Honeybee Development Research Articles

Minibrain plays a role in the adult brain development of honeybee (Apis mellifera) workers.

The brain of adult honeybee (Apis mellifera) workers is larger than that of queens, facilitating behavioural differentiation between the castes. This brain diphenism develops during the pharate-adult stage and is driven by a caste-specific gene expression cascade in response to unique hormonal milieus. Previous molecular screening identified minibrain (mnb; DYRK1A) as a potential regulator in this process. Here, we used RNAi approach to reduce mnb transcript levels and test its role on brain diphenism development in honeybees. White-eyed unpigmented cuticle worker pupae were injected with dsRNA for mnb (Mnb-i) or gfp, and their phenotypes were assessed two and 8 days later using classic histological and transcriptomic analyses. After 2 days of the injections, Mnb-i bees showed 98% of downregulation of mnb transcripts. After 8 days, the brain of Mnb-i bees showed reduction in total volume and in the volume of the mushroom bodies (MB), antennal, and optic lobes. Additionally, signs of apoptosis were observed in the Kenyon cells region of the MB, and the cohesion of the brain tissues was affected. Our transcriptomic analyses revealed that 226 genes were affected by the knockdown of mnb transcripts, most of which allowing axonal fasciculation. These results suggest the evolutionary conserved mnb gene has been co-opted for promoting hormone-mediated developmental brain morphological plasticity generating caste diphenism in honeybees.

Early exposure to glyphosate during larval development induces late behavioural effects on adult honey bees

As the most abundant pollinator insect in crops, Apis mellifera is a sentinel species of the pollinator communities. In these ecosystems, honey bees of different ages and developmental stages are exposed to diverse agrochemicals. However, most toxicological studies analyse the immediate effects during exposure. Late effects during adulthood after early exposure to pollutants during larval development are poorly studied in bees. The herbicide glyphosate (GLY) is the most applied pesticide worldwide. GLY has been detected in honey and beebread from hives near treated crops. Alterations in growth, morphogenesis or organogenesis during pre-imaginal development could induce late adverse effects after the emergence. Previous studies have demonstrated that GLY alters honey bee development, immediately affecting survival, growth and metabolism, followed by late teratogenic effects. The present study aims to determine the late impact on the behaviour and physiology of adult bees after pre-imaginal exposure to GLY. For that, we reared brood in vitro or in the hive with sub-chronic exposure to the herbicide with the average detected concentration in hives. Then, all newly emerged bees were reared in an incubator until maturity and tested when they became nurse-aged bees. Three behavioural responses were assessed as markers of cognitive and physiological impairment. Our results show i) decreased sensitivity to sucrose regardless of the rearing procedure, ii) increased choice latency and locomotor alterations during chemotaxis and iii) impaired associative learning. These late toxicity signs could indicate adverse effects on task performance and colony efficiency.

Assessment of Lambda-Cyhalothrin and Spinetoram Toxicity and Their Effects on the Activities of Antioxidant Enzymes and Acetylcholinesterase in Honey Bee (Apis mellifera) Larvae.

Honeybees play a crucial role as agricultural pollinators and are frequently exposed to various pollutants, including pesticides. In this study, we aimed to evaluate the toxicity of lambda-cyhalothrin (LCY) and spinetoram (SPI) in honey bee larvae reared in vitro through single (acute) and repeated (chronic) exposure. The acute LD50 values for LCY and SPI were 0.058 (0.051-0.066) and 0.026 (0.01-0.045) μg a.i./larva, respectively. In chronic exposure, the LD50 values of LCY and SPI were 0.040 (0.033-0.046) and 0.017 (0.014-0.019) μg a.i./larva, respectively. The chronic no-observed-effect dose of LCY and SPI was 0.0125 μg a.i./larva. Adult deformation rates exceeded 30% in all LCY treatment groups, showing statistically significant differences compared to the solvent control group (SCG). Similarly, SPI-treated bees exhibited significantly more deformities than SCG. Furthermore, we examined the activities of several enzymes, namely, acetylcholinesterase (AChE), glutathione-S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD), in larvae, pupae, and newly emerged bees after chronic exposure at the larval stage (honey bee larval chronic LD50, LD50/10 (1/10th of LD50), and LD50/20 (1/20th of LD50)). LCY and SPI induced significant changes in detoxification (GST), antioxidative (SOD and CAT), and signaling enzymes (AChE) during the developmental stages (larvae, pupae, and adults) of honey bees at sublethal and residue levels. Our results indicate that LCY and SPI may affect the development of honey bees and alter the activity of enzymes associated with oxidative stress, detoxification, and neurotransmission. These results highlight the potential risks that LCY and SPI may pose to the health and normal development of honey bees.

Exploring the synergistic toxicity of synthetic pesticides and their impact on development and behavior of Honeybee (Apis mellifera L.)

Exploring the synergistic toxicity of synthetic pesticides and their impact on development and behavior of Honeybee (Apis mellifera L.)

Impact of Apis cerana cerana (Hymenoptera: Apidae) body size on newly built cell size

The influence of cell structure size on the morphological development of honey bees has been widely studied. However, the effect of the honey bees' body size on the cell size they build remains unclear. This study investigated the influence of the body size of Apis cerana cerana on the structure size of newly built honey cells, worker cells, and drone cells. Accordingly, we established colonies of A. c. cerana with large and small body-size workers. We then analyzed the birth weight and seven external morphological characteristics of workers, as well as the depth and diameter of their newly built honey cells, worker cells, and drone cells. We found significant differences in the birth weight, proboscis length, thorax width, right forewing length, femur length, tibia length, and metatarsus length of the right hindleg, and total length of the 3rd and 4th tergite of workers from large-body-size colonies (LBSC) and small-body-size colonies (SBSC). Also, the depth and diameter of the newly built cells by LBSC were significantly larger than those by SBSC. The body size of workers positively correlated with the size of newly built cells. This study emphasizes the influence of workers' body size on the structural dimensions of newly constructed cells, providing insights into the comb-building biology of A. c. cerana.

Toxic effects of acaricide fenazaquin on development, hemolymph metabolome, and gut microbiome of honeybee (Apis mellifera) larvae

Fenazaquin, a potent insecticide widely used to control phytophagous mites, has recently emerged as a potential solution for managing Varroa destructor mites in honeybees. However, the comprehensive impact of fenazaquin on honeybee health remains insufficiently understood. Our current study investigated the acute and chronic toxicity of fenazaquin to honeybee larvae, along with its influence on larval hemolymph metabolism and gut microbiota. Results showed that the acute median lethal dose (LD50) of fenazaquin for honeybee larvae was 1.786 μg/larva, and the chronic LD50 was 1.213 μg/larva. Although chronic exposure to low doses of fenazaquin exhibited no significant effect on larval development, increasing doses of fenazaquin resulted in significant increases in larval mortality, developmental time, and deformity rates. At the metabolic level, high doses of fenazaquin inhibited nucleotide, purine, and lipid metabolism pathways in the larval hemolymph, leading to energy metabolism disorders and physiological dysfunction. Furthermore, high doses of fenazaquin reduced gut microbial diversity and abundance, characterized by decreased relative abundance of functional gut bacterium Lactobacillus kunkeei and increased pathogenic bacterium Melissococcus plutonius. The disrupted gut microbiota, combined with the observed gut tissue damage, could potentially impair food digestion and nutrient absorption in the larvae. Our results provide valuable insights into the complex and diverse effects of fenazaquin on honeybee larvae, establishing an important theoretical basis for applying fenazaquin in beekeeping.

Nurse bees regulate the larval nutrition of developing workers (Apis mellifera) when feeding on various pollen types.

Nutrition has been identified as a key driver of colony health and productivity. Yet, in honey bees, relatively little is known about how the vast variety of natural pollen sources impact larval development. The impact of the nutritional quality of 4 naturally occurring pollen sources, of importance to the Western Australian beekeeping industry, was tested on honey bee (Apis mellifera L.) development. Bee packages consisting of 800 g of bees and a mated sister queen were assigned to 40 nucleus hives and randomly allocated to one of the 4 feed treatments (10 colonies each) of marri (Corymbia calophylla Lindl.), jarrah (Eucalyptus marginata Sm.), clover (Trifolium repens L.), and canola (Brassica napus L.) pollen. Emerging bees were collected once the first bees started hatching on the assigned feed sources. Newly emerging bees were weighed individually, and body composition was measured in batches according to the feed treatment groups. Food consumption was recorded for the duration of the experiment. Nurse bees successfully raised young adult workers from the larval stage until emergence when fed with one of 4 pollen patties with different nutritional qualities. There was no difference in the body composition or weight of emerging bees fed on the different pollen types. However, the body weight of bees increased over time, most likely related to colony size and structure. With the type of pollen patties having little impact on larval development, the availability of pollen may be more important than its composition, providing bees have access to all essential nutrients.

Changes in Vitellogenin, Abdominal Lipid Content, and Hypopharyngeal Gland Development in Honey Bees Fed Diets with Different Protein Sources.

Honey bees play an important role in the pollination of flowering plants. When honey bee colonies are deficient in pollen, one of their main nutrients, protein supplements are required. In this study, the effects of diets with six different protein sources on the physiological characteristics of worker bees (vitellogenin (Vg), abdominal lipid content (ALC), hypopharyngeal gland (HPG)) and consumption were investigated. The protein sources of the diets (diet I, …, diet VI) included pollen, spirulina dust (Arthrospira platensis Gomont), fresh egg yolk, lyophilized lactose-free skimmed milk powder, active fresh yeast, and ApiProtein. It was identified that consumption by worker bees was highest in the diet group supplemented with spirulina (diet II). Although there was no statistical difference regarding the Vg content in the hemolymph, numerically, the highest content was found in diet group IV (lyophilized lactose-free skimmed milk powder) (4.73 ± 0.03 ng/mL). ALC and HPG were highest in the group fed diet II. These results suggest that offering honey bees diets with certain protein sources can support their physiological traits.

Comparative Study of Amino Acids in the Different Stages of Honey Bee

Apiculture is the practice of keeping bees as well as the manufacturing of honey and beeswax. Nectar is the main source of carbohydrates for honey bees. The amount of nectar needed pet colony depends on how concentrated the sugars are in the nectar. A worker bee needs 11 mg of dry sugar each day, which translates to about 22 µl of 50% sugar syrup per bee per day. In order to make honeybees grow satisfactorily enough feed must be provided, and feed should have all the necessary nutrients for the growth of honeybees. The important nutrients such as amino acids, lipids, carbohydrates, and requirements of vitamins and minerals are necessary for the growth and development of honeybees. During development, several structural, physiological, and biochemical changes occur takes place in an organism from stage to stage. There is a variation in the quality of all biochemical components. In the present study, the free amino acids from the eggs, larvae, pupae, and adult honeybee Apis cerana indica and also honey have been analyzed. This work was carried out by adopting the technique of paper chromatography. The developed chromatograms were dried and the spots were identified by spraying with (O.IN) Ninhydrin in acetone. The Rf values were calculated and compared with the Rf values of the standard amino acids. The presence of amino acids in different life stages of honeybee and honey were analyzed and compared.

Assessment of Mitochondrial Function in the AmE-711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects.

There is a growing concern that chronic exposure to fungicides contributes to negative effects on honey bee development, life span, and behavior. Field and caged-bee studies have helped to characterize the adverse outcomes (AOs) of environmentally relevant exposures, but linking AOs to molecular/cellular mechanisms of toxicity would benefit from the use of readily controllable, simplified host platforms like cell lines. Our objective was to develop and optimize an in vitro-based mitochondrial toxicity assay suite using the honey bee as a model pollinator, and the electron transport chain (ETC) modulators boscalid and pyraclostrobin as model fungicides. We measured the effects of short (~30 min) and extended exposures (16-24 h) to boscalid and pyraclostrobin on AmE-711 honey bee cell viability and mitochondrial function. Short exposure to pyraclostrobin did not affect cell viability, but extended exposure reduced viability in a concentration-dependent manner (median lethal concentration = 4175 µg/L; ppb). Mitochondrial membrane potential (MMP) was affected by pyraclostrobin in both short (median effect concentration [EC50] = 515 µg/L) and extended exposure (EC50 = 982 µg/L) scenarios. Short exposure to 10 and 1000 µg/L pyraclostrobin resulted in a rapid decrease in the oxygen consumption rate (OCR), approximately 24% reduction by 10 µg/L relative to the baseline OCR, and 64% by 1000 µg/L. Extended exposure to 1000 µg/L pyraclostrobin reduced all respiratory parameters (e.g., spare capacity, coupling efficiency), whereas 1- and 10-µg/L treatments had no significant effects. The viability of AmE-711 cells, as well as the MMP and cellular respiration were unaffected by short and extended exposures to boscalid. The present study demonstrates that the AmE-711-based assessment of viability, MMP, and ETC functionality can provide a time- and cost-effective platform for mitochondrial toxicity screening relevant to bees. Environ Toxicol Chem 2024;43:976-987. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

An engineered bacterial symbiont allows noninvasive biosensing of the honey bee gut environment.

The honey bee is a powerful model system to probe host-gut microbiota interactions, and an important pollinator species for natural ecosystems and for agriculture. While bacterial biosensors can provide critical insight into the complex interplay occurring between a host and its associated microbiota, the lack of methods to noninvasively sample the gut content, and the limited genetic tools to engineer symbionts, have so far hindered their development in honey bees. Here, we built a versatile molecular tool kit to genetically modify symbionts and reported for the first time in the honey bee a technique to sample their feces. We reprogrammed the native bee gut bacterium Snodgrassella alvi as a biosensor for IPTG, with engineered cells that stably colonize the gut of honey bees and report exposure to the molecules in a dose-dependent manner through the expression of a fluorescent protein. We showed that fluorescence readout can be measured in the gut tissues or noninvasively in the feces. These tools and techniques will enable rapid building of engineered bacteria to answer fundamental questions in host-gut microbiota research.

The Effect of Comb Cell Size on the Development of Apis mellifera Drones.

The growth and development of honeybees are influenced by many factors, one of which is the cell size of the brood comb. Larger worker bees can be obtained by being raised in bigger cells. However, whether cell size has the same effect on drone development is still unknown. Here, using 3D-printed foundations, we observed the development of drones kept in comb cells of different sizes from the late larval stage through eclosion. The results showed that drones in larger cell-size combs had heavier body weights, longer body lengths, and larger head widths, thorax widths, and abdomen widths compared to those in smaller cell-size combs. Furthermore, regardless of developmental stages, the drones' body weights increased linearly with the comb's cell size. However, the other morphological changes of drones in different developmental stages were out of proportion to the cell-size changes, resulting in smaller cells with a higher fill factor (thorax width/cell width). Our findings confirm that comb cell size affects the development of honeybees; drones become bigger when raised in large cells.

Productive qualities of bees of the Central Russian and Carpathian breeds

The study evaluates the winter hardiness, spring development, and honey production of Central Russian honey bees (Apis mellifera mellifera) and Carpathian honey bees (Apis mellifera carpatica) in the Chelyabinsk region. The research analyzes key parameters of wintering bee colonies, including colony strength in autumn, food consumption, level of chalkbrood disease, and varroa mite resistance. Results indicate that Central Russian honeybees exhibit high winter hardiness and disease resistance, contributing to their resilience during cold winter periods. Despite originating from warmer climates, Carpathian honey bees also demonstrated good winter hardiness, albeit with some susceptibility to chalkbrood disease. Both bee breeds showed comparable spring development, directly influencing overall honey production. Comparing honey productivity data revealed that Central Russian honey bees significantly outperformed Carpathian bees in terms of honey yield per harvesting period. Thus, the study underscores the importance of selecting appropriate bee breeds to ensure successful wintering and high honey productivity in harsh climates, crucial for effective beekeeping in the Chelyabinsk region and similar environments.

Diverse Regulatory Manners and Potential Roles of lncRNAs in the Developmental Process of Asian Honey Bee (Apis cerana) Larval Guts.

Long non-coding RNAs (lncRNAs) are crucial modulators in a variety of biological processes, such as gene expression, development, and immune defense. However, little is known about the function of lncRNAs in the development of Asian honey bee (Apis cerana) larval guts. Here, on the basis of our previously obtained deep-sequencing data from the 4-, 5-, and 6-day-old larval guts of A. cerana workers (Ac4, Ac5, and Ac6 groups), an in-depth transcriptome-wide investigation was conducted to decipher the expression pattern, regulatory manners, and potential roles of lncRNAs during the developmental process of A. cerana worker larval guts, followed by the verification of the relative expression of differentially expressed lncRNAs (DElncRNAs) and the targeting relationships within a competing endogenous RNA (ceRNA) axis. In the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, 527 and 498 DElncRNAs were identified, respectively, which is suggestive of the dynamic expression of lncRNAs during the developmental process of larval guts. A cis-acting analysis showed that 330 and 393 neighboring genes of the aforementioned DElncRNAs were respectively involved in 29 and 32 functional terms, such as cellular processes and metabolic processes; these neighboring genes were also respectively engaged in 246 and 246 pathways such as the Hedgehog signaling pathway and the Wnt signaling pathway. Additionally, it was found that 79 and 76 DElncRNAs as potential antisense lncRNAs may, respectively, interact with 72 and 60 sense-strand mRNAs. An investigation of competing endogenous RNA (ceRNA) networks suggested that 75 (155) DElncRNAs in the Ac4 vs. Ac5 (Ac5 vs. Ac6) comparison group could target 7 (5) DEmiRNAs and further bind to 334 (248) DEmRNAs, which can be annotated to 33 (29) functional terms and 186 (210) pathways, including 12 (16) cellular- and humoral-immune pathways (lysosome pathway, necroptosis, MAPK signaling pathway, etc.) and 11 (10) development-associated signaling pathways (Wnt, Hippo, AMPK, etc.). The RT-qPCR detection of five randomly selected DElncRNAs confirmed the reliability of the used sequencing data. Moreover, the results of a dual-luciferase reporter assay were indicative of the binding relationship between MSTRG.11294.1 and miR-6001-y and between miR-6001-y and ncbi_107992440. These results demonstrate that DElncRNAs are likely to modulate the developmental process of larval guts via the regulation of the source genes' transcription, interaction with mRNAs, and ceRNA networks. Our findings not only yield new insights into the developmental mechanism underlying A. cerana larval guts, but also provide a candidate ceRNA axis for further functional dissection.

Host Preference Evaluation of Varroa destructor on Apis mellifera by Air-Flow Olfactometer

Four arm air-flow Olfactometer was used to study the behavior and host preference of Varroa destructor on different stages of Apis mellifera L. Four arm Olfactometer studies revealed that among adult castes, V. destructor exhibited strong orientation towards drone and recorded maximum mite population and percentage preference followed by nurse bees with no significant difference (p= 0.158). However, varroa mites were less attracted to foragers accounting mite population of 10.5 with percentage preference of 21%. Further, it was determined that the among all the larval stages, the odor produced by drone larvae elicited strong orientation behavior by recording a mite population of 18.5 mites with percentage preference of 37% followed by 18-20 hr old larvae with 12.25 mite population and were significantly different (p=0.015). Our results showed that the newly-emerged 2-3 hr old worker larvae were less attractive to mites. Based on the present studies it was evaluated that drone brood was preferred by Varroa mites among all the hosts. Drone brood trap method can be used for the efficient management of varroa in an integrated approach for the sustainable development and wellbeing of honeybees by reducing the application of chemical methods and resistance development in this mite.

Cellular heterogeneity of the developing worker honey bee (Apis mellifera) pupa: a single cell transcriptomics analysis.

It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behavior, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination, and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study, we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15) and sought to determine their gene expression signatures. To identify cell-type populations, we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 17 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene coexpression analysis. We combined this analysis with literature mining, the honey bee protein atlas, and gene ontology analysis to determine cell cluster identity. Of the cell clusters identified, 17 were related to the nervous system and sensory organs, 7 to the fat body, 19 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes, and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single-cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology at the cellular level.

Development Strategy and Existing Qualities of Honeybee in Beekeepers Group Badung Regency-Bali

The cultivation of local honeybees is one of the new alternative jobs for the local community in Badung Regency, Bali to survive their life during Covid-19. This research aims to identify and determine of development strategy and to evaluate the quality existing of honeybees. The total sample was used 50 respondents consisting of 22 members of beekeepers of the “Sarining Trigona Pertiwi” group, 4 samples from local government, 4 from related institution government, and 20 local people. The SWOT method is used to formulate appropriate strategies and IE matrix is to analyze the condition of the honeybee-tourism strategy. The results of the analysis showed that internal factors have a total value of 3.20, with the highest factor being strategic partners in product innovation development. External factor analysis has a total value of 2.88 with the highest factor being increasingly diverse product diversification. Based on the IE matrix, tourism in quadrant IV shows the potential development of honeybees in Badung Regency, Bali is in a position to grow and develop with the appropriate intensive and integrated strategy. Based on the SWOT matrix were obtained strategies to conduct socialization of the cultivation program, having a research program regularly, developing products based on consumer demand, collaborating with cooperatives and Banks in credit services, conducting marketing and good market expansion, making SOP in the process, manufacture and storage of products, government policies on reforestation and forest area protection, human resource training, preparing market analysis and strategies, alternative food sources for the honeybees, and increasing product development. However, the qualities of Tetragonula laevicep honeybees produced by the beekeeper of “Sarining Trigona Pertiwi” are still lower than Indonesia National Standard (SNI) therefore; need to increase the quality in the future.

The microbiome and gene expression of honey bee workers are affected by a diet containing pollen substitutes.

Pollen is the primary source of dietary protein for honey bees. It also includes complex polysaccharides in its outer coat, which are largely indigestible by bees but can be metabolized by bacterial species within the gut microbiota. During periods of reduced availability of floral pollen, supplemental protein sources are frequently provided to managed honey bee colonies. The crude proteins in these supplemental feeds are typically byproducts from food manufacturing processes and are rarely derived from pollen. Our experiments on the impact of different diets showed that a simplified pollen-free diet formulated to resemble the macronutrient profile of a monofloral pollen source resulted in larger microbial communities with reduced diversity, reduced evenness, and reduced levels of potentially beneficial hive-associated bacteria. Furthermore, the pollen-free diet sharply reduced the expression of genes central to honey bee development. In subsequent experiments, we showed that these shifts in gene expression may be linked to colonization by the gut microbiome. Lastly, we demonstrated that for bees inoculated with a defined gut microbiota, those raised on an artificial diet were less able to suppress infection from a bacterial pathogen than those that were fed natural pollen. Our findings demonstrate that a pollen-free diet significantly impacts the gut microbiota and gene expression of honey bees, indicating the importance of natural pollen as a primary protein source.

Expression Profile, Regulatory Network, and Putative Role of microRNAs in the Developmental Process of Asian Honey Bee Larval Guts.

MiRNAs, as a kind of key regulators in gene expression, play vital roles in numerous life activities from cellular proliferation and differentiation to development and immunity. However, little is known about the regulatory manner of miRNAs in the development of Asian honey bee (Apis cerana) guts. Here, on basis of our previously gained high-quality transcriptome data, transcriptome-wide identification of miRNAs in the larval guts of Apis cerana cerana was conducted, followed by investigation of the miRNAs' differential expression profile during the gut development. In addition to the regulatory network, the potential function of differentially expressed miRNAs (DEmiRNAs) was further analyzed. In total, 330, 351, and 321 miRNAs were identified in the 4-, 5-, and 6-day-old larval guts, respectively; among these, 257 miRNAs were shared, while 38, 51, and 36 ones were specifically expressed. Sequences of six miRNAs were confirmed by stem-loop RT-PCR and Sanger sequencing. Additionally, in the "Ac4 vs. Ac5" comparison group, there were seven up-regulated and eight down-regulated miRNAs; these DEmiRNAs could target 5041 mRNAs, involving a series of GO terms and KEGG pathways associated with growth and development, such as cellular process, cell part, Wnt, and Hippo. Comparatively, four up-regulated and six down-regulated miRNAs detected in the "Ac5 vs. Ac6" comparison group and the targets were associated with diverse development-related terms and pathways, including cell, organelle, Notch and Wnt. Intriguingly, it was noticed that miR-6001-y presented a continuous up-regulation trend across the developmental process of larval guts, implying that miR-6001-y may be a potential essential modulator in the development process of larval guts. Further investigation indicated that 43 targets in the "Ac4 vs. Ac5" comparison group and 31 targets in the "Ac5 vs. Ac6" comparison group were engaged in several crucial development-associated signaling pathways such as Wnt, Hippo, and Notch. Ultimately, the expression trends of five randomly selected DEmiRNAs were verified using RT-qPCR. These results demonstrated that dynamic expression and structural alteration of miRNAs were accompanied by the development of A. c. cerana larval guts, and DEmiRNAs were likely to participate in the modulation of growth as well as development of larval guts by affecting several critical pathways via regulation of the expression of target genes. Our data offer a basis for elucidating the developmental mechanism underlying Asian honey bee larval guts.

The mushroom body development and learning ability of adult honeybees are influenced by cold exposure during their early pupal stage.

The honeybees are the most important pollinator in the production of crops and fresh produce. Temperature affects the survival of honeybees, and determines the quality of their development, which is of great significance for beekeeping production. Yet, little was known about how does low temperature stress during development stage cause bee death and any sub-lethal effect on subsequent. Early pupal stage is the most sensitive stage to low temperature in pupal stage. In this study, early pupal broods were exposed to 20°C for 12, 16, 24, and 48h, followed by incubation at 35°C until emergence. We found that 48h of low temperature duration cause 70% of individual bees to die. Although the mortality at 12 and 16h seems not very high, the association learning ability of the surviving individuals was greatly affected. The brain slices of honeybees showed that low temperature treatment could cause the brain development of honeybees to almost stop. Gene expression profiles between low temperature treatment groups (T24, T48) and the control revealed that 1,267 and 1,174 genes were differentially expressed respectively. Functional enrichment analysis of differentially expressed genes showed that the differential expression of Map3k9, Dhrs4, and Sod-2 genes on MAPK and peroxisome signaling pathway caused oxidative damage to the honeybee head. On the FoxO signal pathway, InsR and FoxO were upregulated, and JNK, Akt, and Bsk were downregulated; and on the insect hormone synthesis signal pathway, Phm and Spo genes were downregulated. Therefore, we speculate that low temperature stress affects hormone regulation. It was detected that the pathways related to the nervous system were Cholinergic synapse, Dopaminergic synapse, GABAergic synapse, Glutamatergic synapse, Serotonergic synapse, Neurotrophin signaling pathway, and Synaptic vesicle cycle. This implies that the synaptic development of honeybees is quite possibly greatly affected by low temperature stress. Understanding how low temperature stress affects the physiology of bee brain development and how it affects bee behavior provide a theoretical foundation for a deeper comprehension of the temperature adaptation mechanism that underlies the "stenothermic" development of social insects, and help to improve honeybee management strategies to ensure the healthy of colony.