiving aggression reduces juvenile hormone (JH) titers. As a result, immediately after contests, inaccurate signalers have lower JH titers than controls. Ornaments also directly influence rival JH titers. Three hours after contests, wasps who interacted with rivals signaling high fighting ability have higher JH titers than wasps who interacted with rivals signaling low fighting ability. Therefore, ornaments influence hormone titers of both signalers and receivers. We demonstrate that relationships between hormones and ornaments are flexible and bidirectional rather than static and unidirectional. Dynamic relationships among ornaments, behavior, and physiology may be an important, but overlooked factor in the evolution of honest communication.

Why do Varroa mites prefer nurse bees?

The Varroa mite, Varroa destructor, is an acarine ecto-parasite on Apis mellifera. It is the worst pest of Apis mellifera, yet its reproductive biology on the host is not well understood. In particular, the significance of the phoretic stage, when mites feed on adult bees for a few days, is not clear. In addition, it is not clear whether the preference of mites for nurses observed in the laboratory also happens inside real colonies. We show that Varroa mites prefer nurses over both newly emerged bees and forgers in a colony setting. We then determined the mechanism behind this preference. We show that this preference maximizes Varroa fitness, although due to the fact that each mite must find a second host (a pupa) to reproduce, the fitness benefit to the mites is not immediate but delayed. Our results suggest that the Varroa mite is a highly adapted parasite for honey bees.

Low-Temperature Stress during Capped Brood Stage Increases Pupal Mortality, Misorientation and Adult Mortality in Honey Bees.

Honey bees (Apis mellifera) are key pollinators, playing a vital role in ecosystem maintenance and stability of crop yields. Recently, reduced honey bee survival has attracted intensive attention. Among all other honey bee stresses, temperature is a fundamental ecological factor that has been shown to affect honey bee survival. Yet, the impact of low temperature stress during capped brood on brood mortality has not been systematically investigated. In addition, little was known about how low temperature exposure during capped brood affects subsequent adult longevity. In this study, capped worker broods at 12 different developmental stages were exposed to 20°C for 12, 24, 36, 48, 60, 72, 84 and 96 hours, followed by incubation at 35°C until emergence. We found that longer durations of low temperature during capped brood led to higher mortality, higher incidences of misorientation inside cells and shorter worker longevity. Capped brood as prepupae and near emergence were more sensitive to low-temperature exposure, while capped larvae and mid-pupal stages showed the highest resistance to low-temperature stress. Our results suggest that prepupae and pupae prior to eclosion are the most sensitive stages to low temperature stress, as they are to other stresses, presumably due to many physiological changes related to metamorphosis happening during these two stages. Understanding how low-temperature stress affects honey bee physiology and longevity can improve honey bee management strategies.